Application of SYK inhibitor R406 and BAY-61-3606 in preparation of medicine for treating retinoblastoma
阅读说明：本技术 Syk抑制剂r406和bay-61-3606在制备视网膜母细胞瘤治疗药物上的应用 (Application of SYK inhibitor R406 and BAY-61-3606 in preparation of medicine for treating retinoblastoma ) 是由 金子兵 刘慧� 华梓淇 于 2019-09-16 设计创作，主要内容包括：一种SYK抑制剂R406和BAY-61-3606在制备视网膜母细胞瘤治疗药物上的应用,以人胚胎干细胞来源的体外3D人Rb模型为基础,结合转录组学、表观遗传学及蛋白组学等研究,发现了SYK在Rb肿瘤发生发展过程中的重要作用,SYK可作为Rb治疗的新靶点,其效果主要包括：抑制肿瘤细胞增殖,具体体现在SYK抑制剂作用后的肿瘤模型,根据流式细胞术结果和免疫荧光染色结果,其内Ki67阳性的细胞数要明显少于未用SYK抑制剂处理的模型,提示有增殖能力的细胞、即肿瘤细胞数量变少；促进肿瘤细胞凋亡。(An application of SYK inhibitors R406 and BAY-61-3606 in preparation of retinoblastoma therapeutic drugs is based on an in vitro 3D human Rb model derived from human embryonic stem cells, and combined with researches such as transcriptomics, epigenetics, proteomics and the like, an important role of SYK in the process of Rb tumorigenesis and development is discovered, the SYK can be used as a new target for Rb therapy, and the effects mainly comprise: inhibiting tumor cell proliferation, specifically reflecting in tumor model after SYK inhibitor action, according to flow cytometry result and immunofluorescence staining result, the number of Ki67 positive cells in the tumor model is obviously less than that of the model without SYK inhibitor treatment, which indicates that the number of cells with proliferation capacity, i.e. tumor cells, is less; promoting the apoptosis of tumor cells.)
1. Use of SYK inhibitors R406 and BAY-61-3606 in preparation of medicine for treating retinoblastoma is provided.
2. Use of a SYK inhibitor R406 and BAY-61-3606 according to claim 1, in the manufacture of a medicament for the treatment of retinoblastoma, wherein the final concentration of SYK inhibitor R406 in said medicament for the treatment of retinoblastoma is greater than or equal to 5 μ M.
3. The use of a SYK inhibitor R406 and BAY-61-3606 as claimed in claim 1, wherein said SYK inhibitor BAY-61-3606 is present in a final concentration of greater than or equal to 5 μ M in said medicament for the treatment of retinoblastoma.
4. Use of a SYK inhibitor R406 and BAY-61-3606 for the manufacture of a medicament for the treatment of retinoblastoma according to claim 1, wherein said SYK inhibitor R406 and BAY-61-3606 inhibit tumor cell proliferation by reducing Ki67 expression.
5. The use of a SYK inhibitor R406 and BAY-61-3606 for the manufacture of a medicament for the treatment of retinoblastoma, as claimed in claim 1, wherein said SYK inhibitor R406 and BAY-61-3606 promote apoptosis in tumor cells by increasing the expression of Cleaveccaspase 3.
6. The use of a SYK inhibitor R406 and BAY-61-3606 as claimed in claim 1, wherein said SYK inhibitor R406 has the chemical formula:
7. the use of a SYK inhibitor R406 and BAY-61-3606 as claimed in claim 1, wherein said SYK inhibitor BAY-61-3606 has the chemical formula:
Technical Field
The invention particularly relates to the technical field of medicine and life science, and particularly relates to application of SYK inhibitors R406 and BAY-61-3606 in preparation of a medicine for treating retinoblastoma.
