阅读说明：本技术 研究aml外泌体对aml化疗抵抗作用和机制的方法 (Method for researching AML exosome resistance effect and mechanism on AML chemotherapy ) 是由 俞珏华 程禹 于 2020-06-03 设计创作，主要内容包括：本发明是研究AML外泌体对AML化疗抵抗作用和机制的方法,包括以下步骤：1)通过体内外实验证实JAK/STAT3信号通路调控MSC对AML细胞化疗抵抗的作用；2)通过实验证实AML来源的外泌体对MSCs中JAK/STAT3信号通路的上调作用,以及在介导MSC对AML细胞化疗的抵抗作用；3)通过体外实验研究AML来源的外泌体调控JAK/STAT3信号通路的作用机理。本发明的优点：通过研究JAK/STAT3信号通路以及外泌体在AML化疗抵抗中的作用机制,为临床治疗AML提供了新的治疗靶点,具有重要的临床意义。(The invention relates to a method for researching the resistance effect and mechanism of AML exosome to AML chemotherapy, which comprises the following steps: 1) in vivo and in vitro experiments prove that JAK/STAT3 signal channels regulate the effect of MSC on the resistance of AML cell chemotherapy; 2) experiments prove that the exosome from AML has the up-regulation effect on JAK/STAT3 signal channels in MSCs and the resistance effect on AML cell chemotherapy in mediated MSC; 3) the mechanism of action of an AML-derived exosome for regulating a JAK/STAT3 signal pathway is researched through an in vitro experiment. The invention has the advantages that: by researching the JAK/STAT3 signal pathway and the action mechanism of exosome in AML chemotherapy resistance, a new treatment target point is provided for clinically treating AML, and the method has important clinical significance.)

1. A method for studying the resistance and mechanism of AML exosomes to AML chemotherapy, comprising the steps of:

1) in vivo and in vitro experiments prove that JAK/STAT3 signal channels regulate the effect of MSC on the resistance of AML cell chemotherapy;

2) experiments prove that the exosome from AML has the up-regulation effect on JAK/STAT3 signal channels in MSCs and the resistance effect on AML cell chemotherapy in mediated MSC;

3) the mechanism of action of an AML-derived exosome for regulating a JAK/STAT3 signal pathway is researched through an in vitro experiment.

2. The method for studying the effect and mechanism of AML exosomes on AML chemotherapy according to claim 1, characterized in that said step 1) comprises:

1.1) in vitro experiments: respectively separating and culturing MSCs and AML cells, activating or inhibiting JAK/STAT3 signal channels in the MSCs, co-culturing the MSCs and the AML cells after intervention treatment and the MSCs without any treatment, and observing survival conditions and proliferation conditions of the AML cells under the condition of chemotherapeutic drug intervention in the three groups of co-cultured cells;

1.2) in vivo experiments: constructing an AML mouse model; 1.1) MSCs respectively activated, inhibited and untreated in vitro JAK/STAT3 signal paths are injected into AML mice through tail veins, and the general growth condition, the routine detection of peripheral blood, the blood smear analysis, the mouse bone marrow image analysis and the liver pathology detection of the AML mice are observed.

3. The method for studying the effect and mechanism of AML exosomes on AML chemotherapy according to claim 1 or 2, characterized in that said step 2) comprises:

2.1) extracting exosomes in AML cells and exosomes in normal cells, adding the exosomes into MSCs cell culture solution, and detecting the protein expression level of a JAK/STAT3 signal channel;

2.2) co-culturing the MSCs cultured respectively with the AML cell exosomes and the normal cell exosomes in the step 2.1) with the AML cells, adding the same amount of the chemotherapeutic drug cytarabine, and checking the proliferation condition and the survival condition of the AML cells.

4. The method for studying the effect and mechanism of AML exosomes on AML chemotherapy according to claim 3, characterized in that said step 3) comprises:

3.1) extracting the total protein content in MSCs cultured by AML extracellular exosomes and normal extracellular exosomes respectively in 2.1), detecting the protein expression levels of VEGF, MMPs, IL-1 beta and TGF beta, and researching the action mechanism of the AML-derived exosomes for regulating a JAK/STAT3 signal pathway;

3.2) after 3.1), carrying out expression intervention or overexpression on a clear signal molecule, and detecting whether a JAK/STAT3 signal channel in the MSCs is inhibited or activated.

Technical Field

The invention relates to a method for researching the effect and mechanism of AML exosome on AML chemotherapy resistance, in particular to a method for researching the effect and mechanism of AML exosome activation of JAK/STAT3 signal channel in marrow stromal cells to further cause AML chemotherapy resistance, belonging to the technical field of biological medicine.

