Culture method of mesenchymal stem cells and application of mesenchymal stem cells in treatment of leukemia

Abstract

The invention relates to the technical field of cells, in particular to a culture method of mesenchymal stem cells and application of the mesenchymal stem cells in treatment of leukemia. The mesenchymal stem cells are cultured by adopting a UC-MSCs culture medium, the UC-MSCs culture medium takes a DMEM / F12 culture medium as a basic component, a fermentation extract is added, and preparation raw materials of the fermentation extract comprise the following components in parts by weight: 1-2 parts of radix puerariae, 2-3 parts of ligusticum wallichii, 1-2 parts of selfheal, 1-1.5 parts of cacumen biotae, 0.8-1.2 parts of echinacea purpurea and 0.5-1 part of mangnolia officinalis.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and particularly to a method for culturing mesenchymal stem cells and their application in the treatment of leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy, which is mainly characterized by the massive accumulation of malignant primitive and immature myeloid cells in the bone marrow and blood. These primitive leukemia cells are arrested at an early stage of differentiation and proliferate clonally in large numbers, seriously interfering with normal hematopoiesis and immune function. Currently, the main treatment for AML is high-dose chemotherapy combined with hematopoietic stem cell transplantation (HSCT). However, due to the use of high-dose chemotherapy, serious side effects still occur. At the same time, HSCT requires finding a suitable match to avoid immune rejection reactions, which also greatly limits the clinical application of HSCT. Mesenchymal stem cells (MSCs) exist in almost all tissues. Currently, it is possible to isolate and prepare MSCs from tissues such as bone marrow, adipose tissue, umbilical cord, placenta, and amniotic fluid. MSCs not only have strong self-renewal ability and multi-directional differentiation potential, but also have characteristics such as immune regulation ability and low immunogenicity, and are regenerative source cells with broad application prospects in the field of transplantation. Clinically, the most commonly used are bone marrow-derived mesenchymal stem cells (BM-MSCs), generally mainly for autologous transplantation, and can be used to treat various chronic diseases. However, the BM-MSCs of AML patients have lost their strong self-renewal ability and also lost their role in maintaining the growth and function of normal hematopoietic stem cells, and instead support the growth and proliferation of AML cells. And there are research reports that BM-MSCs derived from AML patients can protect AML cells from the killing effect of chemotherapeutic drugs. Therefore, autologous transplantation of BM-MSCs cannot achieve the effect of alleviating the course of AML. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the present invention provides a method for culturing mesenchymal stem cells and their application in the treatment of leukemia.

[0004] The present invention is achieved by the following technical solutions: A method for culturing mesenchymal stem cells, comprising the following steps: S1. Use umbilical cord tissue to isolate and prepare umbilical cord mesenchymal stem cells (UC-MSCs), resuspend them with UC-MSCs medium, and adjust the seeding density to 2×10 6cells / mL, and placed in an incubator with 5% CO2 at 37°C for culture. The culture medium was changed every 2 days. S2. When the cell fusion reached 80%, it was digested and passaged at a ratio of 1:3 with 0.25% trypsin-EDTA solution, and cultured continuously using UC-MSCs medium in an incubator with 5% CO2 at 37°C. The culture medium was changed once every 2 days during the culture period.

[0005] Furthermore, the UC-MSCs medium uses DMEM / F12 medium as the basic component, and the DMEM / F12 medium also includes the following components: PALL serum substitute 5 v / v%, fermentation extract 4 g / L, L-glutamine 10 mM, vitamin C 200 μM, transferrin 3 μg / mL, bFGF 10 ng / mL, insulin 12 μg / mL, β-mercaptoethanol 50 μM, EGF 20 ng / mL.

[0006] Furthermore, the raw materials for preparing the fermentation extract include the following components in parts by weight: 1-2 parts of Pueraria lobata, 2-3 parts of Ligusticum chuanxiong, 1-2 parts of Prunella vulgaris, 1-1.5 parts of Platycladus orientalis, 0.8-1.2 parts of Echinacea purpurea, and 0.5-1 part of Magnolia officinalis.