Background
Retinoblastoma (Rb) is the most common intraocular malignancy that originates from the retina in infants and young children, accounting for 2% -4% of children's malignancies, with about 95% of cases occurring before the age of 5. Rb patients are usually in middle and late stages when seeing a doctor, are difficult to treat, are easy to relapse and transfer, and seriously harm the eyesight and the life of children patients. Mutations or deletions in the RB1 allele are a direct cause of RB occurrence. The RB1 gene is located on human chromosome 13q14.2, the coded protein consists of 928 amino acids and is an important cell cycle regulatory protein, after mutation of RB1 allele, cells lose normal RB protein function, and cell differentiation is out of control, so that tumors are formed.
At present, the international treatment method for Rb is mainly systemic chemotherapy assisted by local intervention of ophthalmic artery. However, most of these chemotherapy drugs are traditional broad-spectrum tumor cytotoxic drugs, and have obvious disadvantages, such as that different individual patients have obviously different reactions to the same chemotherapy scheme, part of patients are too sensitive to the drugs to cause significant adverse reactions, and other patients have drug resistance to affect the curative effect. In addition, when the tumor is diagnosed late and the tumor volume is too large and the risk of metastasis exists, an eyeball removal treatment is needed. In China, patients are mostly in high-risk late stage, and the eye ball removal is still a mainstream treatment method. At present, for the treatment of Rb, a specific treatment means aiming at the etiology (occurrence and development of Rb) is still lacked, and a new effective gene therapy target is urgently needed to be searched.
Recently, the research finds that a retina Organoid (Organoid) model which has a structure and components similar to those of human retina, is complete in structure, mature in function and sensitive to light can be cultured in vitro by utilizing human embryonic stem cells (hES cells), and the research such as disease pathogenesis, drug screening and the like can be carried out on the retina model from which a patient induces the differentiation of the pluripotent stem cells. In the early stage, the RB1 gene mutation or knockout hES cell with the same genetic background as the patient is utilized to spontaneously form an artificial Rb tumor organoid model in the in-vitro retinal tissue development process, so that the tumor growth environment and the tumor generation and development process of the patient can be simulated more truly, and a more reliable model is provided for researching tumor pathogenesis, drug development and treatment.
Spleen tyrosine kinase (SYK) is a kinase encoded by SYK gene and expressed in various cells such as B cells. It is involved in mediating the signaling pathways of a variety of membrane receptors, including CD74, Fc receptors, and integrins, among others. Furthermore, abnormalities in the SYK pathway are associated with a variety of tumors such as B-cell-associated tumors and epithelial cell carcinomas. Clinically, SYK inhibitors are also used to treat B-cell associated tumors and a variety of autoimmune diseases, such as cerdulatinib, entospletinib, fostamatinib, and the like. In addition, conventional SYK inhibitors such as R406, Piceatannol, MNS, TAK-659, BAY61-3606, and the like are available. There are no reports of SYK inhibitors that can be used to treat retinoblastoma.
Disclosure of Invention
In order to solve the defects of the prior art, provide a new gene target and a medicine for treating retinoblastoma, improve the effectiveness of Rb treatment and provide reference for clinical medication, the invention provides application of SYK inhibitors R406 and BAY-61-3606 in preparation of a medicine for treating retinoblastoma.
The technical solution adopted by the invention is as follows: use of SYK inhibitors R406 and BAY-61-3606 in preparation of medicine for treating retinoblastoma is provided.
The final concentration of SYK inhibitor R406 in the retinoblastoma therapeutic drug is greater than or equal to 5 μ M.
The final concentration of the SYK inhibitor BAY-61-3606 in the medicine for treating the retinoblastoma is more than or equal to 5 mu M.
The SYK inhibitors R406 and BAY-61-3606 inhibit the proliferation of tumor cells by reducing the expression of Ki 67.
