阅读说明：本技术 重楼皂苷ⅶ在制备msn蛋白抑制剂中的应用 (Application of paris polyphylla saponin VII in preparation of MSN protein inhibitor ) 是由 刘静 王海勤 萧小鹃 朱林 李珍珍 易慧 于 2020-12-05 设计创作，主要内容包括：本发明公开了重楼皂苷Ⅶ在制备MSN蛋白抑制剂中的应用,通过系列实验,证明了重楼皂苷Ⅶ具有抗多发性骨髓瘤的活性。且重楼皂苷Ⅶ通过直接抑制MSN蛋白水平而靶向多发性骨髓瘤干细胞样细胞侧群细胞而发挥了抗多发性骨髓瘤的活性。重楼皂苷Ⅶ可以作为MSN蛋白的抑制剂达到治疗因MSN蛋白质表达升高引起的疾病如多发性骨髓瘤等,有望被开发为新型的抗肿瘤药物。(The invention discloses application of paris polyphylla saponin VII in preparation of an MSN protein inhibitor, and series of experiments prove that the paris polyphylla saponin VII has activity of resisting multiple myeloma. And the paris saponin VII can target cells of a cell side population of stem cells of multiple myeloma by directly inhibiting the MSN protein level so as to exert the activity of resisting multiple myeloma. The paris saponin VII can be used as an inhibitor of MSN protein to treat diseases caused by the expression increase of the MSN protein, such as multiple myeloma and the like, and is expected to be developed into a novel anti-tumor medicament.)

1. Application of rhizoma paridis saponin VII in preparing MSN protein inhibitor is provided.

2. Application of rhizoma paridis saponin VII in preparing medicine for treating multiple myeloma is provided.

3. The use according to claim 2, wherein the use of Paris saponin VII is for the manufacture of a medicament for the treatment of relapsed/refractory multiple myeloma.

4. The application of the paris saponin VII in preparing the tumor stem cell inhibitor.

Technical Field

The invention relates to a new application of paris saponin VII, in particular to an application of paris saponin VII in preparation of an MSN protein inhibitor. Belongs to the technical field of medicine.

Background

Msn (moesin) is a member of the ezrin-radixin-moesin (erm) family of proteins, a protein that links the actin cytoskeleton to transmembrane receptors. The physiological role of ERM proteins is related to the architecture of the cell cortex and is involved in cell differentiation and morphogenesis. MSN is upregulated in a variety of human cancers, including breast, prostate, pancreatic, and lung and melanoma, among others, and is the ERM protein that is predominant in lymphocytes and is involved in T and B cell efflux from secondary lymphoid organs. CD44 can provide a link between the plasma membrane and the actin cytoskeleton by binding to ERM proteins (Ezrin, radixin, moesin). The MSN-CD44 interaction is a potential therapeutic target for glioblastoma, and the Wnt/beta-catenin pathway is a specific signal downstream of the MSN-CD44 interaction. In pancreatic ductal adenocarcinoma, Ezrin can regulate the characteristics of tumor stem cells and may be a new target for therapy. MSN is closely related to tumor stem cells and multidrug resistance. No specific inhibitors of MSN are currently available.

Despite recent advances in MM treatment, there remains no cure, with patients who are resistant and relapsed/refractory. Bortezomib (BTZ) drug resistance is a common type of drug resistance and is a problem that needs to be solved urgently in clinic. Multiple myeloma stem cell-like cells (MMSCs) are defined as a cell within malignant tissue that has the ability to self-renew and differentiate into the major lineage of myeloma plasma cells. MMSC is considered to be a root cause of drug resistance, and inhibition of MMSC becomes a new therapeutic strategy for MM. Currently, typical characteristics of MMSCs include increase in side population cells (SP cells), expression of classical stem cell genes, and clonogenic potential. The side population of cells is a population of cells that is low-stained with Hoechst33342, away from the main population of cells. Its phenotype is mainly associated with the ABC transporter family, a member of the ATP-dependent cell surface proteins. The ABC transporter family includes ABCG2, ABCB1, ABCC1 and the like. Genes closely related to sternness include Oct4, Nanog, Sox2, Lin28, Klf4 and the like, and the genes are highly expressed in stem cells, so that MMSC has stronger colony forming capability and tumorigenic capability.

In recent years, compound libraries have become indispensable tools in the process of new drug discovery, helping scientists to find lead compounds for new drug development. Cancer is a major health problem worldwide and natural products are considered as a new, reliable therapy for the treatment of different types of human cancers. More and more researches show that the discovery of the drug with obvious anti-tumor activity and the target protein thereof has important significance for promoting clinical treatment. Paris polyphylla saponin VII (Polyphyllin VII, PP7) is one of bioactive components of Paris polyphylla, and plays an important role in antitumor, antibacterial, antiviral, immunoregulation, hemostasis, etc. in modern medical research. The existing research shows that PP7 can inhibit the activity of various tumors, including lung cancer, nasopharyngeal carcinoma, liver cancer, breast cancer, oral cancer, colon cancer and the like, and the anti-tumor action mechanism comprises: induction of apoptosis, cell cycle inhibition and autophagy inhibition. In conclusion, PP7 is a broad-spectrum antitumor drug with important clinical development potential, but the role of PP7 in multiple myeloma and tumor stem cells and the role of target proteins are not clear. The paris saponin VII is widely existed in plants such as paris polyphylla and the like, and the natural medicine resources are rich, so the paris polyphylla saponin VII has great potential value in clinical treatment of tumors such as MM resistance and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the paris saponin VII in preparing the MSN protein inhibitor.

In order to achieve the purpose, the invention adopts the following technical scheme:

1. application of rhizoma paridis saponin VII in preparing MSN protein inhibitor is provided.

2. Application of rhizoma paridis saponin VII in preparing medicine for treating multiple myeloma is provided.

Preferably, the application of the paris saponin VII in preparing the medicine for treating relapsed/refractory multiple myeloma is provided.