[0007] Furthermore, the preparation method of the fermentation extract includes the following steps: L1. Wash Pueraria lobata, Ligusticum chuanxiong, Prunella vulgaris, Platycladus orientalis, Echinacea purpurea, and Magnolia officinalis, dry them, crush them through an 80-100 mesh sieve, mix them with 10-15 times the weight-volume of deionized water, soak for 1 h, perform ultrasonic treatment at 80-90°C and 500-600 W for 1 h, cool to room temperature, and filter to obtain filter residue and filtrate; L2. Dry the filter residue obtained in step L1, pass it through an 80-100 mesh sieve, mix it with 10-15 times the weight-volume of deionized water, sterilize it at 121°C, and inoculate the activated Lactobacillus rhamnosus and Bacillus subtilis at inoculation amounts of 5×10 6 CFU / mL and 1×10 6 CFU / mL respectively, and culture them on a shaker at 30°C and 60-80 r / min for 48 h, then centrifuge at 5000-6000 rpm for 20 min to obtain supernatant I; L3. Sterilize the filtrate obtained in step L1 at 121°C, and use 5×10 6Inoculate the activated Lactobacillus plantarum at an inoculum size of CFU / mL, culture it in a shaker at 30 °C and 60 - 80 r / min for 72 h, centrifuge at 10000 rpm for 10 - 15 min, mix the supernatant with the supernatant I obtained in step L2, perform vacuum distillation at 72 °C and a vacuum of 800 mbar until no liquid drips continuously, add activated carbon for injection at 5 mg / mL, sterilize at 115 °C for 30 min, cool to 30 °C, filter through a titanium rod filter, and freeze-dry to obtain a fermentation extract.

[0008] Further, in step L2, the Lactobacillus rhamnosus and Bacillus subtilis are purchased from the China General Microbiological Culture Collection Center, with the numbers CGMCC 1.8882 and CGMCC 1.821.

[0009] Further, in step L3, the Lactobacillus plantarum is purchased from the China General Microbiological Culture Collection Center, with the number CGMCC 1.572.

[0010] Further, the present invention also provides the application of the mesenchymal stem cells prepared by the culture method of the mesenchymal stem cells in the preparation of a drug for treating leukemia.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for culturing mesenchymal stem cells and its application in the treatment of leukemia. Through the synergistic effect of specific culture medium components, the proliferation ability of mesenchymal stem cells and the effect in the treatment of leukemia are significantly improved. The culture medium components of the present invention have a synergistic effect to enhance the proliferation ability of mesenchymal stem cells. The present invention uses DMEM / F12 as the basal medium, adds a fermentation extract (prepared by co-fermenting specific traditional Chinese medicine components such as Pueraria lobata, Ligusticum chuanxiong, and Prunella vulgaris with Lactobacillus rhamnosus, Bacillus subtilis, and Lactobacillus plantarum), and is compounded with components such as a serum substitute and growth factors to form a unique culture system, significantly improving the culture effect of umbilical cord mesenchymal stem cells (UC-MSCs). The active ingredients in traditional Chinese medicine can release small molecule substances that are more easily absorbed by cells after fermentation by probiotics, combined with the metabolites of the bacteria, to improve the cell microenvironment. The fermentation extract and each component act synergistically to effectively enhance the proliferation ability and biological activity of umbilical cord mesenchymal stem cells. The components of the fermentation extract of the present invention have a synergistic effect. After fermenting traditional Chinese medicine components such as Pueraria lobata, Ligusticum chuanxiong, and Prunella vulgaris with Lactobacillus rhamnosus and Bacillus subtilis, a variety of bioactive substances are released to regulate the activity of UC-MSCs and enhance their ability to inhibit leukemia cells. Moreover, after the traditional Chinese medicine components are fermented, the bioavailability is improved, which can promote UC-MSCs to secrete hematopoietic factors (such as GM-CSF), improve the hematopoietic inhibition state of the bone marrow microenvironment, enhance the inhibitory effect on leukemia cells, and reduce the side effects of chemotherapy. The fermentation extract of the present invention adopts a "water extraction - two-stage fermentation" process. First, the filter residue is fermented, and Lactobacillus rhamnosus and Bacillus subtilis degrade plant fibers to release active substances. Then, the filtrate is fermented, and Lactobacillus plantarum further metabolizes water-soluble components to stabilize the structure of active substances, while enhancing the antioxidant ability and increasing the yield and bioavailability of active ingredients. The present invention uses a PALL serum substitute to replace animal serum, avoiding the risk of heterologous component contamination and improving the safety of clinical application. Brief Description of the Drawings