The SYK inhibitor R406 and BAY-61-3606 promote tumor cell apoptosis by increasing the expression of cleared
The chemical structural formula of the SYK inhibitor R406 is as follows:
the chemical structural formula of the SYK inhibitor BAY-61-3606 is as follows:
the invention has the beneficial effects that: the invention provides an application of SYK inhibitor R406 and BAY-61-3606 in preparing a medicine for treating retinoblastoma, an Rb tumor organoid model obtained by inducing and differentiating 3D retina in vitro of hES cells is utilized, the key role of SYK in the generation and development of Rb tumor is discovered, and a new treatment target is provided for treating Rb; the inhibitors R406 and BAY61-3606 aiming at SYK targets are found for the first time to be capable of obviously inhibiting the growth of retinoblastoma, promoting the apoptosis of tumor cells and effectively treating the retinoblastoma. The invention aims to provide a new gene target and a medicine for treating retinoblastoma, improve the effectiveness of Rb treatment and provide reference for clinical medication.
Drawings
FIG. 1 shows the differential expression of genes in Rb tumor organoids versus normal retinal organoids on different days.
FIG. 2 is a signal pathway based on transcriptome analysis showing significant differences between tumor and normal controls.
FIG. 3 shows the difference in genome-wide methylation between Rb tumor organoid and normal retinal organoid.
FIG. 4 is a comparison of the transcription and methylation levels of the SYK gene in Rb tumor organoids and normal retinal organoids.
FIG. 5 is a protein showing significant differential changes between the Rb tumor organoid group and the normal retinal organoid control group.
FIG. 6 shows the results of Ki67 and SYK staining in Rb tumor and normal retinal organoids.
FIG. 7 is a general experimental flow chart for drug testing using Rb-like organs.
Figure 8 is the flow cytometry results for Ki67 in Rb tumor organoids after drug treatment.
FIG. 9 is a comparison of Ki67 and caspase3 staining in Rb tumor and normal retinal organoids.
Detailed Description
Technical scheme
1. Constructing an Rb1 gene mutant hES cell line, and obtaining an Rb organoid model by using an in-vitro 3D retina induced differentiation technology;
2. the method for detecting the key gene and signal channel change condition in the process of Rb tumor occurrence and development by using a transcriptome sequencing technology (RNA-Seq) comprises the following specific steps:
(1) preparing Rb and normal retina organoid models in different developmental stages in vitro, collecting Rb and normal retina organoid samples at different time points (
(2) and analyzing the detection result of the RNA-Seq, combining the early-stage determined key time period (60 th to 120 th) of tumorigenesis, analyzing significant difference genes of a tumor group and a normal group in the time period, and comprehensively analyzing key signal paths related to tumorigenesis development.
3. Detecting the change condition of key differential genes in protein level and methylation level, and specifically comprising the following steps:
(1) cell immunofluorescence staining detects significant changes of key differential genes at the protein level;
(2) proteomics detects significant changes of key differential genes at the protein level;
(3) genome-wide methylation detects methylation changes in key differential genes.
4. Various SYK inhibitors and other antineoplastic agents were separately targeted to Rb tumors, as follows:
(1) dividing Rb organs into eight groups, and culturing each group for 60 days for administration;
(2) each group was given a clinical traditional treatment: vincristine (5nM), etoposide (0.5. mu.M), carboplatin (10. mu.M), topotecan (10nM), inhibitors against the PI3K/AKT signaling pathway: rapamycin (10 μ M), and inhibitors targeting SYK: r406 (5. mu.M) and BAY-61-3606 (5. mu.M), with DMSO as a solvent control;
(3) the administration time was 7 days, and the culture of each group of pellets was continued after the administration.
5. Collecting Rb organoids, and detecting the result of drug treatment, the steps are as follows:
(3) continuously culturing each group of Rb organoids for 120 days
(4) Each group was prepared by selecting a part of Rb-like organs, fixing, embedding and freezing
(5) Observation of Marker expression by immunofluorescence staining
(6) Each group of picked partial Rb-like organs was digested into single cells
(7) After incubation with fluorescent signal, single cells were filtered out and the condition of fluorescent signal was analyzed by flow cytometry
Experimental Material
Cellular material
Human embryonic stem cell line (H9) from WiCell Research Institute (Madison, Wis.)
