Cultivation method of mesenchymal stem cells, mesenchymal stem cell population and application thereof

Abstract

The present invention belongs to the technical field of cell culture, and in particular relates to a method for culturing mesenchymal stem cells by using cannabidiol, obtaining a mesenchymal stem cell group that can highly express the anti-inflammatory factors TGF-β1, IL-10 and TSG6, and providing the use of the mesenchymal stem cell group in the preparation of a drug for treating autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a method for culturing mesenchymal stem cells by using cannabidiol, thereby obtaining a mesenchymal stem cell group that can highly express the anti-inflammatory factors TGF-β1, IL-10 and TSG6, and provides the use of the mesenchymal stem cell group in the preparation of drugs for treating autoimmune diseases. Background Art

[0002] Compared with traditional immunosuppressants, bone marrow mesenchymal stem cells (MSCs) do not express co-stimulatory molecules, have low immunogenicity, and do not require human leukocyte antigen (HLA) matching for cell therapy, making them more effective and less prone to side effects. The therapeutic effect of BMSCs is mainly anti-inflammatory, including promoting the secretion of anti-inflammatory factors TGF-β1, IL-10, TSG6 and inhibiting the secretion of pro-inflammatory factors IL-1a, IL-6, IL-10, IL-23, etc.

[0003] Cannabidiol (CBD) is a non-psychoactive cannabinoid extracted from cannabis that has antioxidant, anti-inflammatory, neuroprotective and immunomodulatory effects. [1-2] In addition, CBD can restore intestinal epithelial mucosal damage caused by various stimuli. However, it is still unclear whether CBD can promote the expression of anti-inflammatory factors in BMSCs.

[0004] [1]Thiele EA, Bebin EM, Filloux F, et al. Long-term cannabidiol treatment for seizures in patients with tuberous sclerosis complex: An open-labelextension trial[J]. Epilepsia, 2022, 63(2): 426-439.

[0005] [2]]Nouri K, Anooshe M, Karimi-Haghighi S, et al.Involvement ofHippocampal D1-Like Dopamine Receptors in the Inhibitory Effect ofCannabidiol on Acquisition and Expression of Methamphetamine-InducedConditioned Place Preference[J].Neurochem Res, 2021, 46(8): 2008-2018. Summary of the invention

[0006] The present invention provides a mesenchymal stem cell population with enhanced paracrine function, as well as a method for culturing and producing the above-mentioned mesenchymal stem cell population and the use of the mesenchymal stem cell population in preparing a drug for treating autoimmune diseases. The above-mentioned findings have not been reported yet.

[0007] According to one of the invention purposes of the present invention, the use of cannabidiol in the preparation of mesenchymal stem cells that highly express anti-inflammatory factors is provided. The inventors found that bone marrow mesenchymal stem cells treated with CBD highly expressed anti-inflammatory factors, and the highly expressed anti-inflammatory factors included: TGF-β1, IL-10 and TSG6.

[0008] The study found that these anti-inflammatory factors increased exponentially in the supernatant of stem cell culture after incubation with a specific concentration of CBD. However, the above characteristics only appear within a specific concentration range, which is any concentration value between 1-100nmol / L and 10-100nmol / L. More specifically, the optimal concentration range is 10-100nmol / L, and the optimal concentration value is 10nmol / L. Specifically, the results of the ELISA test showed that after treatment with 10nmol / LCBD, the ability of mesenchymal stem cells to secrete TGF-β1 was 2.55 times that of untreated cells; the ability to secrete IL-10 was 2.48 times that of untreated cells; and the ability to secrete TSG6 was 3.06 times that of untreated cells. Prior to this, the above characteristics of cannabidiol had not been discovered.

[0009] According to another object of the present invention, a method for preparing mesenchymal stem cells that highly express anti-inflammatory factors is provided, comprising: the step of culturing mesenchymal stem cells in a culture medium containing cannabidiol.

[0010] In the above method, the concentration of cannabidiol in the culture medium is any concentration value between 1 and 100 nmol / L or 10 and 100 nmol / L, more particularly, the optimal concentration range is 10 to 100 nmol / L, and the optimal concentration value is 10 nmol / L. Specifically, the ability of mesenchymal stem cells treated with 10 nmol / LCBD to secrete TGF-β1 is 2.55 times that of untreated cells; the ability to secrete IL-10 is 2.48 times that of untreated cells; and the ability to secrete TSG6 is 3.06 times that of untreated cells.