[0012] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0013] Figure 1 Growth conditions of the mesenchymal stem cells described in Example 1 and Comparative Examples 1-3 of the present invention; Figure 2 Effect of the mesenchymal stem cells described in Example 1 and Comparative Examples 1-3 of the present invention on GM-CSF in mouse serum; Figure 3 Effect of the mesenchymal stem cells described in Example 1 and Comparative Examples 1-3 of the present invention on the proportion of CD45+ cells in mice. Detailed implementation manners

[0014] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in combination with specific embodiments. However, the present invention is not limited to the following embodiments. It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0015] Example 1: A method for culturing mesenchymal stem cells, comprising the following steps: S1. Umbilical cord mesenchymal stem cells (UC-MSCs) are isolated and prepared from umbilical cord tissue, resuspended with UC-MSCs medium, and the seeding density is adjusted to 2×10 6 cells / mL, and placed in an incubator at 5% CO2 and 37 °C for culture, and the medium is changed every 2 days; S2. When the cell confluence reaches 80%, digestion and passage are carried out with 0.25% trypsin-EDTA solution at a ratio of 1:3, and UC-MSCs medium is used for continuous culture at 5% CO2 and 37 °C. The medium is changed once every 2 days during the culture period.

[0016] The UC-MSCs medium uses DMEM / F12 medium as the basic component, and the DMEM / F12 medium further includes the following components: PALL serum substitute 5 v / v%, fermentation extract 4 g / L, L-glutamine 10 mM, vitamin C 200 μM, transferrin 3 μg / mL, bFGF 10 ng / mL, insulin 12 μg / mL, β-mercaptoethanol 50 μM, EGF 20 ng / mL.

[0017] The preparation method of the fermentation extract includes the following steps: L1. 2 g of Pueraria lobata, 3 g of Ligusticum chuanxiong, 2 g of Prunella vulgaris, 1.5 g of Platycladus orientalis, 1.2 g of Echinacea purpurea, and 1 g of Magnolia officinalis are washed, dried, pulverized and passed through a 100-mesh sieve, mixed with 15 times the weight-to-volume of deionized water, soaked for 1 h, sonicated at 90 °C and 600 W for 1 h, cooled to room temperature, and filtered to obtain filter residue and filtrate; L2. The filter residue obtained in step L1 is dried, passed through a 100-mesh sieve, mixed with 15 times the weight-to-volume of deionized water, sterilized at 121 °C, and respectively at 5×10 6 CFU / mL and 1×10 6Inoculate the activated Lactobacillus rhamnosus and Bacillus subtilis at an inoculum concentration of CFU / mL, culture them in a shaker at 30 °C and 80 r / min for 48 h, centrifuge at 6000 rpm for 20 min to obtain supernatant I; the Lactobacillus rhamnosus and Bacillus subtilis are purchased from the China General Microbiological Culture Collection Center, with the numbers CGMCC 1.8882 and CGMCC 1.821; L3. Sterilize the filtrate obtained in step L1 at 121 °C, inoculate the activated Lactobacillus plantarum at an inoculum concentration of 5×10 6 CFU / mL, culture it in a shaker at 30 °C and 80 r / min for 72 h, centrifuge at 10000 rpm for 15 min, mix the supernatant with supernatant I obtained in step L2, perform vacuum distillation at 72 °C and a vacuum degree of 800 mbar until no liquid drips continuously, add activated carbon for injection at 5 mg / mL, sterilize at 115 °C for 30 min, cool to 30 °C, filter through a titanium rod filter, and freeze-dry to obtain a fermentation extract; the Lactobacillus plantarum is purchased from the China General Microbiological Culture Collection Center, with the number CGMCC 1.572.

[0018] Example 2: A method for culturing mesenchymal stem cells, comprising the following steps: S1. Isolate and prepare umbilical cord mesenchymal stem cells (UC-MSCs) from umbilical cord tissue, resuspend them with UC-MSCs medium, adjust the inoculation density to 2×10 6 cells / mL, place them in an incubator with 5% CO2 at 37 °C for culture, and change the medium every 2 days; S2. When the cell fusion reaches 80%, digest and passage them with a 0.25% trypsin-EDTA solution at a ratio of 1:3, use UC-MSCs medium, and continue to culture at 5% CO2 and 37 °C. During the culture period, change the medium once every 2 days.