The drugs used in this test
(1) Vinchristine, Carboplatin, Rapamycin, R406 were purchased from Selleck;
(2) etoposide, Topotecan, available from J & K;
(3) BAY-61-3606 available from MedChemexpress;
(4) DMSO (dimethyl sulfoxide) was purchased from Sigma.
Enzyme, reagent consumables and kit
(1) Specific experimental modes such as establishment of RB1 gene mutation vector and mutation hES cell line thereof, acquisition of Rb tumor model and the like;
(2) cell culture: TeSR-E8 medium (stem cell technologies); stemspan (stem cell technologies); reproTeSR (Stem cell technologies); fetal bovine serum (GIBCO); matrigel (becton dickinson); 0.5 μ M EDTA solution (GIBCO); 10 μ M Y-27632 (Selleck); six well cell culture plates (Nunc);
(3) the reagent materials for retinal organoid differentiation are GMEM (Gibco), KSR serum replacement (Gibco), NEAA nonessential amino acids (Gibco), pyruvate (Gibco), β -mercaptoethanol (Gibco), penicillin (Gibco), streptomycin (Gibco), IWR1e (Sigma), hBMP4(Sigma), fetal bovine serum (Gibco), SAG (Sigma), DMEM/F12(Gibco), N2 supplement (Gibco), retinoic acid, IMDM (Gibco), Hams F12(Gibco), GlutaMax (Gicoo), thioglycerol (Sigma), V-Lance Knibfe (ALCON SURGICAL).
(4) Rb organoid maintenance medium: DMEM/F12 containing 10% fetal bovine serum, 1% N2 supplement, 0.5 μ M retinoic acid, 100U/ml penicillin, 100mg/ml streptomycin;
(5) RNA extraction and sequencing: RNeasy Mini Kit (Qiagen); NEB Next Ultra RNA library Prep Kit for Illumina (NEB); HiSeq PE Cluster Kit v4-cBot-HS (Illumina);
(6) DNA extraction and methylation analysis: EZ DNA Methylation-Gold Kit (Zymo Research); DNeasy Blood & Tissue Kit (QIAGEN);
(7) proteomics analysis: 2D Quant kit (GE Co.);
(8) fixing, embedding and section staining of Rb organoids: paraford (beyotimebiotechnology); NEG-50FROZEN section medium (Thermo Fisher Scientific); anti-Ki67 antibody (Abcam); anti-cleared Caspase3 antibody (Cell Signaling Technology); dapi (thermofisher scientific); anti-SYK antibody (Cell Signaling Technology);
(9) rb organoid digestion, flow cytometry: 0.25% Trypsin-EDTA, Phenol red (ThermoFisher Scientific); alexa Fluor 647Mouse anti-Ki-67 Clone B56(BD Biosciences); alexa Fluor 488Mouse anti-Oct3/4Clone 40/Oct-3(BD Biosciences).
Experimental methods
The preparation and verification of RB1 gene mutation hES cells, the identification of in vitro 3D retina induced differentiation and 3D retinoblastoma and other methods specifically comprise the following steps: (a) establishing a RB1 allele mutation or knockout human pluripotent stem cell line by performing gene editing on a human retinoblastoma RB1 allele in a human embryonic stem cell or reprogramming a somatic cell of a patient carrying RB1 allele mutation Rb into a human induced pluripotent stem cell, and further screening and identifying; (b) inducing RB1 allele mutation or knockout human pluripotent stem cells to differentiate into a human retinoblastoma model by using an in vitro three-dimensional retinal differentiation system.
Comparison of expression differences in SYK between Rb tumor organoids and Normal retinal organoids
RNA-seq detection and analysis
(1) Culturing Rb tumor organoids and normal retinal organoids at different time points (
(2) randomly selecting organoids of different days, washing the organoids for 3 times by PBS, adding TRIzol, and shaking to dissolve the organoids to extract total RNA of cells;
(3) RNA was purified using the RNeasy Mini Kit from Qiagen and the purity of the RNA was checked using the software NanoDrop 2000;
(4) constructing an RNA Library with an Annoroad Gene Technology and performing RNA sequencing, including creating a sequence Library using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB), and clustering the Library using HiSeq PECluster Kit v4-cBot-HS (Illumina);
(5) sequencing the library on an Illumina platform according to a 150bp fragment;
(6) preliminary data analysis was performed with bmlcoud.