Background

Acute Myelogenous Leukemia (AML), a type of malignant disease of myeloid hematopoietic stem cells, is characterized mainly by abnormal proliferation of primitive and immature myeloid cells in bone marrow and peripheral blood, clinically manifested by anemia, hemorrhage, infection and fever, organ infiltration, metabolic abnormalities, etc., is one of ten common high-grade malignant tumors in adults, and even in early treatment, complete remission is difficult to achieve. Currently, the incidence of AML is on the rise, and clinically, the treatment mode of AML includes chemotherapy and hematopoietic stem cell transplantation, but the transplantation cost is expensive and a suitable ligand is difficult to find, so that the AML is still mainly treated by the chemotherapy, but the chemotherapy resistance exists, so that the AML is difficult to cure, and the reason of the chemotherapy resistance is not completely clear at present. There is therefore a great need to find a mechanism for AML chemotherapy resistance to promote the therapeutic effect of chemotherapy on AML.

In recent years, there has been increasing evidence that the myeloid microenvironment, niche (niche), of leukemic patients plays an important role in AML and other types of leukemia, and thus, for the treatment of AML, attention is paid not only to the tumor cells themselves, but also to their microenvironment. The bone marrow microenvironment is a three-dimensional structure body with stromal cells as a main body and a plurality of adhesion molecules, growth factors and chemotactic factors participating together, and the functions of the bone marrow microenvironment play a role of a complex network system needing multiple cells and polyphones. The microenvironment of the tumor includes tumor cells and surrounding stromal cells, and the stromal cells of bone Marrow (MSCs) in the stromal cells have a particularly prominent effect on the growth, infiltration and metastasis of the tumor, for example, they can support the survival of leukemia cells in the bone marrow by secreting cytokines and chemical factors and activating some signal pathways related to cell survival. However, it is not completely clear how AML cells promote leukemia cell growth and resist chemotherapy by affecting the niches, i.e. MSCs. Research shows that exosomes, namely vesicles with the size of 30-200 nm secreted by normal cells or tumor cells, are key molecules for regulating cell communication. The exosome derived from the tumor cell can also promote the growth of the tumor and has a destructive effect on the stability of healthy tissues. Exosomes secreted by Chronic Myelogenous Leukemia (CML) cells can promote the production of cytokine IL-8 by MSCs and promote the survival of leukemia cells. In exosomes extracted from peripheral blood of AML patients, it was found that the exosomes contain leukemia-associated antigens, and the circulating exosomes also contain a number of signaling molecules capable of antagonizing the anti-tumor effect of immunoreceptor cells by suppressing them. Exosomes in AML cells may therefore be involved in anti-chemotherapy processes of AML cells, possibly through certain signaling pathways.

Recent studies have reported that tumor cells from AML patients are very resistant to anticancer drugs such as ATO (arsenic trioxide, FDA approved for the treatment of acute promyelocytic leukemia APL). ATO can induce AML cell apoptosis and inhibit AML cell proliferation by enhancing Reactive Oxygen Species (ROS) level, and has therapeutic effect on AML, but as JAK2/STAT3 signal channel in AML can inhibit apoptosis of cells by reducing ROS level, and has antagonistic effect on anticancer effect of ATO, and administration of an antagonist Ruxolitinib of JAK can enhance sensitivity of AML cells to ATO treatment.

STAT3 (signal transducer and activator of transcription 3) is an important transcriptional regulator that plays an important role in a variety of biological processes, such as cell survival, migration, proliferation, metabolism, and inflammation. The signaling pathway mediated by STAT3, and its upstream JAKs, is abnormally increased in expression in a variety of cancers. The JAK/STAT3 signal channel can induce the production of some cytokines such as VEGF, promote the formation of blood vessels and provide convenience for the proliferation and migration of tumor cells; promoting the synthesis of MMPs (matrix metalloproteinases), and facilitating the invasion and remote metastasis of tumor cells; promote the secretion of inflammatory factors IL-1 beta and TGF beta, and inhibit the immune response of immune cells to tumor cells. In addition to the above direct effects on tumor cells, the JAK/STAT3 signaling pathway can also inhibit some activities on tumor cells infiltrating related immune cells, such as neutrophils, NK cells, effector T cells and dendritic cells, thereby reducing the immune response of the body.