[0019] The UC-MSCs medium uses DMEM / F12 medium as the basic component, and the DMEM / F12 medium also includes the following components: PALL serum substitute 5 v / v%, fermentation extract 4 g / L, L-glutamine 10 mM, vitamin C 200 μM, transferrin 3 μg / mL, bFGF 10 ng / mL, insulin 12 μg / mL, β-mercaptoethanol 50 μM, EGF 20 ng / mL.

[0020] The preparation method of the fermentation extract includes the following steps: L1. Wash 1 g of Pueraria lobata, 2 g of Ligusticum wallichii, 1 g of Prunella vulgaris, 1 g of Platycladus orientalis, 0.8 g of Echinacea purpurea, and 0.5 g of Magnolia officinalis, dry them in an oven, crush them through an 80-mesh sieve, mix them with 10 times the weight-to-volume of deionized water, soak for 1 h, perform ultrasonic treatment at 80 °C and 500 W for 1 h, cool to room temperature, filter to obtain filter residue and filtrate; L2. Dry the filter residue obtained in step L1, pass it through an 80-mesh sieve, mix it with 10 times the weight-to-volume of deionized water, sterilize at 121 °C, and inoculate the activated Lactobacillus rhamnosus and Bacillus subtilis at inoculation amounts of 5×10 6 CFU / mL and 1×10 6 CFU / mL respectively, culture in a shaker at 30 °C and 60 r / min for 48 h, centrifuge at 5000 rpm for 20 min to obtain supernatant I; the Lactobacillus rhamnosus and Bacillus subtilis are purchased from the China General Microbiological Culture Collection Center, with the numbers CGMCC 1.8882 and CGMCC1.821; L3. Sterilize the filtrate obtained in step L1 at 121 °C, inoculate the activated Lactobacillus plantarum at an inoculation amount of 5×10 6 CFU / mL, culture in a shaker at 30 °C and 60 r / min for 72 h, centrifuge at 10000 rpm for 10 min, mix the supernatant with supernatant I obtained in step L2, perform vacuum distillation at 72 °C and a vacuum degree of 800 mbar until no liquid drips continuously, add activated carbon for injection at 5 mg / mL, sterilize at 115 °C for 30 min, cool to 30 °C, filter through a titanium rod filter, and freeze-dry to obtain a fermentation extract; the Lactobacillus plantarum is purchased from the China General Microbiological Culture Collection Center, with the number CGMCC 1.572.

[0021] Example 3: A method for culturing mesenchymal stem cells, comprising the following steps: S1. Isolate and prepare umbilical cord mesenchymal stem cells (UC-MSCs) from umbilical cord tissue, resuspend them with UC-MSCs medium, adjust the seeding density to 2×10 6 cells / mL, place them in an incubator with 5% CO2 at 37 °C for culture, and change the medium every 2 d; S2. When the cell confluence reaches 80%, digest and passage them with 0.25% trypsin-EDTA solution at a ratio of 1:3, use UC-MSCs medium, and continue to culture at 5% CO2 and 37 °C. Change the medium once every 2 days during the culture period.

[0022] The UC-MSCs culture medium uses DMEM / F12 medium as the basic component, and the DMEM / F12 medium also includes the following components: PALL serum substitute 5 v / v%, fermentation extract 4 g / L, L-glutamine 10 mM, vitamin C 200 μM, transferrin 3 μg / mL, bFGF 10 ng / mL, insulin 12 μg / mL, β-mercaptoethanol 50 μM, EGF 20 ng / mL.

