A method for culturing mesenchymal stem cells and its application in treating leukemia

Abstract

The present invention relates to the field of cell technology, and more particularly to a method for culturing mesenchymal stem cells and its use in treating leukemia. The mesenchymal stem cells are cultured in a UC-MSCs culture medium, which is based on DMEM / F12 medium and supplemented with a fermentation extract. The fermentation extract is prepared by adding the following raw materials, in parts by weight: 1-2 parts of Pueraria root, 2-3 parts of Chuanxiong rhizome, 1-2 parts of Prunella vulgaris, 1-1.5 parts of Platycladus orientalis leaves, 0.8-1.2 parts of Echinacea purpurea, and 0.5-1 parts of Magnolia officinalis.

Description

Technical Field

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

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the accumulation of malignant primitive and immature myeloid cells in the bone marrow and blood. These primitive leukemic cells are arrested in the early stages of differentiation and undergo extensive clonal proliferation, severely impairing ongoing hematopoietic and immune function. Currently, the mainstay of treatment for AML is high-dose chemotherapy combined with hematopoietic stem cell transplantation (HSCT). However, the use of high-dose chemotherapy still results in severe side effects. Furthermore, HSCT requires an appropriate match to avoid immune rejection, which significantly limits its clinical application. Mesenchymal stem cells (MSCs) are present in nearly all tissues. Currently, MSCs can be isolated and prepared from tissues such as bone marrow, adipose tissue, umbilical cord, placenta, and amniotic fluid. MSCs possess not only robust self-renewal capacity and multipotential differentiation potential, but also immunomodulatory abilities and low immunogenicity, making them promising regenerative cell sources for transplantation. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are most commonly used clinically, typically via autologous transplantation, to treat a variety of chronic diseases. However, BM-MSCs from AML patients lose their robust self-renewal capacity and their role in maintaining the growth and function of normal hematopoietic stem cells, instead supporting the growth and proliferation of AML cells. Studies have also reported that BM-MSCs from AML patients can protect AML cells from the damaging effects of chemotherapy drugs. Therefore, autologous transplantation of BM-MSCs is ineffective in alleviating the course of AML. Summary of the Invention

[0003] In view of the deficiencies of the existing technology, the present invention provides a method for culturing mesenchymal stem cells and its application in treating leukemia.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for culturing mesenchymal stem cells comprises the following steps:

[0006] S1. Umbilical cord mesenchymal stem cells (UC-MSCs) were prepared by isolating umbilical cord tissue and resuspending in UC-MSCs culture medium. The seeding density was adjusted to 2×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0007] S2. When cells reach 80% confluency, digest and passage them using 0.25% trypsin-EDTA solution at a 1:3 ratio. Continue culturing in UC-MSCs medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0008] Furthermore, the UC-MSCs culture medium uses DMEM / F12 culture medium as a basic component, and the DMEM / F12 culture medium also includes the following components: PALL serum replacement 5v / v%, fermentation extract 4 g / L, L-glutamine 10mM, vitamin C 200 μM, transferrin 3 μg / mL, bFGF 10 ng / mL, insulin 12 μg / mL, β-mercaptoethanol 50 μM, and EGF 20 ng / mL.

[0009] Furthermore, the raw materials for preparing the fermentation extract include the following components in parts by weight: 1-2 parts of Pueraria root, 2-3 parts of Chuanxiong, 1-2 parts of Prunella Vulgaris, 1-1.5 parts of Platycladus orientalis leaves, 0.8-1.2 parts of Echinacea purpurea, and 0.5-1 part of Magnolia officinalis.

[0010] Furthermore, the method for preparing the fermentation extract comprises the following steps:

[0011] L1. Wash and dry Pueraria root, Chuanxiong, Prunella vulgaris, Platycladus orientalis leaves, Echinacea, and Magnolia officinalis, crush through an 80-100 mesh sieve, mix with 10-15 times the weight volume of deionized water, soak for 1 h, ultrasonically treat at 80-90°C, 500-600W for 1 h, cool to room temperature, and filter to obtain a residue and filtrate;

[0012] 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 then add 5×10 6 CFU / mL and 1×10 6 The activated Lactobacillus rhamnosus and Bacillus subtilis were inoculated at an inoculum size of 100 CFU / mL, cultured at 30°C and 60-80 rpm for 48 h, and centrifuged at 5000-6000 rpm for 20 min to obtain supernatant I;

[0013] L3. Sterilize the filtrate obtained in step L1 at 121°C and add 5×10 6The activated Lactobacillus plantarum was inoculated at an inoculum size of 100 CFU / mL, cultured on a shaker at 30°C and 60-80 r / min for 72 h, centrifuged at 10,000 rpm for 10-15 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was 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 to obtain a fermentation extract.