In previous studies, it was found that the supernatant of extracted AML cells (AML-CM) enhanced the phosphorylation level of STAT3 in MSCs. In the cell extraction process, exosomes are distributed in supernatant due to small molecular weight, so the early-stage pre-experimental result indicates that AML exosomes can be related to STAT3 signal pathways of MSCs.

Through further preliminary experiments, an exosome inhibitor GW4869 was added to AML-CM and was found to be capable of reducing phosphorylation levels of STAT3 in MSCs; meanwhile, the exosome inhibitor is also added into a system for co-culturing AML cells and MSCs, and the protective effect of the MSCs on the AML cells is obviously weakened, and the apoptosis rate of AML is obviously increased. The results of the above preliminary experiments suggest that exosomes of AML cells can enhance the association between AML cells and MSCs by modulating the STAT3 signaling pathway in the bone marrow microenvironment of AML.

Based on the above preliminary experimental results, the following hypothesis is proposed: the exosomes derived from AML can up-regulate JAK/STAT3 signal channels in the MSCs, thereby promoting the protective effect of the MSCs on AML cells, enhancing chemotherapy resistance and finally leading to the formation of drug resistance.

Therefore, the invention researches the JAK/STAT3 signal channel and the action mechanism of exosome in AML chemotherapy resistance, provides a new treatment target for clinically treating AML, and has important clinical significance.

Disclosure of Invention

The invention provides a method for researching the resistance effect and mechanism of AML exosome to AML chemotherapy, aiming at filling the blank in the prior art and providing a new therapeutic target point for clinically treating AML.

The technical solution of the invention is as follows: a method for studying the effect and mechanism of AML exosome resistance to AML chemotherapy comprising the steps of:

1) in vivo and in vitro experiments prove that JAK/STAT3 signal channels regulate the effect of MSC on the resistance of AML cell chemotherapy;

2) experiments prove that the exosome from AML has the up-regulation effect on JAK/STAT3 signal channels in MSCs and the resistance effect on AML cell chemotherapy in mediated MSC;

3) the mechanism of action of an AML-derived exosome for regulating a JAK/STAT3 signal pathway is researched through an in vitro experiment.

Preferably, the step 1) comprises:

1.1) in vitro experiments: respectively separating and culturing MSCs and AML cells, activating or inhibiting JAK/STAT3 signal channels in the MSCs, co-culturing the MSCs and the AML cells after intervention treatment and the MSCs without any treatment, and observing survival conditions and proliferation conditions of the AML cells under the condition of chemotherapeutic drug intervention in the three groups of co-cultured cells;

1.2) in vivo experiments: constructing an AML mouse model; 1.1) MSCs respectively activated, inhibited and untreated in vitro JAK/STAT3 signal paths are injected into AML mice through tail veins, and the general growth condition, the routine detection of peripheral blood, the blood smear analysis, the mouse bone marrow image analysis and the liver pathology detection of the AML mice are observed.

Preferably, the step 2) comprises:

2.1) extracting exosomes in AML cells and exosomes in normal cells, adding the exosomes into MSCs cell culture solution, and detecting the protein expression level of a JAK/STAT3 signal channel;

2.2) co-culturing the MSCs cultured respectively with the AML cell exosomes and the normal cell exosomes in the step 2.1) with the AML cells, adding the same amount of the chemotherapeutic drug cytarabine, and checking the proliferation condition and the survival condition of the AML cells.

Preferably, the step 3) comprises:

3.1) extracting the total protein content in MSCs cultured by AML extracellular exosomes and normal extracellular exosomes respectively in 2.1), detecting the protein expression levels of VEGF, MMPs, IL-1 beta and TGF beta, and researching the action mechanism of the AML-derived exosomes for regulating a JAK/STAT3 signal pathway;

3.2) after 3.1), carrying out expression intervention or overexpression on a clear signal molecule, and detecting whether a JAK/STAT3 signal channel in the MSCs is inhibited or activated.

The invention has the advantages that: on the basis of preliminary experiments, the following studies were continued:

1) the in vivo and in vitro experiments further prove that the JAK/STAT3 signal channel regulates the function of MSC on the chemotherapy resistance of AML cells;

2) the up-regulation effect of the exosome from AML on a JAK/STAT3 signal channel in MSCs and the resistance effect of MSC on AML cell chemotherapy are proved;

3) the mechanism of action of an AML-derived exosome for regulating a JAK/STAT3 signal pathway is researched through an in vitro experiment.

By researching the JAK/STAT3 signal pathway and the action mechanism of exosome in AML chemotherapy resistance, a new treatment target point is provided for clinically treating AML, and the method has important clinical significance.