[0023] The preparation method of the fermentation extract includes the following steps: L1. Wash 1.5 g of Pueraria lobata, 2.5 g of Ligusticum chuanxiong, 1.5 g of Prunella vulgaris, 1.2 g of Platycladus orientalis, 1 g of Echinacea purpurea, and 0.8 g of Magnolia officinalis, dry them in an oven, crush them through a 90-mesh sieve, mix them with 12 times the weight-volume of deionized water, soak for 1 h, ultrasonicate at 85 °C and 550 W for 1 h, cool to room temperature, filter to obtain filter residue and filtrate; L2. Dry the filter residue obtained in step L1, pass it through a 90-mesh sieve, mix it with 12 times the weight-volume of deionized water, sterilize at 121 °C, and inoculate the activated Lactobacillus rhamnosus and Bacillus subtilis at inoculation amounts of 5×10 6 CFU / mL and 1×10 6 CFU / mL, culture in a shaker at 30 °C and 70 r / min for 48 h, centrifuge at 5500 rpm for 20 min to obtain supernatant I; the Lactobacillus rhamnosus and Bacillus subtilis are purchased from the China General Microbiological Culture Collection Center, with the numbers CGMCC 1.8882 and CGMCC 1.821; L3. Sterilize the filtrate obtained in step L1 at 121 °C, inoculate the activated Lactobacillus plantarum at an inoculation amount of 5×10 6 CFU / mL, culture in a shaker at 30 °C and 70 r / min for 72 h, centrifuge at 10000 rpm for 12 min, mix the supernatant with supernatant I obtained in step L2, perform vacuum distillation at 72 °C and a vacuum of 800 mbar until no liquid drips continuously, add activated carbon for injection at 5 mg / mL, sterilize at 115 °C for 30 min, cool to 30 °C, filter through a titanium rod filter, and freeze-dry to obtain the fermentation extract; the Lactobacillus plantarum is purchased from the China General Microbiological Culture Collection Center, with the number CGMCC 1.572.

[0024] The difference between Comparative Example 1 and Example 1 is only that the fermentation extract is not added.

[0025] The difference between Comparative Example 2 and Example 1 is only that the supernatant I obtained in step L2 is subjected to vacuum distillation at 72 °C and a vacuum degree of 800 mbar until no liquid drips continuously, activated carbon for injection is added at 5 mg / mL, sterilized at 115 °C for 30 min, cooled to 30 °C, filtered through a titanium rod filter, and freeze-dried, and the obtained product is used to replace the fermentation extract.

[0026] The difference between Comparative Example 3 and Example 1 is only that the filtrate obtained in step L1 is sterilized at 121 °C, and the activated Lactobacillus plantarum is inoculated at an inoculation amount of 5×10 6 CFU / mL, cultured in a shaker at 30 °C and 80 r / min for 72 h, centrifuged at 10000 rpm for 15 min, the supernatant is subjected to vacuum distillation at 72 °C and a vacuum degree of 800 mbar until no liquid drips continuously, activated carbon for injection is added at 5 mg / mL, sterilized at 115 °C for 30 min, cooled to 30 °C, filtered through a titanium rod filter, and freeze-dried, and the obtained product is used to replace the fermentation extract.

[0027] Experimental Example 1: The P3-generation umbilical cord mesenchymal stem cells cultured in Example 1 and Comparative Examples 1-3 were collected and prepared into a cell suspension of 1×10 4 cells / mL with the corresponding UC-MSCs medium, inoculated into a 24-well plate, 1 mL per well, and cultured with the media corresponding to Example 1 and Comparative Examples 1-3 respectively, and placed in an incubator at 37 °C and 5% CO2. The number of cells was calculated on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days of culture respectively, and the cell growth curve was plotted. The results are as Figure 1 shown.

[0028] Figure 1 The results showed that compared with Example 1, Comparative Examples 1-3 changed the components in the medium, and the cell proliferation rate showed a downward trend, lower than that of Example 1. The above results indicate that the fermentation extract in the medium of the Example 1 group acts together with the various components in the medium, thereby effectively enhancing the proliferation ability of umbilical cord mesenchymal stem cells.

[0029] Experimental Example 2: Prepare a 10 mg / mL cytarabine (Ara-C) solution with sterile normal saline. Collect the leukemia cells of patients with high white blood cell acute myeloid leukemia (FBA classification M2 type), select 5-week-old B-NDG mice, irradiate the whole body with 1.5 Gy after one week of adaptive feeding, and inject 1×10 human leukemia cells into the tail vein within 24 h after irradiation 7On day 0, a human leukemia mouse model was constructed. After successful construction, the mice were randomly divided into 5 groups of 6 mice each, namely the AML + Ara - C group, the Example 1 group, Comparative Example 1, Comparative Example 2 group, and Comparative Example 3 group. Each mouse was intraperitoneally injected with 60 mg / kg of Ara - C for 3 consecutive days (from Day 40 to Day 42). One day after the end of Ara - C administration (Day 43), 10 6 P4 - generation UC - MSCs were injected into the tail vein of each mouse in the Example 1 and Comparative Example 1 - 3 groups. One week after the start of Ara - C administration (Day 46), blood was collected from the tail vein, serum was collected, and the GM - CSF content in the serum was measured by ELISA. The results are as Figure 2 shown.