[0014] Furthermore, in step L2, the Lactobacillus rhamnosus and Bacillus subtilis were purchased from China General Microorganism Culture Collection Center with the numbers CGMCC 1.8882 and CGMCC 1.821.

[0015] Furthermore, in step L3, the Lactobacillus plantarum was purchased from China General Microorganism Culture Collection Center with the number CGMCC 1.572.

[0016] Furthermore, the present invention also provides the use of mesenchymal stem cells obtained by the mesenchymal stem cell culture method in the preparation of drugs for treating leukemia.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a method for culturing mesenchymal stem cells and its application in treating 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 synergize to enhance the synergy and enhance the proliferation ability of mesenchymal stem cells. The present invention uses DMEM / F12 as the basal culture medium, adds fermentation extract (containing specific Chinese medicine components such as Pueraria lobata, Chuanxiong, and Prunella vulgaris, and is prepared by co-fermentation with Lactobacillus rhamnosus, Bacillus subtilis, and Lactobacillus plantarum), and compounded with serum substitutes, growth factors and other ingredients to form a unique culture system, which significantly improves the culture effect of umbilical cord mesenchymal stem cells (UC-MSCs). The active ingredients in traditional Chinese medicine can release small molecules that are more easily absorbed by cells after fermentation by probiotics, combine with bacterial metabolites, improve the cell microenvironment, and the fermentation extract synergizes with each component to effectively enhance the proliferation ability and biological activity of umbilical cord mesenchymal stem cells. The composition synergistic effect of the fermentation extract of the present invention, after the Chinese medicinal ingredients such as Pueraria lobata, Chuanxiong, and Prunella vulgaris are fermented by Lactobacillus rhamnosus and Bacillus subtilis, a variety of bioactive substances are released, UC-MSC activity is regulated, and its ability to inhibit leukemia cells is enhanced, and after fermentation, the bioavailability of the Chinese medicinal ingredients is improved, and UC-MSCs can be promoted to secrete hematopoietic factors (such as GM-CSF), improve the hematopoietic suppression state of the bone marrow microenvironment, enhance the inhibitory effect on leukemia cells, and reduce chemotherapy side effects. The fermentation extract of the present invention adopts "water extraction-two-stage fermentation" process, first the filter residue is fermented, Lactobacillus rhamnosus and Bacillus subtilis degrade plant fiber, release active substances, and then carry out filtrate fermentation, plant lactobacillus further metabolizes water-soluble components, stabilizes active substance structure, improves antioxidant capacity, and improves active ingredient yield and bioavailability. The present invention adopts PALL serum substitute to replace animal serum, avoids the risk of heterologous component contamination, and improves clinical application safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The growth conditions of the mesenchymal stem cells described in Example 1 and Comparative Examples 1-3 of the present invention;

[0021] Figure 2 The effect of the mesenchymal stem cells described in Example 1 and Comparative Examples 1-3 of the present invention on mouse serum GM-CSF;

[0022] Figure 3The effects 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 are shown. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples, but the present invention is not limited to the following examples. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0024] Example 1: A method for culturing mesenchymal stem cells, comprising the following steps:

[0025] S1. Umbilical cord mesenchymal stem cells (UC-MSCs) were prepared by isolating umbilical cord tissue and resuspending in UC-MSCs culture medium. The seeding density was adjusted to 2×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0026] S2. When cells reach 80% confluency, digest and passage them using 0.25% trypsin-EDTA solution at a 1:3 ratio. Continue culturing in UC-MSCs medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0027] The UC-MSCs culture medium is based on DMEM / F12 medium, which also contains the following ingredients: PALL serum replacement 5v / v%, fermentation extract 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, and EGF 20ng / mL.