Drawings

FIG. 1 is a schematic flow chart of example 1.

FIG. 2 is a schematic flow chart of example 2.

FIG. 3 is a schematic flow chart of example 3.

Detailed Description

The present invention will be described in further detail with reference to examples and specific embodiments.

The microenvironment of the tumor includes tumor cells and surrounding stromal cells, and the stromal cells of bone Marrow (MSCs) in the stromal cells have a prominent effect on the growth, infiltration and metastasis of the tumor. Exosomes are key molecules for regulating cell communication, and are found to contain leukemia-associated antigens in exosomes extracted from peripheral blood of AML patients, and circulating exosomes also contain a plurality of signal molecules which can antagonize the anti-tumor effect of immunoreceptor cells by inhibiting the immunoreceptor cells, so that the exosomes in the AML cells are suggested to participate in the anti-chemotherapy process of the AML cells through certain signal pathways.

As a result of previous studies, it was found that the supernatant of extracted AML cells (AML-CM) enhanced the phosphorylation level of STAT3 in MSCs. After further study, the addition of the exosome inhibitor GW4869 to AML-CM was found to reduce the phosphorylation level of STAT3 in MSCs; meanwhile, the exosome inhibitor is also added into a system for co-culturing AML cells and MSCs, and the protective effect of the MSCs on the AML cells is obviously weakened, and the apoptosis rate of AML is obviously increased. The results of the above preliminary experiments suggest that exosomes of AML cells can enhance the association between AML cells and MSCs by modulating the STAT3 signaling pathway in the bone marrow microenvironment of AML.

Based on the above preliminary experimental results, the following hypothesis is proposed: the exosomes derived from AML can up-regulate JAK/STAT3 signal channels in the MSCs, thereby promoting the protective effect of the MSCs on AML cells, enhancing chemotherapy resistance and finally leading to the formation of drug resistance. On the basis of preliminary experiments, the following studies were therefore carried out:

1) further proves the function of JAK/STAT3 signal channel for regulating and controlling MSC to resist AML cell chemotherapy in vitro and in vivo experiments;

2) the up-regulation effect of the exosome from AML on a JAK/STAT3 signal channel in MSCs and the resistance effect of MSC on AML cell chemotherapy are proved;

3) the mechanism of action of AML-derived exosomes in modulating the JAK/STAT3 signaling pathway was studied in vitro experiments.

The research method is designed from the three aspects, and the regulation and control effect and mechanism of an exosome from AML on a JAK/STAT3 signal pathway in MSCs are determined, so that a new treatment target point is provided for clinically treating AML.

Specifically, a method for studying the resistance and mechanism of AML exosomes to AML chemotherapy comprises the following steps:

1) the in vivo and in vitro experiments prove that the JAK/STAT3 signal channel regulates the function of MSC on resisting AML cell chemotherapy:

1.1) in vitro experiments: and (3) respectively separating and culturing the MSCs and the AML cells, activating or inhibiting JAK/STAT3 signal channels in the MSCs, co-culturing the MSCs after intervention treatment and the MSCs without any treatment with the AML cells, and observing survival and proliferation of the AML cells under the condition of chemotherapy drug intervention in the three groups of co-cultured cells.

1.2) in vivo experiments: constructing an AML mouse model; 1.1) MSCs respectively activated, inhibited and untreated in vitro JAK/STAT3 signal paths are injected into AML mice through tail veins, and the general growth condition, the routine detection of peripheral blood, the blood smear analysis, the mouse bone marrow image analysis and the liver pathology detection of the AML mice are observed.

2) Experiments prove that the exosome from AML has up-regulation effect on JAK/STAT3 signal channels in MSCs and resistance effect on AML cell chemotherapy by mediated MSC:

2.1) extracting exosomes in AML cells and exosomes in normal cells, adding the exosomes into MSCs cell culture solution, and detecting the protein expression level of a JAK/STAT3 signal channel.

2.2) co-culturing the MSCs cultured respectively with the AML cell exosomes and the normal cell exosomes in the step 2.1) with the AML cells, adding the same amount of the chemotherapeutic drug cytarabine, and checking the proliferation condition and the survival condition of the AML cells.

3) The mechanism of action of an AML-derived exosome for regulating a JAK/STAT3 signal pathway is researched through an in vitro experiment:

3.1) extracting the total protein content in MSCs cultured by AML extracellular exosomes and normal extracellular exosomes respectively in 2.1), detecting the protein expression levels of VEGF, MMPs, IL-1 beta and TGF beta, and researching the action mechanism of the AML-derived exosomes for regulating JAK/STAT3 signal pathways. (i.e., to determine which molecule or molecules the AML exosomes affect the JAK/STAT3 signaling pathway in MSCs by affecting the expression level of that molecule or molecules.)