3. The application of the paris saponin VII in preparing the tumor stem cell inhibitor.

The invention has the beneficial effects that:

the invention discloses a new application of paris polyphylla saponin VII in preparation of an MSN protein inhibitor. The invention proves that the paris saponin VII has the activity of resisting multiple myeloma through a series of experiments. And the paris saponin VII can target cells of a cell side population of stem cells of multiple myeloma by directly inhibiting the MSN protein level so as to exert the activity of resisting multiple myeloma. The paris saponin VII can be used as an inhibitor of MSN protein to treat diseases caused by the expression increase of the MSN protein, such as multiple myeloma and the like, and is expected to be developed into a novel anti-tumor medicament.

In vitro activity experiments of the paris saponin VII show that the paris saponin VII has obvious multiple pith resistance activity and small cytotoxicity. The paris saponin VII can directly inhibit the level of MSN protein in MM cells, has the function of selectively inhibiting the proliferation of various MM cells, has low cytotoxicity, can be used as a specific inhibitor of the MSN protein, can target side group cells to play the activity of resisting multiple myeloma by reducing the level of the MSN protein, achieves the aim of treating diseases such as MM and the like caused by abnormal expression of the MSN protein, and is expected to be developed into a novel anti-tumor targeted medicament.

Drawings

FIG. 1 shows the results of the comparison of Paris saponin VII on MM cell lines, normal human PBMC cells and MM patient primary CD138⁺The effect of cell viability; wherein, A is the influence of 2 mu M paris polyphylla saponin VII (PP7), paris polyphylla saponin VI (PP6) and paris polyphylla saponin I (PP1) on the survival rate of MM cell lines detected by a CCK-8 method; b, detecting the influence of the paris polyphylla saponin VII with different concentrations on the cell survival rate of immortalized B lymphocytes and MM cell lines by a CCK-8 method; c is CCK-8 method for detecting different concentrations of paris polyphylla saponin VII on normal human Peripheral Blood Mononuclear Cells (PBMC) and primary CD138 of MM patient⁺Effect of cell survival ARP1, KMS11, MM.1S, RPMI-8226, ANBL6 and ANBL6-BR cells were different MM cell lines. PBMC cells are normal human peripheral blood mononuclear cells, MM patient is primary CD138 of MM patient⁺A cell. ns: p > 0.05<0.05，**p<0.01，***p<0.001。

FIG. 2 shows the saponin of Paris polyphyllaVII inhibiting multiple myeloma stem cell-like cells and targeting side population cells; wherein, A is the influence of the flow detection of the Paris saponin VII on the cell ratio of ARP1 and RPMI-8226 cell side population; b, qPCR (quantitative polymerase chain reaction) is used for detecting the influence of the paris polyphylla saponin VII on ARP1 and RPMI-8226 cell dryness genes Oct4, Nanog, Sox2, Lin28 and Klf 4; c, after the side population and the main population of the ARP1 cells are separated in a flow mode, detecting the influence of the paridis saponin VII on the survival rate of the side population cells and the main population cells by a CCK-8 method; d is the influence of the paridis saponin VII on the clone forming capability of the side population cells and the main population cells detected by a soft agar clone forming experiment after the side population and the main population cells of the ARP1 cells are flow-sorted. NC is a control, PP7 is paris polyphylla saponin VII, V is Verapamil (Verapamul), and the Verapamil group is a negative control group. SP is the side population cell, MP is the main population cell. ns: p > 0.05<0.05，**p<0.01，***p<0.001; e-flow detection of Paris saponin VII on MM patient primary CD138⁺Influence of the ratio of cell-side population cells.

FIG. 3 shows that Paris saponin VII inhibits MSN protein; wherein, A is DARTS (target point stability method of drug affinity reaction) for searching target protein of Paris saponin VII. pronase is pronase, protease: total protein represents the protein mass ratio of the two; b is the change of the MSN protein detected by WB after the DARTS method is used for processing the sample; c is the change of MSN protein detected by WB after the sample is processed by CETSA (cell thermal transition analysis); d is WB to detect the difference of MSN expression in immortalized B lymphocyte and MM cell line of normal human, GAPDH is glyceraldehyde-3-phosphate dehydrogenase as reference; e is the change of MSN protein after detecting the treatment of ARP1, MM.1S, ANBL6 and ANBL6-BR cells by the Paris saponin VII by WB. B cell is immortalized lymphocyte of normal human, MM.1R, U266 are MM cell line. Beta-actin is beta actin, acting as an internal reference.

FIG. 4 is a graph of the ratio of interfering with the expression of MSN to inhibit the growth of MM cells and side population cells; wherein, A is the change of MSN in protein level in ARP1 cell detected by WB after the interference of shRNA added with MSN. GAPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal reference; b, after adding shRNA interference of MSN, cell counting detects the growth change of ARP1 cells; c is the change of SP ratio of ARP1 cells detected by flow after shRNA interference of MSN is added. Control sequences for shRNA p <0.05, p <0.01, p < 0.001.

FIG. 5 is a graph of promoting growth of MM cells and increasing the rate of lateral population cells after overexpression of MSN; among them, after adding MSN over-expression slow virus vector, WB detects MSN change at protein level in ARP1 cell. GAPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal reference; b, after an overexpression lentiviral vector of MSN is added, cell counting is carried out to detect the growth change of the ARP1 cells; c, after an overexpression lentiviral vector of MSN is added, the change of the cell ratio of the ARP1 cell side population is detected in a flow mode; d is primary CD138 of MM patients detected by WB after adding MSN over-expression lentiviral vector⁺Changes in MSN at the protein level in the cells. GAPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal reference; e is the primary CD138 of MM patients detected by CCK-8 after adding an over-expression lentivirus vector of MSN⁺Change in cell viability. EV is lentivirus empty vector, OE-MSN is lentivirus vector after over-expression of MSN, Verapamil (Verapamul) group is negative control group, p<0.01。

FIG. 6 shows that Paris saponin VII inhibits ANGL 6-BR cell neoplasia; wherein, A is a picture taken by a camera after a tumor under the skin of the mouse is taken out; b is a mouse tumor volume growth curve; c is the weight change curve of the mouse; d is the isolated volume of the mouse tumor; e is the mouse tumor in vitro body weight; f is immunohistochemical assay Ki 67; g is a Bortezomib (BTZ) drug-resistant cell strain for immunohistochemical detection that MSN ANBL6-BR cells are MM cell strain ANBL 6.