[0030] Figure 2 The results showed that the GM - CSF in the peripheral blood serum of the mice in the Example 1 and Comparative Example 1 - 3 groups was significantly higher than that in the AML + Ara - C group, and the effect of Example 1 was the best, indicating that UC - MSCs can promote hematopoietic function reconstruction by increasing the content of hematopoietic factors such as GM - CSF in the serum. Infusing the UC - MSCs cultured by the method of the present invention into mice can effectively improve the treatment effect of leukemia.

[0031] Experimental Example 3: An Ara - C (cytarabine) solution with a concentration of 10 mg / mL was prepared with sterile normal saline. Leukemia cells of patients with hyperleukocytic acute myeloid leukemia (FBA classification M2 type) were collected. Five - week - old B - NDG mice were selected. After one - week of adaptive feeding, they were whole - body irradiated with 1.5 Gy. Within 24 h after irradiation, 1×10 7 human leukemia cells were injected into the tail vein (Day 0) to construct a human leukemia mouse model. After successful construction, the mice were randomly divided into 5 groups of 6 mice each, namely the AML + Ara - C group, the Example 1 group, Comparative Example 1, Comparative Example 2 group, and Comparative Example 3 group. Each mouse was intraperitoneally injected with 60 mg / kg of Ara - C for 3 consecutive days (from Day 40 to Day 42). One day after the end of Ara - C administration (Day 43), 10 6 P4 - generation UC - MSCs were injected into the tail vein of each mouse in the Example 1 and Comparative Example 1 - 3 groups. One week after the start of Ara - C administration (Day 46), 3 mice in each group were randomly sacrificed, and the content of human CD45+ cells in the mouse bone marrow was detected by flow cytometry. The results are as Figure 3 shown.

[0032] Figure 3The results showed that the proportion of CD45+ cells in Example 1 and Comparative Examples 1-3 was lower than that in the AML+Ara-C group, and the effect of Example 1 was the best, indicating that after culturing umbilical cord mesenchymal stem cells with the addition of fermentation extract in the culture medium, the present invention can synergistically enhance the effect with chemotherapeutic drugs and improve the treatment effect.

[0033] Those of ordinary skill in the art should understand that the discussion of any above embodiment is only exemplary and not intended to imply that the scope of the present invention is limited to these examples; under the idea of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, and they are not provided in detail for the sake of brevity.

Claims

1. A method for culturing mesenchymal stem cells, characterized in that, Including the following steps: S1. Culturing UC-MSCs with UC-MSCs medium; S2. Digesting and subculturing UC-MSCs, and continuing to culture with UC-MSCs medium; The UC-MSCs medium uses DMEM / F12 medium as the basic component, and the DMEM / F12 medium also includes the following components: 5 v / v% PALL serum substitute, 4 g / L fermentation extract, 10 mM L-glutamine, 200 μM vitamin C, 3 μg / mL transferrin, 10 ng / mL bFGF, 12 μg / mL insulin, 50 μM β-mercaptoethanol, 20 ng / mL EGF; The raw materials for preparing the fermentation extract include the following components in parts by weight: 1-2 parts of Pueraria lobata, 2-3 parts of Ligusticum chuanxiong, 1-2 parts of Prunella vulgaris, 1-1.5 parts of Platycladus orientalis, 0.8-1.2 parts of Echinacea purpurea, 0.5-1 part of Magnolia officinalis; The preparation method of the fermentation extract includes the following steps: L1. Weighing Pueraria lobata, Ligusticum chuanxiong, Prunella vulgaris, Platycladus orientalis, Echinacea purpurea, and Magnolia officinalis according to parts by weight, performing water extraction and then filtration to obtain filter residue and filtrate; L2. Inoculating the filter residue obtained in step L1 with Lactobacillus rhamnosus and Bacillus subtilis, culturing, and centrifuging to obtain supernatant I; L3. Inoculating the filtrate obtained in step L1 with Lactobacillus plantarum, culturing, centrifuging, mixing the supernatant with supernatant I obtained in step L2, performing vacuum distillation, adding activated carbon, sterilizing, filtering, and freeze-drying to obtain the fermentation extract; Use of the mesenchymal stem cells prepared by the method in the preparation of a drug for treating leukemia.

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