3.2) after 3.1), carrying out expression intervention or overexpression on a clear signal molecule, and detecting whether a JAK/STAT3 signal channel in the MSCs is inhibited or activated. (by this procedure, the mechanism of the signal molecule in regulation of the JAK/STAT3 signaling pathway in MSCs by AML-derived exosomes was verified.)

Example 1

As shown in FIG. 1, the effects of JAK/STAT3 signaling pathway in modulating MSC on resistance to chemotherapy of AML cells was demonstrated

1.1 at the cellular level, the effect of JAK/STAT3 signaling pathway in regulating MSC on the resistance of AML cell chemotherapy

1.1.1 extraction of AML cells from AML patients and monocytes from healthy controls

Peripheral blood of an AML patient is extracted and treated for the first time, lymphocyte separation liquid is used for separating peripheral blood mononuclear cells, smear and Ruter-Giemsa staining are carried out, and the AML cell proportion is more than or equal to 90 percent through classification and counting. With 10% fetal bovine serum (fetai bo)vine serum, FBS) to adjust the concentration of AML cells to 1xl0⁶Perml, put in 6-well plates. At 37 deg.C, 5% CO₂After culturing for 24h in the incubator, centrifuging and collecting the culture supernatant with a 0.4 μm filter screen to remove impurities, and storing at-20 ℃ for later use as marked pAML-S. The same method is adopted to separate and culture the peripheral blood mononuclear cells (1x 10) of healthy people⁶mL), the collected culture supernatant was filtered through a 0.4 μm sieve to remove impurities, labeled Ctrl-S, and stored at-20 ℃ for use as an experimental control.

1.1.2 bone marrow isolation culture of human MSCs in AML patients and healthy controls

And (3) evenly packaging the collected bone marrow into two sterile tubes of 50ml, marking as tubes A and B, respectively adding 3ml of 6% hydroxyl starch, uniformly mixing, and standing at room temperature for 30 min. Transferring the plasma containing the white blood cells at the upper part of the A, B tube to a new centrifuge tube C, centrifuging at the rotation speed of 400g for 10min, and transferring the supernatant to a new centrifuge tube D; heating the tube D in a 56 deg.C water bath for 30min to inactivate complement, centrifuging at 800g for 20min to remove flocculent precipitate, transferring the supernatant to a new centrifuge tube E, and adding the supernatant as autologous plasma in cell culture. Adding 15ml of normal saline into a tube C, and fully and uniformly mixing; taking two new centrifuge tubes, adding 15ml of human lymphocyte separation liquid, slowly adding 15ml of plasma diluted by normal saline into the upper layer of the separation liquid by using a sterile pipette, centrifuging at 23 ℃ at the rotating speed of 750g for 20 minutes, sucking a middle white substance into 50ml of new centrifuge tubes by using a 1ml pipette gun, adding 4 times of normal saline in volume, uniformly mixing, centrifuging at the rotating speed of 1500rpm, centrifuging for 10 minutes, and then discarding the supernatant. Collecting and resuspending the centrifuged cell pellet with 10ml of physiological saline, centrifuging at 1000rpm for 5 minutes, and repeating the resuspension step once; sucking 10 mu L of the cells resuspended in the last step, adding 4g/L trypan blue staining solution in the same volume, uniformly mixing, passing through a blood cell counting plate, and counting under an inverted optical microscope; adopting MEM culture medium, adding 10% autologous serum, and performing sterile experiment; adjusting the suspended cell density to 10 with the culture medium⁵Adding autologous serum to reach serum concentration of 10%, inoculating the cells into a culture bottle, culturing for 3 days, changing the culture solution every 24 hours, and discarding non-adherent cells(ii) a When the cells fused to 80-90%, they were digested with 0.25% pancreatin containing EDTA at 4000 cells/cm²The density of the cells is passed, amplified in vitro for 4-5 generations, digested by pancreatin, and washed by normal saline after termination, and the adult bone marrow MSC cell suspension is diluted by 22ml of normal saline.

1.1.3STAT3 overexpressing lentiviruses and siRNA treatment

STAT3 overexpression lentivirus LV5-STAT3 and control lentivirus LV5-Mock were purchased from Shanghai Jima pharmaceutical technology, Inc. STAT3-siRNA was designed and synthesized by Ruibo, Guangzhou, and the synthetic sequence was as follows: siRNA1: CCGTGGAACCATACACAAA, siRNA 2: GATACGACTGAGGCGCCTA, siRNA 3: GCACCTTCCTGCTAAGATT are provided.