FIG. 7 shows that the Paris saponin VII or the knock-down MSN inhibits ARP1 cell neoplasia; wherein A is the photograph taken by a small animal imager after injecting 100mg/kg D-fluorescein into the abdominal cavity of each mouse; b, taking out subcutaneous tumors of the mice, and taking pictures by using a camera; c is a mouse tumor volume growth curve; d is a mouse weight change curve; e is mouse tumor ex vivo body weight.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.

The purity of the paris saponin VII related to the invention is 98 percent (mass percentage).

Example 1: paris saponin VII was identified as an ideal natural compound monomer for the treatment of multiple myeloma

Paris saponin VII (PP7), Paris saponin VI (PP6), and Paris saponin I (PP1) (all from Selleck Biotechnology Co., Ltd., USA) have antitumor activity. We first treated MM cells mm.1s (from ATCC), ARP1, ANBL6 and ANBL6-BR cells (all from professor zhong hei tumor institute, university of south and central university) with these 3 monomers at a concentration of 2 μ M, and expected to obtain the natural product monomer with the best antitumor activity in four cells. After the paris polyphylla saponin VII is proved to have the most remarkable anti-tumor effect, the paris polyphylla saponin VII is detected by a CCK-8 method to immortalize B lymphocytes (from the genetics laboratory of the institute of Life sciences of the university of Central and south China) and MM cell lines, normal PBMC cells (from the Hunan Yao hospital of the university of Central and south China) and primary CD138 of MM patients⁺Influence of cell survival (from tumor hospital, Hunan province) to assess the sensitivity of different cell types to Paris saponin VII.

1. CCK-8 method for detecting influence of 2 mu M of 3 types of paris polyphylla saponin on cell survival rate of MM.1S, ARP1, ANBL6 and ANBL6-BR

(1) Mm.1s, ARP1, ANBL6 and ANBL6-BR cells were grown to log phase, resuspended in a 15mL centrifuge tube and blown out to mix. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(2) A plate group of wells containing no cells but complete medium (RPMI-1640 medium + 10% BI fetal bovine serum + 1% double antibody) and a control group containing no drug, only cells and complete medium were set, and 4 duplicate wells were set for each concentration.

(3) Adding 2 μ M of each of 3 kinds of rhizoma paridis saponin, inoculating cell (about 8 × 10 per well in 96-well plate)³Individual cells) were grown in 96-well plates, 4 wells per group, for 24 h.

(4) 1/10 volumes of Cell Counting Kit-8(CCK-8) were added directly to the Cell culture medium and mixed thoroughly to ensure homogeneity of color in the wells and avoid the formation of bubbles as much as possible. For a 96-well plate, 10. mu.l of the activity detection reagent was added per 100. mu.l of the culture medium.

(5) And (5) continuing to culture for 3 hours until the color becomes orange, reading the light absorption value at 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

Cell viability [ (%) [ a (medicated) -a (blank) ]/[ a (0 medicated) -a (blank) ] × 100

A (dosing): absorbance of wells with cells, CCK8 solution and drug solution

A (blank): absorbance of wells with medium and CCK8 solution without cells

A (0 dosing): absorbance of wells with cells, CCK8 solution, but no drug solution

Cell viability (%): cell proliferation Activity or cytotoxic Activity

2. CCK-8 method for detecting influence of different concentrations of paris saponin VII on cell survival rate of MM cell line

(1) Normal human immortalized lymphocytes, ARP1, KMS11, MM.1S, RPMI-8226 and ANBL6 cells were grown to log phase, resuspended in 15mL centrifuge tubes, and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(2) A plate group of wells containing no cells but complete medium (RPMI-1640 medium + 10% BI fetal bovine serum + 1% double antibody) and a control group containing no drug, only cells and complete medium were set, and 4 duplicate wells were set for each concentration.

(3) Adding 0.5 μ M, 1 μ M and 2 μ M rhizoma paridis saponin VII, respectively, and inoculating cells (about 8 × 10 per well in 96-well plate)³Individual cells) were grown in 96-well plates, 4 wells per group, for 24 h.

(4) 1/10 volumes of Cell Counting Kit-8(CCK-8) were added directly to the Cell culture medium and mixed thoroughly to ensure homogeneity of color in the wells and avoid the formation of bubbles as much as possible. For a 96-well plate, 10. mu.l of the activity detection reagent was added per 100. mu.l of the culture medium.

(5) And (5) continuing to culture for 3 hours until the color becomes orange, reading the light absorption value at 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

3. CCK-8 method for detecting paris polyphylla saponin VII with different concentrations on normal human PBMC cells and primary CD138 of MM patients⁺Effect of cell survival

(1) Bone marrow mononuclear cells of MM patients were isolated from lymphocyte isolates.

(2) Sorting CD138 from mononuclear cells by CD138 magnetic beads⁺A cell.

(3) Will CD138⁺The primary cells were resuspended in a 15mL centrifuge tube and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(4) A plate group of wells containing no cells but complete medium (RPMI-1640 medium + 10% BI fetal bovine serum + 1% double antibody) and a control group containing no drug, only cells and complete medium were set, and 4 duplicate wells were set for each concentration.

(5) Adding 0.5 μ M, 1 μ M and 2 μ M rhizoma paridis saponin VII, respectively, and inoculating cells (about 8 × 10 per well in 96-well plate)³Individual cells) were grown in 96-well plates, 4 wells per group, for 24 h.

(6) 1/10 volumes of Cell Counting Kit-8(CCK-8) were added directly to the Cell culture medium and mixed thoroughly to ensure homogeneity of color in the wells and avoid the formation of bubbles as much as possible. For a 96-well plate, 10. mu.l of the activity detection reagent was added per 100. mu.l of the culture medium.

(7) The culture was continued for 12 hours until the color became orange, and the absorbance at 450nm was read with a microplate reader to calculate the cell survival rate.