Methylation detection and analysis
(1) Selecting Rb and normal retina organoids at day 120, extracting DNA with DNeasy Blood & tissue kit (QIAGEN), and determining DNA content with software NanoDrop 2000;
(2) constructing a WGBS (white-genome bisulfate sequencing) library with 1. mu.g of DNA, including bisulfite conversion of DNA using EZ DNA Methylation-Gold Kit (Zymo Research);
(3) methylated CpGs were extracted by Bismark methylation extraction and analyzed by SMART2 software for the gene.
Proteomics detection and analysis
(1) Selecting Rb and normal retina organoids on day 90, and washing with PBS for several times; the pellet was carefully dissociated into a single cell suspension using 0.25% trypsin-EDTA and the cells collected by centrifugation (> 1X10^ 6); then carrying out liquid nitrogen grinding, washing with 10% TCA acetone, washing with acetone and air drying;
(2) adding appropriate amount of 8M urea buffer (containing 5mM DTT,2mM EDTA, 3. mu.M TSA, 10% protease inhibitor cocktail III, 30mM nicotinamide) to extract protein, and performing ultrasonic treatment for 3min each time for 2 times;
(3) taking 3 mu l of protein extracting solution, determining the protein concentration according to a 2D Quant kit (GE company), taking 20 mu g of protein extracting solution according to a quantitative result, and observing whether the concentration determination is accurate and the protein extraction is good or not by SDS-PAGE;
(4) adding 2mM DTT with final concentration, incubating at 37 deg.C for 2h for reduction, adding 25mM IAM with final concentration, incubating at room temperature for 55min for alkylation, adding 1/5 volume of TCA dropwise, and precipitating protein at 4 deg.C for more than 2h for precipitation;
(5) washing with acetone: 16000g, centrifuging at 4 deg.C for 5min, discarding supernatant, adding 1ml acetone pre-cooled at-20 deg.C into precipitate, washing for 3 times, and standing at-20 deg.C for 30min each time.
(6) Air drying: 16000g, centrifuging at 4 deg.C for 5min, discarding acetone, opening the tube cover, air drying for 10min to volatilize residual acetone completely.
(7) Dissolving: the pellet was resuspended in protein at an appropriate amount of 0.1M TEAB.
(8) Enzymolysis: adding trypsine according to the mass ratio of 1:20, and performing enzymolysis at 37 ℃ overnight.
(9) And (3) draining: 16000g, centrifuging at 4 deg.C for 5min, collecting supernatant, vacuum concentrating, drying, and storing at-20 deg.C
(10) Desalting: data X C18 (Phenomenex) column was desalted and dried by vacuum concentration on a desk.
(11) HPLC peptide fragment: the samples were divided into two portions, and one portion was mixed in 100. mu.g aliquots and pre-separated into 18 fractions by HPLC for quantification of the whole albumin fraction.
(12) MS identification and advanced bioinformatics analysis: peptide fragments after HPLC pre-separation are identified by Q extraction (Thermo Scientific), and data obtained by MS identification are further analyzed by advanced bioinformatics.