1.1.4 cell Co-culture and chemotherapy resistance Observation

Bone marrow MSCs of healthy controls isolated and cultured in 1.1.2 were divided into three groups, and LV-5-STAT3, control lentivirus, STAT3-siRNA, and bone marrow MSCs of AML patients were administered separately and co-cultured with AML cells of AML patients extracted in 1.1.1, respectively.

Cytarabine concentration experiments: the extracted AML cells are planted in a 48-well plate and divided into 5 groups, 0 mug/L, 5 mug/L, 10 mug/L, 30 mug/L and 50 mug/L cytarabine are respectively given to the groups, each group is subjected to 3 repeated wells, the proliferation condition of the AML cells is detected after the treatment of cytarabine with different concentrations by a CCK8 method, and the minimum cytarabine concentration capable of obviously inhibiting the proliferation of the AML cells is selected for subsequent experiments.

The CCK8 method comprises the following detection steps: the CCK8 kit from Engreen was purchased with the following steps:

1) 100 μ l of cell suspension was prepared in a 96-well plate, and the plate was pre-incubated in an incubator for 24 hours (at 37 ℃, 5% CO)₂Under the conditions of (a).

2) To the plates 10. mu.l of the test substances were added at different concentrations.

3) The plates were incubated in an incubator for 48 hours.

4) To each well was added 10 μ l CCK8 solution (note that no air bubbles were formed in the wells, preventing readings that affected the OD values).

5) The plates were incubated in an incubator for 3 hours.

6) Absorbance at 450nm was measured with a microplate reader.

The co-cultured cells were treated with cytarabine at the optimum concentration as described above, and proliferation of AML cells in different groups was examined by the CCK8 method and survival of AML cells was examined by the MTT method.

1.2 at the animal level, it was demonstrated that the JAK/STAT3 signaling pathway regulates the effects of MSCs in resistance to chemotherapy of AML cells

The AML cell line HL-60 was purchased from Shanghai Haisheng industries, Ltd. Taking out the frozen AML cell strain HL-60, quickly melting, thawing at 37 deg.C, introducing 5% CO into cell culture box₂Thawing at 37 deg.C, inoculating HL-60 into RPMI-1640 culture medium containing 100mg/L streptomycin, 100U/mL penicillin G and 10% fetal calf serum, and culturing. Conventional subculture for 3 days, injecting into centrifuge tube after EDTA digestion, centrifuging at low speed of 1000r/min for 5min, collecting AML cell strain HL-60, and modulating into cell number density of 1 × 10⁷The cell suspension of (a) is ready for use.

AML mouse model establishment: the SCID mice are 60 in SPF grade, half in male and female, 7-8 weeks old and 20-22 g in body mass. The constant temperature of the animal room is required to be 20-22 ℃, the constant humidity is 60-70%, 5 animals are fed in 1 cage, the animals are naturally illuminated, purified water is freely drunk, and food materials are sterilized under high pressure. Performing intraperitoneal injection pretreatment on cyclophosphamide with the body mass of 100mg/kg on a super-clean bench, injecting 1 time per day for 2 days, taking the standby AML cell strain HL-60 cell suspension on the 3 rd day, and injecting the suspension from the right side of the mouse into subcutaneous single points (1 × 10)⁷Mice), blank control group was not treated. Tumor infiltration in the liver and spleen and peripheral blood leukocyte (4.03 +/-1.92) x10 appear in 3 weeks after injection⁹the/L is the standard of tumor formation.

MSCs given LV-5-STAT3, control lentivirus, STAT3-siRNA, respectively, at 1.1.4 were injected tail vein into AML and control mice. And the following observations and tests were performed for each group of mice:

general conditions of the mice: mice were observed during the experiment at a frequency of 3 times per week for mental, appetite, mobility, hair condition, abdominal mass, body mass, etc. and recorded.

Routine detection of peripheral blood: before the mice die on day 25, the eyeballs are picked up to take blood, the blood collection amount is 1 mL/mouse, EDTA-K2 is anticoagulated, and a full-automatic blood routine detector is used for detecting the routine peripheral blood.

Blood smear analysis: preparing blood smear, staining with Ruhl-Giemsa, drying, performing microscopic examination, and sealing with neutral gum.