The experimental result is shown in figure 1, wherein A is the effect of 2 mu M paris saponin VII (PP7), paris saponin VI (PP6) and paris saponin I (PP1) on the survival rate of the MM cell line detected by a CCK-8 method; b, detecting the influence of the paris polyphylla saponin VII with different concentrations on the cell survival rate of immortalized B lymphocytes and MM cell lines by a CCK-8 method; c is CCK-8 method for detecting different concentrations of paris polyphylla saponin VII on normal human Peripheral Blood Mononuclear Cells (PBMC) and primary CD138 of MM patient⁺Effect of cell survival ARP1, KMS11, MM.1S, RPMI-8226, ANBL6 and ANBL6-BR cells were different MM cell lines. PBMC cells are normal human peripheral blood mononuclear cells, MM patient is primary CD138 of MM patient⁺A cell. ns: p > 0.05<0.05，**p<0.01，***p<0.001. Parsaponin VII is most active in four MM cells, and in four MM cellsThe IC50 was less than 2 μ in all species. The paris saponin VII can obviously inhibit the proliferation of MM cell lines ARP1, KMS11, MM.1S, RPMI-8226, ANBL6 and ANBL6-BR cells, but has lower inhibition rate on normal human B lymphocytes. Paris saponin VII capable of remarkably inhibiting CD138⁺The proliferation of primary cells is small in inhibition and proliferation effects on normal human PBMC, so that the anti-MM peptide has remarkable anti-MM activity, small side effects on normal cells and strong selectivity.

Example 2: parsaponin VII inhibits multiple myeloma stem cell-like cells and targets side population cells.

The influence of the paris polyphylla saponin VII on the cell ratio of ARP1 and RPMI-8226 cell side population is detected by adopting a flow type, the influence of the paris polyphylla saponin VII on ARP11 and RPMI-8226 cell dryness genes Oct4, Nanog, Sox2, Lin28 and Klf4 is detected by qPCR, after the side population and main population cells of ARP1 cells are sorted by flow type, the influence of the paris polyphylla saponin VII on the survival rate of the side population cells and the main population cells is detected by CCK-8, and the influence of the paris polyphylla saponin VII on the clonogenic capacity of the side population cells and the main population cells is detected by a soft agar clonogenic experiment. And detecting primary CD138 of MM patient by using rhizoma paridis saponin VII through flow type⁺Influence of the cell side population cell ratio.

The experimental procedure was as follows:

1. flow detection of influence of Paris saponin VII on ARP1 and RPMI-8226 cell side population cell ratio

(1) Cells were harvested by centrifugation at 800rpm for 5min, with cells in each sample > 5X 10⁵

(2) The cells were resuspended (cell density about 10) in RPMI-1640 medium containing 2% by volume fetal bovine serum, 10mM HEPES buffer, 1% by volume penicillin-bis-antibody⁶one/mL)

(3) Incubating the cell fluid with 5. mu.g/mL Hoechst33342 in a water bath at 37 ℃ for 90min (after staining, the cell fluid is kept at 4 ℃)

(4) Centrifuging at 1000rpm for 10min at 4 deg.C

(5) The cells were resuspended in phosphate buffer containing 2% fetal bovine serum, 10mM HEPES by volume.

(6) Adding PI with final concentration of 2ug/mL before flow detection, staining for 5min, and detecting on flow cytometer.

2. qPCR (quantitative polymerase chain reaction) detection of influence of paris polyphylla saponin VII on ARP1 and RPMI-8226 cell dryness genes Oct4, Nanog, Sox2, Lin28 and Klf4

(1) ARP1 and RPMI-8226 cells were grown to log phase, and cells were treated with 1. mu.M Paris Saponin VII. The cells were resuspended in a 15mL centrifuge tube and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(2) RNA extraction: centrifuging at 1000rpm for 5min, removing supernatant, adding 1mL TRIzol into cell precipitate, mixing, and standing on ice for 10min to dissolve cells in TRIzol completely.

(3) To TRIzol was added 300. mu.L of chloroform, and after mixing by inversion, it was left at room temperature for 15 min. The colorless clear upper aqueous phase was pipetted into a new 1.5mL centrifuge tube. Adding isopropanol with the same volume as the upper layer water phase, reversing, mixing, and standing at room temperature for 15 min. Centrifuge at 12000rpm at 4 ℃ for 10min and discard the supernatant.

(4) To the cell pellet, 1mL of 75% ethanol solution was added, centrifuged at 8000rpm and 4 ℃ for 5min, and the supernatant was discarded.

(5) After filtration, DEPC water is added for dissolving, and the RNA concentration is measured.

(6) Reverse transcription: the RNA is subjected to reverse transcription by using a reverse transcription kit to obtain cDNA.

(7) qPCR 10. mu.L of qPCR reaction system, 3. mu.l of cDNA, 5. mu.l of 2 XSSYBR Green Mix, 1. mu.l of Forward Primer and 1. mu.l of Reverse Primer were prepared, and qPCR experiments were performed under the following reaction conditions:

3. after flow-sorting side population and main population cells of ARP1 cells, CCK-8 was used to determine the effect of paridis saponin VII on the survival rate of side population cells and main population cells

(1) When ARP1 was grown to log phase, the side and main populations were sorted using flow cytometry

(2) The cells were resuspended in a 15mL centrifuge tube and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(3) The plate group with medium without cells and the DMSO control group with proper concentration without drug are set, and each concentration is set with 4 multiple wells.

(4) Adding 0.5 μ M, 1 μ M and 2 μ M rhizoma paridis saponin VII, respectively, and inoculating cells (about 8 × 10 per well in 96-well plate)³Individual cells) were grown in 96-well plates, 4 wells per group, for 24 h.

(5) 1/10 volumes of Cell Counting Kit-8(CCK-8) were added directly to the Cell culture medium and mixed thoroughly to ensure homogeneity of color in the wells and avoid the formation of bubbles as much as possible. For a 96-well plate, 10. mu.l of the activity detection reagent was added per 100. mu.l of the culture medium.

(6) And (5) continuing to culture for 3 hours until the color becomes orange, reading the light absorption value at 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

4. After the side population and the main population of the ARP1 cells are subjected to flow sorting, a soft agar clone formation experiment is carried out to detect the influence of the paris saponin VII on the clone formation capability of the side population cells and the main population cells.

(1) When ARP1 grew to log phase, the side and main population cells were sorted using flow cytometric sorting.

(2) Respectively preparing agarose with the mass concentration of 3.5 percent and the mass concentration of 1.66 percent, sterilizing the agarose under high pressure, and standing the agarose at room temperature for later use.

(3) The agarose prepared in step 1 was melted in a microwave oven, and 3.5% and 1.66% agarose were placed in 42-degree and 37-degree water baths, respectively, while RPMI-1640 medium containing 20% by volume fetal bovine serum and 1% by volume penicillin was placed in a 42-degree water bath.