[0028] The method for preparing the fermentation extract comprises the following steps:

[0029] L1. Wash and dry 2 g of Pueraria root, 3 g of Chuanxiong rhizome, 2 g of Prunella vulgaris, 1.5 g of Platycladus orientalis leaves, 1.2 g of Echinacea purpurea, and 1 g of Magnolia officinalis. Grind through a 100-mesh sieve and mix thoroughly with 15 times the weight by volume of deionized water. Soak for 1 hour, sonicate at 90°C at 600W for 1 hour, cool to room temperature, and filter to obtain the residue and filtrate.

[0030] L2. Dry the filter residue obtained in step L1, pass it through a 100-mesh sieve, mix it with 15 times the weight volume of deionized water, sterilize it at 121°C, and then add 5×10 6 CFU / mL and 1×10 6The activated Lactobacillus rhamnosus and Bacillus subtilis were inoculated with an inoculum size of 100 CFU / mL, cultured in a shaking incubator at 30°C and 80 rpm for 48 h, and centrifuged at 6000 rpm for 20 min to obtain supernatant I; the Lactobacillus rhamnosus and Bacillus subtilis were purchased from the China General Microbiological Culture Collection Center with the numbers CGMCC 1.8882 and CGMCC1.821;

[0031] L3. Sterilize the filtrate obtained in step L1 at 121°C and add 5×10 6 CFU / mL was inoculated with activated Lactobacillus plantarum, cultured on a shaker at 30°C and 80 r / min for 72 h, centrifuged at 10,000 rpm for 15 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was 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 to obtain a fermentation extract; the Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center and is numbered CGMCC 1.572.

[0032] Example 2: A method for culturing mesenchymal stem cells, comprising the following steps:

[0033] S1. Umbilical cord mesenchymal stem cells (UC-MSCs) were prepared by isolating umbilical cord tissue and resuspending in UC-MSCs culture medium. The seeding density was adjusted to 2×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0034] S2. When cells reach 80% confluency, digest and passage them using 0.25% trypsin-EDTA solution at a 1:3 ratio. Continue culturing in UC-MSCs medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0035] The UC-MSCs culture medium is based on DMEM / F12 medium, which also contains the following ingredients: PALL serum replacement 5v / v%, fermentation extract 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, and EGF 20ng / mL.

[0036] The method for preparing the fermentation extract comprises the following steps:

[0037] L1. Wash and dry 1 g of Pueraria root, 2 g of Chuanxiong rhizome, 1 g of Prunella vulgaris, 1 g of Platycladus orientalis leaves, 0.8 g of Echinacea purpurea, and 0.5 g of Magnolia officinalis. Grind through an 80-mesh sieve, mix with 10 times the weight by volume of deionized water, soak for 1 hour, sonicate at 80°C and 500W for 1 hour, cool to room temperature, and filter to obtain the residue and filtrate.

[0038] L2. Dry the filter residue obtained in step L1, pass it through an 80-mesh sieve, mix it with 10 times the weight volume of deionized water, sterilize it at 121°C, and add 5×10 6 CFU / mL and 1×10 6 The activated Lactobacillus rhamnosus and Bacillus subtilis were inoculated with an inoculum size of 100 CFU / mL, cultured in a shaking incubator at 30°C and 60 rpm for 48 h, and centrifuged at 5000 rpm for 20 min to obtain supernatant I; the Lactobacillus rhamnosus and Bacillus subtilis were purchased from the China General Microbiological Culture Collection Center with the numbers CGMCC 1.8882 and CGMCC1.821;

[0039] L3. Sterilize the filtrate obtained in step L1 at 121°C and add 5×10 6 CFU / mL was inoculated with activated Lactobacillus plantarum, cultured on a shaker at 30°C and 60 r / min for 72 h, centrifuged at 10,000 rpm for 10 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was 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 to obtain a fermentation extract; the Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center and is numbered CGMCC 1.572.

[0040] Example 3: A method for culturing mesenchymal stem cells, comprising the following steps:

[0041] S1. Umbilical cord mesenchymal stem cells (UC-MSCs) were prepared by isolating umbilical cord tissue and resuspending in UC-MSCs culture medium. The seeding density was adjusted to 2×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0042] S2. When cells reach 80% confluency, digest and passage them using 0.25% trypsin-EDTA solution at a 1:3 ratio. Continue culturing in UC-MSCs medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0043] The UC-MSCs culture medium is based on DMEM / F12 medium, which also contains the following ingredients: PALL serum replacement 5v / v%, fermentation extract 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, and EGF 20ng / mL.