Mouse bone marrow image analysis: aseptically isolated mice were bilateral femurs and tibias and the marrow cavity was flushed with PBS to a whitish color. And blowing and beating the cell suspension, centrifuging the cell suspension by using a centrifuge, washing the cell suspension for 1 time by using PBS (phosphate buffer solution), and preparing a cell climbing sheet. Staining with Rue-Giemsa, drying, examining under microscope, and sealing with neutral gum.

Detection of liver pathology: and (3) killing the mice on the 25 th day, taking fresh livers, fixing the livers in paraformaldehyde, dehydrating in sequence according to a set ethanol concentration gradient, performing transparency in dimethylbenzene, embedding tissue blocks in paraffin, performing HE staining after frozen sections, performing xylene transparency after ethanol dehydration, and performing microscopic examination and mounting after drying in the air.

Example 2

As shown in FIG. 2, it was confirmed that the AML-derived exosomes up-regulate the JAK/STAT3 signaling pathway in MSCs and mediate the resistance of MSCs against chemotherapy of AML cells

2.1 extraction of exosomes from AML cells and Normal cells

Plasma exosomes of AML patients and healthy controls in 1.1.1 were extracted using Total Exosome Isolation Kit Exosome extraction Kit and operated according to the instructions, detailed steps were as follows:

(1) dissolving frozen plasma samples in a water bath at 25 ℃, and placing on ice;

(2) centrifuging 500ul plasma at 4 deg.C at 2000r/min for 20min, sucking supernatant with pipette to dispose new sterile and enzyme-free EP tube, and discarding tube bottom cell and debris;

(3) centrifuging the supernatant at 4 deg.C at 10000r/min for 20min, sucking the supernatant with a pipette to dispose a new sterile enzyme-free EP tube, and discarding the tube bottom debris;

(4) adding 250ul sterile, enzyme-free PBS (phosphate buffer saline), and mixing by vortexing;

(5) adding 25ul protease K, mixing by vortex, and incubating at 37 deg.C for 10 min;

(6) adding 150ul Exosome precipitation Reagent (Exosome Isolation Reagent), vortexing, mixing, and incubating at 4 deg.C for 30 min;

(7) centrifuging at room temperature at 10000r/min for 5min, removing supernatant, retaining the tube bottom granule-like precipitate as exosome, and freezing and storing the precipitate at 80 deg.C.

2.2 Regulation of the JAK/STAT3 Signal pathway in MSCs by exosomes

The exosomes in the plasma of the AML patient and the healthy control extracted in 2.1 were co-cultured with the bone marrow MSCs of the healthy control extracted in 1.1.2, and the expression levels of JAK protein and STAT3 protein in the co-culture system were detected by western blot.

Western blot experiment steps: protein extraction: adherent cells were trypsinized to suspended cells, centrifuged, supernatant removed, cells collected, 1ml PBS added, cells transferred to 1.5ml EP tubes at room temperature 400rmp, centrifuged for 3.5min, supernatant removed, washed 2 times with PBS, cells obtained by centrifugation were added to Western cell lysate (RIPA) produced in cloudy days per tube, placed on ice for half an hour for lysis, and then sonicated on a sonicator for 3 times for 15s each time.

And (3) measuring the protein concentration: the BCA protein concentration determination kit of Biyun day is adopted, and the concentration of the standard substance is 0.5 ug/ul. Three multiple wells were set for each concentration. According to the number of samples, a proper amount of BCA working solution is prepared by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and the mixture is fully mixed. Standards were added to standard wells of a 96-well plate at 0,1,2,4,8,12,16,20ul and PBS was added to make up to 20 ul. Add 2ul of sample to the sample wells of a 96 well plate and standard dilutions to 20 ul. 200ul of BCA working solution was added to each well, and the mixture was left at 37 ℃ for 30 min. A570 was measured on a microplate reader. And drawing a standard curve according to the result, and calculating the protein concentration of the sample.

Electrophoresis: for electrophoresis, 85v low voltage constant voltage electrophoresis was used for the top layer gel, and 150v high voltage constant voltage electrophoresis was used for the bromophenol blue into the bottom layer gel. According to the electrophoresis of the prestained protein molecular weight standard, the electrophoresis is expected to be stopped after the target protein is properly separated.

Film transfer: PVDF membrane is selected, and methanol is needed for activation before use. The standard wet-type membrane transfer device of Bio-Rad is used, the membrane transfer current can be set to be 200mA, and the membrane transfer time can be set to be 45-60 min. The membrane can be dyed by ponceau red dyeing liquid to observe the actual membrane transferring effect.