(4) Preparing a lower layer adhesive: 3.5% agarose and medium were mixed according to 1: 5, the agarose is added into the culture medium, and no air bubbles are generated when the agarose is mixed. Immediately after the mixing, the mixture was dropped into a 12-well plate (from the center of the well to the bottom) at 1 ml/well and allowed to stand at room temperature for 10 minutes.

(5) Preparing upper layer glue: diluting the cells and the paris saponin VII in a culture medium according to a certain density, and then mixing 1.66% agarose and the culture medium containing the cells according to the proportion of 1: 5, taking care that the agarose is added into the culture medium, dripping 0.5mL of the mixture onto the lower layer gel after uniform mixing, taking care that air bubbles are not generated, standing for 5 minutes at room temperature, and then transferring the 12-hole plate to a cell incubator for culture. 12 orifice plate: 3000 cells/well.

(6) And (4) dropwise adding a drop of fresh culture medium into the hole every 2-3 days, photographing after 2-3 weeks, and calculating the clone formation rate.

5. Flow-type detection of primary CD138 of Paris polyphylla saponin VII on MM patient⁺Effect of cell side population cell ratio

(1) Bone marrow mononuclear cells of MM patients were isolated from lymphocyte isolates.

(2) Sorting CD138 from mononuclear cells by CD138 magnetic beads⁺A cell.

(3) Will CD138⁺The primary cells were resuspended in a 15mL centrifuge tube and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(4) The cells were collected by centrifugation at 1500rpm for 5min, and the number of cells in each sample was 5X 10⁵

(5) The cells were resuspended (cell density about 10) in RPMI-1640 medium containing 2% by volume fetal bovine serum, 10mM HEPES buffer, 1% by volume penicillin-bis-antibody⁶one/mL)

(6) Incubating the cell fluid with 5. mu.g/mL Hoechst33342 in a water bath at 37 ℃ for 90min (after staining, the cell fluid is kept at 4 ℃)

(7) Centrifuging at 1500rpm for 10min at 4 deg.C

(8) The cells were resuspended in phosphate buffer containing 2% fetal bovine serum, 10mM HEPES by volume.

(9) Adding PI with final concentration of 2ug/mL before flow detection, staining for 5min, and detecting on flow cytometer.

The experimental result is shown in figure 2, wherein A is the influence of the flow detection of the paridis saponin VII on the cell ratio of ARP1 and RPMI-8226 cell side population; b, qPCR (quantitative polymerase chain reaction) is used for detecting the influence of the paris polyphylla saponin VII on ARP1 and RPMI-8226 cell dryness genes Oct4, Nanog, Sox2, Lin28 and Klf 4; c is flow sorting of side and main population details of ARP1 cellsAfter the cells are treated, the influence of the Paris saponin VII on the survival rate of the side group cells and the main group cells is detected by a CCK-8 method; d is the influence of the paridis saponin VII on the clone forming capability of the side population cells and the main population cells detected by a soft agar clone forming experiment after the side population and the main population cells of the ARP1 cells are flow-sorted. NC is a control, PP7 is paris polyphylla saponin VII, V is Verapamil (Verapamul), and the Verapamil group is a negative control group. SP is the side population cell, MP is the main population cell. ns: p > 0.05<0.05，**p<0.01，***p<0.001; e-flow detection of Paris saponin VII on MM patient primary CD138⁺Influence of the ratio of cell-side population cells. In ARP1 and RPMI-8226 cells, the paris saponin VII can obviously inhibit the ratio of lateral population cells and obviously reduce the expression of the sternness genes Oct4, Sox2, Nanog, Lin28 and Klf 4. And after the side population and the main population of the ARP1 cells are separated, the paris saponin VII can still obviously inhibit the ratio of the side population cells, but the inhibition rate of the main population cells is far less than that of the side population cells under the same concentration, and the paris saponin VII also obviously inhibits the ratio of the side population cells in primary cells, which shows that the paris saponin VII can play a role in inhibiting proliferation through the side population cells.

Example 3: paris saponin VII for inhibiting MSN protein

And (3) searching target protein of the paris polyphylla saponin VII by using DARTS (target point stability method of drug affinity reaction). And treating the sample by a DARTS method and a CETSA (cell thermal transition analysis) method, verifying whether the MSN protein is a target protein of the paris polyphylla saponin VII by WB, detecting the difference of MSN expression in an immortalized B lymphocyte and an MM cell line of a normal human by WB, and directly detecting the change of the MSN protein after the paris polyphylla saponin VII is treated by ARP1 and MM.1S cells by WB. Researches show that small molecule drugs can stabilize the conformation morphology of the protein after being combined with the protein, so that the small molecule drugs can resist the hydrolysis of protease, and the DARTS method is invented based on the principle. The CETSA method is invented based on the principle that the drug ligand also induces the thermal stability of the target protein.

1. Target protein for searching paris polyphylla saponin VII by DARTS method

(1) Collecting 2X 10⁷Mm.1s cells in log phase of growth. Lysis of cells with appropriate volumes of M-PER supplemented with protease and phosphatase inhibitorsCell lysis was performed at room temperature for 5 min.

(2) Centrifuge at 13800rpm for 15 minutes at 4 ℃. The supernatant was transferred to a medium containing 10 XTNC buffer (500mM Tris-HCl (pH8.0), 500mM NaCl, 100mM CaCl₂) In the new tube of (2). The BCA method measures protein concentration.

(3) The lysate was aliquoted in several EP tubes. The appropriate concentration of drug or an equal volume of solvent is added. Incubate in water bath for 60min at 25 ℃.

(4) The incubated samples were proteolyzed with different concentrations of pronase (pronase) and incubated in a water bath at 25 ℃ for 30 min.

(5) Adding protease inhibitor and phosphatase inhibitor, adding 2 × loading buffer, boiling protein at 95 deg.C for 10min, and loading.

(6) SDS-PAGE, and the remaining samples were stored at-80 ℃.

(7) And (3) dyeing or silver dyeing by using Coomassie brilliant blue R250, decoloring by using a decoloring solution, and photographing after decoloring.