[0044] The method for preparing the fermentation extract comprises the following steps:

[0045] L1. Wash and dry 1.5 g of Pueraria root, 2.5 g of Chuanxiong rhizome, 1.5 g of Prunella vulgaris, 1.2 g of Platycladus orientalis leaves, 1 g of Echinacea purpurea, and 0.8 g of Magnolia officinalis. Grind through a 90-mesh sieve and mix thoroughly with 12 times their weight by volume of deionized water. Soak for 1 hour, sonicate at 85°C and 550W for 1 hour, cool to room temperature, and filter to obtain the residue and filtrate.

[0046] L2. The filter residue obtained in step L1 was dried, passed through a 90-mesh sieve, mixed with 12 times the weight volume of deionized water, sterilized at 121°C, and 5×10 6 CFU / mL and 1×10 6 The activated Lactobacillus rhamnosus and Bacillus subtilis were inoculated with an inoculum size of 100 CFU / mL, cultured in a shaking incubator at 30°C and 70 rpm for 48 h, and centrifuged at 5500 rpm for 20 min to obtain supernatant I; the Lactobacillus rhamnosus and Bacillus subtilis were purchased from the China General Microbiological Culture Collection Center with the numbers CGMCC 1.8882 and CGMCC1.821;

[0047] L3. Sterilize the filtrate obtained in step L1 at 121°C and add 5×10 6 CFU / mL was inoculated with activated Lactobacillus plantarum, cultured on a shaker at 30°C and 70 r / min for 72 h, centrifuged at 10,000 rpm for 12 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was 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 to obtain a fermentation extract; the Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center and is numbered CGMCC 1.572.

[0048] The only difference between Comparative Example 1 and Example 1 is that no fermentation extract is added.

[0049] The only difference between Comparative Example 2 and Example 1 is that the supernatant I obtained in step L2 is distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously drips out, 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. The resulting product replaces the fermentation extract.

[0050] The only difference between Comparative Example 3 and Example 1 is that the filtrate obtained in step L1 is sterilized at 121°C and heated to 5×10 6 The activated Lactobacillus plantarum was inoculated with an inoculum size of 100 CFU / mL, cultured in a shaking incubator at 30°C and 80 rpm for 72 h, centrifuged at 10,000 rpm for 15 min, and the supernatant was distilled under reduced pressure at 72°C and 800 mbar until no liquid continuously dripped out. Injectable activated carbon was 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. The resulting product replaced the fermentation extract.

[0051] Experimental Example 1: The P3 umbilical cord mesenchymal stem cells obtained by culture in Example 1 and Comparative Examples 1-3 were collected and prepared into 1×10 4 Cell suspensions of 100 cells / mL were inoculated into 24-well plates, 1 mL per well, and cultured using the culture medium corresponding to Example 1 and Comparative Examples 1-3, respectively. The plates were placed in an incubator at 37°C and 5% CO2. The number of cells was counted on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th day of culture, and cell growth curves were plotted. The results are shown in Figure 2. Figure 1 shown.

[0052] Figure 1 The results showed that compared with Example 1, the cell proliferation rate of Comparative Examples 1-3, which changed the components in the culture medium, showed a downward trend and was lower than that of Example 1. The above results indicate that the fermentation extract in the culture medium of Example 1 group works together with the various components in the culture medium to effectively enhance the proliferation capacity of umbilical cord mesenchymal stem cells.

[0053] Experimental Example 2: A 10 mg / mL cytarabine (Ara-C) solution was prepared with sterile saline. Leukemia cells were collected from a patient with hyperleukocytic acute myeloid leukemia (FBA classification M2). Five-week-old B-NDG mice were selected and acclimated for one week before being irradiated with 1.5 Gy of whole-body radiation. Within 24 hours after irradiation, 1×10 human leukemia cells were injected into the tail vein. 7(Day 0) to construct a human leukemia mouse model. After successful construction, the mice were randomly divided into 5 groups, 6 in each group. They were AML+Ara-C group, Example 1 group, Comparative Example 1, Comparative Example 2 group, and Comparative Example 3 group. Each mouse was intraperitoneally injected with Ara-C 60 mg / kg for 3 consecutive days (Day 40 to Day 42). One day after the end of Ara-C administration (Day 43), each mouse in Example 1 and Comparative Examples 1-3 groups was injected with 10 6 One week after the start of Ara-C administration (Day 46), blood was collected from the tail vein and serum was collected. The GM-CSF content in the serum was measured by ELISA. The results are as follows: Figure 2 shown.