Cell immunofluorescence detection of expression of SYK protein in Rb-like organs
(1) Fixing and embedding: selecting 2-3 organoid blocks, washing with DPBS for 2 times, adding 1mL of immunostaining fixative, fixing for 60min in a 4 ℃ refrigerator, removing the immunostaining fixative, washing for 2 times with PBS, adding ponceau staining solution, dyeing for 5min, placing the blocks in an embedding agent, and storing in a-80 ℃ refrigerator or performing frozen slicing after the embedding agent is completely solidified;
(2) freezing and slicing: taking out a sample from-80 ℃, quickly placing the sample in a freezing microtome at-22 ℃ to slice the sample at the thickness of 12-14 mm, and placing the sample at-80 ℃ for later use;
(3) and (3) immunofluorescence staining: washing frozen slices with PBS for 3 times, each for 10min, adding appropriate amount of 4% BSA + 0.5% Triton-100 mixed solution for permeation and sealing, and washing slices with PBS for 3 times, each for 10min at room temperature for 1 h; adding an appropriate amount of primary antibody (anti-SYK, anti-Ki67) diluted with 1% BSA + 0.5% Triton-100, incubating overnight at 4 ℃, washing with PBS for 3 times, each for 10min, adding an appropriate amount of secondary antibody diluted with 1% BSA + 0.5% Triton-100, keeping out of the sun, washing at room temperature for 1h, washing with PBS for 3 times, each for 10min, adding an appropriate amount of DAPI diluted to 1X 1X with PBS, incubating for 5-10min, washing with PBS for 3 times, each for 10min, air-drying the sections, dropping an anti-quenching co-solvent, covering with a cover glass, and taking pictures with a laser focal microscope.
Grouping and administration of Rb-like organs
(1) Culturing Rb organoids to 60 days, randomly selecting a plurality of equal organoids with similar growth vigor and good state, and dividing into 8 10cm non-adhesion culture dishes;
(2) adding 8 medicaments into the organoid maintenance medium according to the designed concentration in advance, wherein the corresponding concentration of each medicament is as follows: vincristine (5nM), etoposide (0.5. mu.M), carboplatin (10. mu.M), topotecan (10nM), rapamycin (10. mu.M), R406 (5. mu.M), BAY-61-3606 (5. mu.M);
(3) adding the prepared culture medium containing the medicine into culture dishes of corresponding groups respectively, and
(4) replacing the common maintenance culture medium without the medicine after the medicine treatment is completed for 7 days;
(5) the culture medium was changed every 7 days, and the culture was maintained until day 120.
Rb-class organ sampling and immunofluorescence staining
(1) Randomly selecting a plurality of Rb organs in each group, and carrying out fixation, embedding, freezing section and Ki67 and
(2) confocal microscopy.
Immune flow assay of the effects of drug action on Rb tumor cells Using Ki67 antibody
(1) Washing organoid pellet several times with PBS, carefully dissociating into single cell suspension using 0.25% trypsin-EDTA (Gibco), centrifuging at 1000rpm for 5min at room temperature, and discarding supernatant;
(2) cells were resuspended in culture medium and stained with antibodies in PEB (PBS containing 0.5% BSA and 2mM EDTA) buffer: incubation with Alexa Fluor 647mouse anti-Ki-67 antibody for 30min at 4 ℃;
(3) cells were filtered through a 100 μm nylon mesh and fluorescence was analyzed by flow analysis through a facscan II (Becton Dickinson).
Results and analysis
Differential analysis of SYK expression at transcriptional level in Rb tumor organoids and Normal retinal organoids
Comparing the differential expression of the genes on different days
The gene expression difference between human retinoblastoma organs (hRBOs) and normal retinal organs on different days is compared by using RNA-seq, and the result is shown in figure 1, after 75 days, the expression level of SYK in the hRBOs is obviously increased in comparison with that of a normal retinal control group, and the SYK is closely related to the generation and development of Rb tumors.
Analysis of key signal pathways in tumorigenesis and development process based on transcriptome data
Whether the difference of key signal pathways is significant or not is analyzed according to transcriptome data of the Rb organoid tumor group and the normal retina organoid control group at different days, and the result is shown in figure 2, the expression level of the PI3K-Akt signal pathway activated by SYK is significantly different between the tumor group and the control group after 60-90 days, and the SYK and the signal pathway to which the SYK belongs play an important role in the occurrence and development of retinoblastoma.