2. WB detection of MSN protein changes after sample processing by DARTS method

(1) DARTS method for processing samples, as described above

(2) Extracting total cell protein: the cells were collected and centrifuged at 800rpm at 4 ℃ for 5 min. Adding a certain amount of RIRA lysate containing protease inhibitor cocktail and phosphatase inhibitor, mixing the cells and cell lysate completely, and ice-cooling for 30 min. Sonicate 20 times. Centrifugation was carried out at 13800rpm at 4 ℃ for 15min, and the supernatant was collected.

(3) BCA method for determining protein concentration

(4) Western Blot protein isolation: protein samples were boiled for denaturation for 5 minutes, separated by SDS-PAGE at 10% mass concentration, membrane-transferred with NC membrane, and blocked with blocking solution (5% volume skim milk prepared from PBST solution) at room temperature for 2 hours. Adding primary antibody, incubating for 10-12 hours, washing for 3 times by PBST, incubating for 2 hours at room temperature by using secondary antibody diluted by a sealing solution, and washing for 3 times by using PBST;

(5) changes in expression of the protein MSN were detected by ECL imaging and gel imaging system.

3. WB detection of MSN protein Change after sample treatment by CETSA (cellular thermal transition analysis)

(1) ARP1 cells were plated evenly on 4 dishes and incubated with 1, 2, 4. mu.M Polyphyllin VII or sterile water equivalent, each at 37 ℃ for 2 h.

(2) After washing with PBS, the cells were resuspended in 500. mu.l PBS containing freshly added protease inhibitor and evenly divided into 10 tubes. The cells in each tube were heated at the indicated temperature (41, 44, 47, 50, 53, 56, 59, 62, 65, 68 ℃) for 3 minutes and held at room temperature (25 ℃) for 3 minutes.

(3) The heated cells were lysed by freezing in liquid nitrogen (1 min) and thawing in water at room temperature (1 min).

(4) The cell lysate was centrifuged at 20000g at 4 ℃ for 20 minutes and the soluble fraction was isolated for immunoblot analysis.

(5) The proteins were separated by Western Blot and visualized by ECL, as described above.

4. WB detection of differences in MSN expression in Normal human immortalized B-lymphocyte and MM cell lines

(1) Collecting 2X 10⁶Mm.1s, mm.1r (from professor zhou chou institute of tumor study, university of south and middle school), KMS11, ARP1, RPMI-8226, ANBL6, U266 (from professor zhou chou institute of tumor study, university of south and middle school), and human immortalized B lymphocytes at the log phase of growth.

(2) Total protein was extracted from cells by RIRA, protein concentration was measured by BCA method, and protein was separated by Western Blot and developed by ECL method as described above.

5. WB (B-site binding) detection of MSN protein changes after treatment of ARP1 and MM.1S cells by using Paris saponin VII

(1) ARP1 and MM.1S cells in log phase of growth were treated with 0.5, 1. mu.M polyphyllin VII or sterile water and cultured in an incubator for 24h, respectively.

(2) Collecting 2X 10⁶ARP1 and MM.1S cells, total cell protein extraction by RIRA, protein concentration determination by BCA method, protein separation by Western Blot, development by ECL, the same as described above

The experimental results are shown in fig. 3, wherein a is DARTS (target stability method for drug affinity reaction) to search for the target protein of the paris saponin vii. pronase is pronase, protease: total protein represents the protein mass ratio of the two; b is the change of the MSN protein detected by WB after the DARTS method is used for processing the sample; c is the change of MSN protein detected by WB after the sample is processed by CETSA (cell thermal transition analysis); d is WB to detect the difference of MSN expression in immortalized B lymphocyte and MM cell line of normal human, GAPDH is glyceraldehyde-3-phosphate dehydrogenase as reference; e is WB to detect the change of MSN protein after the paris saponin VII treats ARP1 and MM.1S cells. Both MM.1R and U266 are MM cell lines. Beta-actin is beta actin, acting as an internal reference.

And (3) processing a sample by using a DARTS method, then carrying out Coomassie brilliant blue staining, successfully finding out a specific difference band of the paris polyphylla saponin VII, and carrying out mass spectrometry on the band to find the MSN protein. WB verification is carried out after the sample is treated by DARTS method, and the MSN protein is degraded by pronase less under the condition that the Paris saponin VII exists, which shows that the MSN protein is protected by the Paris saponin VII. Similarly, WB verification was performed after treatment of the samples by the CETSA method, and it was also found that the degradation rate of MSN protein was significantly less than the control in the presence of the paris saponin vii at elevated temperatures. The above two experiments show that MSN is indeed the protein to which the paris saponin VII is directly bound. After WB detects the difference of MSN expression in immortalized B lymphocyte and MM cell line of normal human, MSN is found to be highly expressed in MM cell line, and after treatment of polyphyllin VII, the expression of ARP1 and MM.1S cell MSN protein level is obviously inhibited. In conclusion, the MSN protein is a target protein of the paris polyphylla saponin VII, and the paris polyphylla saponin VII directly inhibits the MSN protein.

Example 4: silencing expression of MSN inhibits growth of MM cells and the ratio of lateral population cells

After reducing the expression of MSN by shRNA, WB detects the change of MSN in ARP1 cells on protein level, cell count detects the change of ARP1 cell growth, and flow detects the change of ARP1 cell side group ratio.

The experimental procedure was as follows:

1. WB measures changes in MSN at the protein level in ARP1 cells following shRNA interference with MSN.

(1) And (3) interfering the MSN expression, namely, uniformly blowing and mixing the cells in the logarithmic phase in the culture dish, respectively taking 0.5ml of cell suspension into four holes of a six-hole plate, respectively marking the cell suspension into a Scramble group (a control group) and an sh1 and sh2 group (a knock-down group), adding shRNA into the knock-down group, and setting one auxiliary hole. The cells in the six-well plate were added 1640 medium (1.5 ml) to each well, and cultured for 24 hours.

(2) And changing the culture solution, and adding 2mL of complete culture medium into each hole for culture.

(3) Extracting total cellular protein, the same method as described above

(4) Protein concentration was measured by BCA method, which was the same as the above method

(5) Western Blot for protein isolation, the same as described above

(6) Development with ECL was carried out in the same manner as described above.