[0054] Figure 2 The results showed that the GM-CSF content in the peripheral blood serum of mice in Example 1 and Comparative Examples 1-3 was significantly higher than that in the AML+Ara-C group, with Example 1 having the best effect. This indicates that UC-MSCs can promote hematopoietic function reconstruction by increasing the content of serum hematopoietic factors such as GM-CSF. UC-MSCs cultured using the method of the present invention can be infused into mice to effectively improve the therapeutic effect of leukemia.

[0055] Experimental Example 3: A 10 mg / mL cytarabine (Ara-C) solution was prepared with sterile saline. Leukemia cells were collected from a patient with hyperleukocytic acute myeloid leukemia (FBA classification M2). Five-week-old B-NDG mice were selected and acclimated for one week before being irradiated with 1.5 Gy of whole-body irradiation. Within 24 hours after irradiation, 1×10 human leukemia cells were injected into the tail vein. 7 (Day 0) to construct a human leukemia mouse model. After successful construction, the mice were randomly divided into 5 groups, 6 in each group. They were AML+Ara-C group, Example 1 group, Comparative Example 1, Comparative Example 2 group, and Comparative Example 3 group. Each mouse was intraperitoneally injected with Ara-C 60 mg / kg for 3 consecutive days (Day 40 to Day 42). One day after the end of Ara-C administration (Day 43), each mouse in Example 1 and Comparative Examples 1-3 groups was injected with 10 6 One week after the start of Ara-C administration (Day 46), three mice in each group were randomly killed and the bone marrow of the mice was collected and the content of human CD45+ cells was detected by flow cytometry. The results are as follows Figure 3 shown.

[0056] 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 Example 1 had the best effect, indicating that after the fermentation extract was added to the culture medium to culture umbilical cord mesenchymal stem cells, the present invention can synergize with chemotherapy drugs to improve the therapeutic effect.

[0057] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. A method for culturing mesenchymal stem cells, characterized in that: The following steps are involved: S1. Culture umbilical cord mesenchymal stem cells (UC-MSCs) in UC-MSCs medium. S2. UC-MSCs were digested and passaged, and then cultured in UC-MSCs medium. The UC-MSCs culture medium uses DMEM / F12 culture medium as a basic component, and further comprises the following components: PALL serum replacement 5v / v%, fermentation extract 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, EGF 20ng / mL; The raw materials for preparing the fermentation extract include the following components in parts by weight: 1-2 parts of Pueraria root, 2-3 parts of Chuanxiong, 1-2 parts of Prunella vulgaris, 1-1.5 parts of Platycladus orientalis leaves, 0.8-1.2 parts of Echinacea purpurea, and 0.5-1 part of Magnolia officinalis. The method for preparing the fermentation extract comprises the following steps: L1 parts by weight of Pueraria root, Chuanxiong, Prunella vulgaris, Platycladus orientalis leaves, Echinacea, Magnolia officinalis water extraction and filtration to obtain a residue and filtrate; L2 The residue obtained in step L1 was inoculated with Lactobacillus rhamnosus and Bacillus subtilis, cultured, and centrifuged to obtain a supernatant I, the Lactobacillus rhamnosus and Bacillus subtilis numbers are CGMCC 1.8882 and CGMCC 1.821; L3 The filtrate obtained in step L1 was inoculated with Lactobacillus plantarum, cultured, centrifuged, and the supernatant obtained in step L2 was mixed with the supernatant I, distilled under reduced pressure, activated carbon was added, sterilized, filtered, and freeze-dried to obtain a fermentation extract, the Lactobacillus plantarum number is CGMCC 1.572; The umbilical cord mesenchymal stem cells prepared by the method are used in the preparation of medicines for treating leukemia.

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