Differential analysis of SYK epigenetic regulatory (methylation modification) levels in Rb tumor organoids and normal retinal organoids
By analyzing the whole genome methylation condition, the difference of the expression level of the same gene in different samples can be compared from the apparent regulation level. FIG. 3 shows the differences in genome-wide methylation between Rb tumor organoids (hRBOs) and normal retinal organoids (hROs), with the red dots on the left indicating that partial genes of hRBOs are significantly less methylated than hROs, and the critical gene SYK is also present. In the case of low methylation level, the gene expression level was high, and thus the result suggests that the expression level of SYK gene of hRBOs was higher at day 120 than that of hROs at the same day. FIG. 4 shows the methylation difference of hRBOs and hROs on SYK gene in a visual way, and it can be seen that the methylation level of hRBOs in the blue bottom promoter region is significantly lower than that of hROs, and this result is also consistent with the expression level of SYK gene reflected by the results of two organoid RNA-seq.
Differential analysis of SYK expression at protein level in Rb tumor organoids and Normal retinal organoids
Proteomics can comprehensively reflect the influence of gene expression difference from the result. As shown in FIG. 5, when the protein expression groups of the Rb tumor organoid at day 90 and the normal retinal organoids on the same day are compared, the expression degree of proteins such as SYK, Ki67 and the like in the Rb tumor organoid is obviously higher than that of the normal retinal organoid, which indicates that the generation and development of the Rb tumor are related to the expression of the SYK protein. In addition, immunofluorescence staining of organoid masses gave the same results, as shown in fig. 6, with Ki67 and the key protein SYK representing tumor proliferation expressed significantly higher in Rb organoids than in normal retinal organoids.
3.1 Effect of SYK inhibitors R406 and BAY-61-3606 on Rb tumor organoids
Design of experimental procedure
To demonstrate that SYK can be an effective target for retinoblastoma therapy and to find new drugs that might be used in clinical therapy, we selected two highly potent SYK inhibitors R406 and BAY-61-3606, and designed a drug trial as shown in figure 7: culturing Rb tumor organoids for 60 days, randomly adding medicine for one week, changing back to a common culture medium for maintaining culture for 120 days, digesting organoid blocks into single cells, performing fluorescent staining flow analysis, and detecting the number of Ki67 positive cells.
Ki67 antibody immune flow analysis of influence of drug on Rb tumor cell proliferation
After flow analysis, the results shown in FIG. 8 were obtained: the percentage of Ki67 positive cells of the DMSO solvent control group is higher, which indicates that the cell proliferation of the Rb tumor organoid is active under the condition of no drug action; except that Etoposide has poor individual difference reaction, the levels of other three groups of Ki67 positive cells are obviously reduced compared with DMSO groups, which indicates that the Rb organs used by the invention can truly reflect the reaction of retinoblastoma to the treatment medicament; furthermore, the effect of the drugs R406 and BAY-61-3606 introduced by the invention is easy to obtain that the drugs have good inhibition effect on the proliferation degree of Rb tumor cells, and the effect is close to or even superior to that of the traditional drugs. The effect of the PI3k/Akt inhibitor Rapamycin is relatively weak, so that the two new drugs introduced by the invention are more suitable for treating retinoblastoma.
Immunofluorescence staining analysis of effects of drugs on proliferation and apoptosis of Rb tumor cells
In addition, the influence of different drugs on Rb organoids is researched by immunofluorescence staining, and as can be seen from FIG. 9, the DMSO-treated solvent control group still has more Ki67 protein expression, which indicates that the proliferation is still active, but the Caspase3 protein reflecting apoptosis is few; the expression of Ki67 of Rb organs treated by other medicines is reduced to a certain extent, and the expression of Caspase3 is improved to a large extent, which indicates that the introduced new medicines R406 and BAY-61-3606 can inhibit the proliferation of Rb tumor cells and promote the apoptosis of the Rb tumor cells like the anticancer medicines traditionally applied in clinic.
The skilled person should understand that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.