2. Cell counting to detect changes in ARP1 cell growth after addition of shRNA to reduce MSN expression

(1) Interfering with MSN expression, the same as described above

(2) The cells were resuspended in a 15mL centrifuge tube and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(3) 3000 cells were added to a 96-well plate, cultured for 1, 3, 5, and 7 days, and counted every two days to draw a growth curve.

3. Flow assay of changes in lateral population cell ratios following addition of shRNA to reduce MSN expression

(1) Interfering with MSN expression, the same as described above

(2) The flow detection of SP is the same as described above.

The experimental results are shown in fig. 4, wherein a is the change of MSN at the protein level in ARP1 cells detected by WB after shRNA interference by adding MSN. GAPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal reference; b, after adding shRNA interference of MSN, cell counting detects the growth change of ARP1 cells; c is the change of SP ratio of ARP1 cells detected by flow after shRNA interference of MSN is added. Control sequences for shRNA p <0.05, p <0.01, p < 0.001. After the interference of the expression of the MSN, the expression of the MSN on the protein level is obviously reduced, the growth of MM cells is obviously inhibited, and the lateral population cell ratio is obviously reduced. Upon knockdown of MSN, the growth of MM cells and the ratio of stem cell-like cells were inhibited.

Example 5: promoting growth of MM cells and increasing lateral population cell ratio after overexpression of MSN

Adding an overexpression lentiviral vector of MSN to increase the expression of MSN, detecting the change of MSN in protein level in ARP1 cells by WB, detecting the change of growth of ARP1 cells by cell count, and detecting the change of cell ratio of ARP1 cell side group by flow.

The experimental procedure was as follows:

1. after addition of the MSN overexpressing lentiviral vector, WB detected changes in MSN at the protein level in ARP1 cells.

(1) Overexpression of MSN: and (3) uniformly blowing and mixing the cells in the logarithmic phase in the culture dish, respectively taking 0.5ml of cell suspension into four wells of a six-well plate, respectively marking the cell suspension into an EV group (no-load control group) and an OEMSN group (over-expression group), adding a no-load lentiviral vector into the no-load control group, adding an over-expression lentiviral vector into the over-expression group, and setting one auxiliary well. 0.5ml of 1640 medium was added to each well containing cells in the six-well plate, and the cells were cultured for 12 hours.

(2) The cells were collected and centrifuged at 800rpm at 4 ℃ for 5 min. The supernatant was discarded, 3mL of medium was added to resuspend the cells, and the cells were cultured in a six-well plate for 48 h.

(3) Extracting total cellular protein, the same method as described above

(4) Protein concentration was measured by BCA method, which was the same as the above method

(5) Western Blot for protein isolation, the same as described above

(6) Development with ECL was carried out in the same manner as described above.

2. Cell counts detected changes in ARP1 cell growth after addition of MSN overexpressing lentiviral vectors

(1) Overexpression of MSN, same as above

(2) The cells were resuspended in a 15mL centrifuge tube and blown out and mixed well. The cell density was calculated by using a cell counting plate in a centrifuge tube in which the cells were diluted 10-fold to 1.5 ml.

(3) 3000 cells were added to a 96-well plate, cultured for 1, 3, 5, and 7 days, and counted every two days to draw a growth curve.

3. After addition of the MSN over-expressing lentiviral vector, changes in the ARP1 cell side population cell ratio were flow-detected.

(1) Overexpression of MSN, as described above

(2) Flow assay for SP was performed as described above.

4. After addition of MSN-overexpressing lentiviral vectors, WB assay for primary CD138 in MM patients⁺Changes in MSN at the protein level in the cells. The same as above.

5. After addition of an over-expressing lentiviral vector for MSN, the MM patients were examined by cell counting for primary CD138⁺A change in cell growth. The same as above.

The results are shown in FIG. 5, where A is an overexpression lentiviral vector with added MSN, and WB detects changes in the protein level of MSN in ARP1 cells. GAPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal reference; b, after an overexpression lentiviral vector of MSN is added, cell counting is carried out to detect the growth change of the ARP1 cells; c, after an overexpression lentiviral vector of MSN is added, the change of the cell ratio of the ARP1 cell side population is detected in a flow mode; d is primary CD138 of MM patients detected by WB after adding MSN over-expression lentiviral vector⁺Changes in MSN at the protein level in the cells. GAPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal reference; e is the primary CD138 of MM patients detected by CCK-8 after adding an over-expression lentivirus vector of MSN⁺Change in cell viability. EV is lentivirus empty vector, OE-MSN is lentivirus vector after over-expression of MSN, Verapamil (Verapamul) group is negative control group, p<0.01. ARP1 cells and MM patients Primary CD138⁺After MSN is over-expressed in the cells, the expression of the MSN on the protein level is obviously improved, the growth capacity of MM cells is obviously enhanced, and the SP ratio is obviously improved. Overexpression of MSN promotes the growth of MM cells and increases the rate of side population cells.

Example 6: paris saponin VII for inhibiting ANBL6-BR cell neoplasia

(1) ANBL6-BR cells were collected using 1640 medium adjusted to 200. mu.l containing 3X 10⁶Cell sap of individual ANBL6-BR cells.

(2) 8 NCG mice, 5-8 weeks old, were selected with weights between 18-22 g. The cells were mixed well in a 1.5mL centrifuge tube, 100. mu.L of the cell fluid was aspirated by a 1mL syringe, and the cell fluid was inoculated into the subcutaneous part of the middle and rear part of the right axilla of the NCG mouse.

(3) When tumors were measurable (i.e., day 14), 8 NCG mice were randomly divided into two groups.

(4) On day 14, treatment included intraperitoneal injection of vehicle (control) or polyphyllin vii (10mg/kg/2 days). Tumor volume and body weight of each mouse were recorded 1 time every 2 days.

(5) The mice were euthanized by day 28 when tumors were very significant in the control mice for 16 consecutive days (total of 8). And (4) drawing a change curve of the tumor volume and the body weight of the mouse, photographing the isolated tumor, and recording the isolated volume and the isolated body weight of the tumor.

(6) The mouse tumor was continuously ground, and after passing through a cell strainer, the cells were collected, and the protein was extracted and Western Blot was performed as described above.