And (3) sealing: preparing 5% sealing liquid from 1g of skimmed milk powder and 20ml of TBST (1 x); immediately placing the protein membrane into a Western washing solution (TBST) prepared in advance after the membrane transfer is finished, and rinsing for 1-2min to wash off the membrane transfer solution on the membrane. Add Western blocking solution, shake slowly on shaker, block for 90min at room temperature.

Primary antibody incubation: after blocking, the membrane was washed 3 times with TBST for 5min, then diluted with Western primary antibody dilution at the appropriate ratio (1:1000) and incubated overnight at 4 ℃ with slow shaking. Recovering the primary antibody. Adding Western washing solution, and slowly shaking and washing on side-shaking table for 3 times, each time for 5 min.

And (3) secondary antibody incubation: with reference to the secondary antibody instructions, horseradish peroxidase (HRP) -labeled secondary antibody was diluted with Western secondary antibody dilution at the appropriate ratio (1:1000) and incubated on a side shaking table for one hour at room temperature with slow shaking. And (5) recovering the secondary antibody. Adding Western washing solution, and slowly shaking and washing on side-shaking table for 3 times, each time for 5 min.

Protein detection: and (3) detecting the protein by using a Western fluorescence detection reagent, preparing a developing solution and exposing.

2.3 exosome-mediated resistance of MSCs to AML cytochemotherapy

The MSCs co-cultured with the AML patient plasma exosomes and the healthy control plasma exosomes in 2.2, respectively, were then co-cultured with the AML cells of the AML patients extracted in 1.1.1, respectively, after exosomes were removed, and then treated with cytarabine solution of a determined concentration in 1.1.4, and proliferation of AML cells was detected in the same manner with reference to the CCK8 experimental procedure in 1.1.4.

MTT assay AML cell survival:

(1) cells grown to log phase were seeded in 96-well plates at a volume of 50 μ L/well and a cell count per well of approximately 5X 10³～1.2×10⁴The holes at the edge are filled with complete medium.

(2) Will be provided withThe cells were incubated at 37 ℃ with 5% CO₂Incubating for 72 hours in the incubator;

(3) adding 20 mu L of MTT into each hole, blowing and beating for several times by using a pipette, placing the mixture into an incubator, and standing for 2-4 hours;

(4) adding 100 mu L of solubilization solution into each hole, blowing and beating for several times by using a pipette, placing in an incubator, and standing overnight;

(5) the light absorption value of each well was measured at a wavelength of 570nm and 650nm with a microplate reader, and the cell viability and IC50 value were calculated for each concentration. The calculation formula is as follows:

survival (viability) × (experimental OD/control OD) × 100%

Example 3

As shown in FIG. 3, the mechanism of action of AML-derived exosomes to modulate the JAK/STAT3 signaling pathway was studied in vitro experiments

3.1 Signaling molecule Studies that may be associated with AML-derived exosomes and the JAK/STAT3 signaling pathway

According to the research of the prior art documents, the JAK/STAT3 signal channel can induce the production of some cytokines such as VEGF, promote the formation of blood vessels and provide convenience for the proliferation and migration of tumor cells; promoting the synthesis of MMPs (matrix metalloproteinases), and facilitating the invasion and remote metastasis of tumor cells; promote the secretion of inflammatory factors IL-1 beta and TGF beta, and inhibit the immune response of immune cells to tumor cells.

Based on the research background, the design experiment aims at VEGF, MMPs, IL-1 beta and TGF beta, and carries out related research on the mechanism of JAK/STAT3 signal pathway regulation by an exosome from AML.

Extracting the total protein content in the MSCs cultured by the AML extracellular exosomes and the normal extracellular exosomes respectively in 2.1, detecting the protein expression levels of VEGF, MMPs, IL-1 beta and TGF beta by using western blot, and determining which molecule or molecules (marked as X) the AML extracellular exosomes influence the expression level of, thereby influencing the JAK/STAT3 signal pathway in the MSCs.

3.2 verification of the mechanism of action of X in the regulation of JAK/STAT3 signaling pathway in MSCs by AML-derived exosomes

After the 3.1 experiment is completed, the X signal molecules in the AML cells are subjected to expression intervention or overexpression, exosomes in the AML cells are extracted and co-cultured with the MSCs, and the expression level of JAK and STAT3 proteins is detected through western blot so as to detect whether the JAK/STAT3 signal channel in the MSCs is inhibited or activated. Through the experiment, the mechanism of the X signal molecule for regulating the JAK/STAT3 signal pathway in MSCs by the exosome derived from AML is verified.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.