(7) Taking fresh tumor tissue of mouse, and performing immunohistochemistry

1) Paraffin section dewaxing to water: putting the slices into xylene I15 min-xylene II15 min-xylene III15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-volume percent 85% ethyl alcohol 5 min-volume percent 75% ethyl alcohol 5 min-distilled water washing in sequence.

2) Antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution (pH9.0) for antigen repairing in a microwave oven, stopping heating for 5min until boiling, stopping heating for 5min, stopping heating for 2min, and turning to medium-low heat for 5min to prevent excessive evaporation of the buffer solution. After natural cooling, the slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

3) Blocking endogenous peroxidase: the sections were placed in 3% by volume hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.

4) And (5) serum blocking, namely dripping 3% BSA (bovine serum albumin) into a histochemical ring to uniformly cover the tissues, and blocking for 30min at room temperature. (Primary antibody was goat-derived blocked with rabbit serum, other sources with BSA)

5) Adding a primary antibody: gently removing the confining liquid, dripping PBS (phosphate buffer solution) on the slices to prepare primary antibodies according to a certain proportion, and flatly placing the slices in a wet box for incubation at 4 ℃ overnight. (Small amount of water added in wet box to prevent evaporation of antibody)

6) Adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section was slightly spun dry, a secondary antibody (HRP-labeled) to the corresponding species was added dropwise to the ring to cover the tissue, and the mixture was incubated at room temperature for 50 min.

7) DAB color development: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.

8) Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.

9) Dewatering and sealing: putting the slices into 75% alcohol by volume for 5min to 85% alcohol by volume for 5min, anhydrous ethanol I5 min to anhydrous ethanol II 5min to xylene I5 min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum.

10) Microscopic examination and image acquisition and analysis.

The experimental results are shown in fig. 6, wherein, a is a photograph taken by a camera after a tumor under the skin of a mouse is taken out; b is a mouse tumor volume growth curve; c is the weight change curve of the mouse; d is the isolated volume of the mouse tumor; e is the mouse tumor in vitro body weight; f is immunohistochemical assay Ki 67; g is a Bortezomib (BTZ) drug-resistant cell strain for immunohistochemical detection that MSN ANBL6-BR cells are MM cell strain ANBL 6. The tumor volume of the mice treated by the paris saponin VII is obviously smaller than that of a control group, the tumor volume growth rate of the mice treated by the paris saponin VII is obviously lower than that of the control group, and in addition, the tumor volume, the tumor body weight and the dyeing degree of Ki67 of the mice treated by the paris saponin VII are all obviously smaller than those of the control group, which shows that the paris saponin VII can obviously inhibit the tumorigenesis of ANBL6-BR cells. In addition, the body weight of the mice treated by the paris polyphylla saponin VII is not obviously changed in the treatment process, which shows that the paris polyphylla saponin VII has no obvious side effect on the mice. And the immunohistochemical result shows that the staining degree of the mouse tumor tissue MSN after the treatment of the paris saponin VII is obviously lower than that of a control group, which indicates that the MSN protein is obviously reduced after the treatment of the paris saponin VII. The results show that the paris saponin VII can overcome the BTZ drug resistance of MM in vivo and in vitro, and can obviously inhibit the expression of MSN protein.

Example 7: paris saponin VII or ARP1 cell tumor inhibition by knocking down MSN

(1) ARP1 cells with luciferase and ARP1 cells with luciferase after MSN knockdown were collected in 1640 medium and adjusted to 200. mu.l containing 1X 10⁶Cell sap of individual ARP1 cells.

(2) 20 NCG mice, 5-8 weeks old, were selected with weights between 18-22 g. The cells were mixed well in a 1.5mL centrifuge tube, 100. mu.L of the cell fluid was aspirated by a 1mL syringe, and the cell fluid was inoculated into the subcutaneous part of the middle and rear part of the right axilla of the NCG mouse.

(3) When tumors were measurable (i.e., day 21), 20 NCG mice were randomly divided into 4 groups.

(4) On day 21, treatment included intraperitoneal injection of vehicle (control) or polyphyllin vii (5 mg/kg/day). Tumor volume and body weight of each mouse were recorded 1 time per day.

(5) After 8 days of continuous treatment (total 8 times), when tumors of the control mice are very significant by day 30, 100mg/kg of D-fluorescein is injected into the abdominal cavity of each mouse, the mice are photographed by a small animal imager after 5min, and one image is photographed every minute within a period of 5min-20 min.

(6) Mice were euthanized. And (5) drawing a change curve of the tumor volume and the body weight of the mouse, photographing the isolated tumor, and recording the isolated weight of the tumor.

The experimental results are shown in fig. 7, wherein, A is a photograph taken by a small animal imager after injecting 100mg/kg D-fluorescein into the abdominal cavity of each mouse; b, taking out subcutaneous tumors of the mice, and taking pictures by using a camera; c is a mouse tumor volume growth curve; d is a mouse weight change curve; e is mouse tumor ex vivo body weight. The tumor volume and the fluorescence imaging degree of the mice treated or knocked down by the paris polyphylla saponin VII are obviously smaller than those of a control group, the tumor volume growth rate of the mice treated or knocked down by the paris polyphylla saponin VII is obviously lower than that of the control group, and in addition, the tumor weight of the mice treated or knocked down by the paris polyphylla saponin VII is obviously smaller than that of the control group, which indicates that the paris polyphylla saponin VII or knocked down by the MSN can obviously inhibit the tumor formation of the ARP1 cells. In addition, the body weight of the mice treated or subjected to MSN knock-down by the paris polyphylla saponin VII is not obviously changed in the treatment process, which indicates that the paris polyphylla saponin VII or the MSN knock-down has no obvious side effect on the mice. The results show that the paris polyphylla saponin VII or the knocked-down MSN can obviously inhibit the development of MM in vivo and in vitro. Compared with mice with the MSN knocked-down group, the mice added with the Paris saponin VII group after the MSN is knocked-down have no significant difference in tumor volume, fluorescence imaging degree, tumor volume growth rate and mouse tumor weight, which shows that the Paris saponin VII hardly exerts drug effect under the condition of knocking-down MSN, and proves that the Paris saponin VII also exerts effect by targeting the MSN in vivo experiments.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto, and various modifications and variations which do not require inventive efforts and which are made by those skilled in the art are within the scope of the present invention.