[0011] The culture medium in the above method can be a serum-containing or serum-free basal culture medium commonly used for culturing cells, such as DMEM, F12, DMEM / F-12 or RPMI 1640 culture medium; specifically, in the present invention, DMEM culture medium can be selected.

[0012] The mesenchymal stem cells described herein are selected from one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and placental mesenchymal stem cells; specifically, although the present invention only explores the enhancing effect of CBD on bone marrow mesenchymal stem cells, it does not rule out the possibility of the same level of effect on other mesenchymal stem cells.

[0013] In addition, the isolation, culture and identification of primary mesenchymal stem cells in the method for obtaining mesenchymal stem cells that highly express anti-inflammatory factors can refer to conventional technical operations in the field. For example, it includes: obtaining primary cell samples, isolating and culturing primary cells, identifying passaged cells, etc. Among them, the primary mesenchymal cell sample can be derived from bone marrow, umbilical cord or placental tissue.

[0014] According to another invention object of the present invention, a mesenchymal stem cell population prepared by the method is provided. The most obvious characteristic of the prepared mesenchymal stem cell population is that the ability to synthesize anti-inflammatory factors is enhanced, specifically including:

[0015] The mesenchymal stem cell population highly expresses TGF-β1, and the ability of the mesenchymal stem cells to secrete TGF-β1 after being treated with 10 nmol / L CBD is 2.55 times that of the untreated group of cells;

[0016] The mesenchymal stem cell population highly expresses IL-10, and the ability of the mesenchymal stem cells to secrete IL-10 after being treated with 10 nmol / L CBD is 2.48 times that of the untreated group of cells;

[0017] The mesenchymal stem cell population highly expressed TSG6, and the TSG6 secretion capacity of the mesenchymal stem cells treated with 10 nmol / L CBD was 3.06 times that of the untreated group of cells.

[0018] According to another object of the present invention, there is provided a use of the mesenchymal stem cell population in the preparation of a drug for treating autoimmune diseases. The autoimmune diseases include: primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), etc.

[0019] The beneficial effects of the present invention are:

[0020] The present invention explores the effects of cannabidiol on the proliferation of bone marrow mesenchymal stem cells and its paracrine secretion, and proposes for the first time that cannabidiol-treated MSCs can obtain a cell population with significantly enhanced immune function. The levels of TGF-β1, IL-10 and TSG6 anti-inflammatory factors synthesized by the treated MSCs are increased several times, providing a reliable cell source for the treatment of autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Relative expression levels of cytokine mRNA in each group. ** P<0.01, *** P<0.001, compared with 0nmol / L group. DETAILED DESCRIPTION

[0022] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] 1. Effects of cannabidiol on MSC proliferation and secretion of inflammatory-related factors

[0024] 1 Materials and Methods

[0025] 1.1 Effect on MSC activity: MSCs in the logarithmic growth phase of the third generation, qualified by flow cytometry, were cultured in DMEM medium to prepare 1×10 5 / ml cell suspension was inoculated in a 96-well plate at 100μl / well, with 6 replicates in each group. Different concentrations of CBD (0.1, 1, 10, 100, 1000nmol / L) were added, and an equal volume of PBS was added to the control group. After incubation for 72h, 10μl CCK-8 working solution was added to each well, and then incubated in a 37℃ incubator for 2h in the dark, and the absorbance (A) was detected at 450nm by an enzyme reader. The results are shown in Table 2.

[0026] 1.2 Cell cycle detection: The 3rd generation MSCs in the logarithmic growth phase, qualified by flow cytometry, were selected and cultured in DMEM medium to prepare 1×10 5 / ml cell suspension, inoculated in 96-well plates at 100μl / well, and when the cells grew to 70-80% confluence, cultured with DMDM ​​culture medium containing 0.1% serum for 12h. Then different concentrations of CBD were added, and the control group added an equal volume of PBS. After incubation for 48h, 0.25% trypsin was used for digestion, cells were collected, and 70% ice ethanol was used to fix the cells for 24h. Washed with PBS, centrifuged, and then 200μl PBS and 5μl RNAase were added, incubated at 37℃ for 30min, PI staining solution was added and stained in the dark for 20min, and detected by flow cytometry. The results are shown in Table 3.

[0027] 1.3 Cytokine mRNA expression level: The 3rd generation MSCs in the logarithmic growth phase and qualified by flow cytometry were taken, and DMEM culture medium was used to prepare a cell suspension of 1×105 / ml, and 100μl / well was inoculated in a 6-well plate, and then different concentrations of CBD were added, and the control group was added with an equal volume of PBS and incubated for 48h. MSCs in each group were collected, and total RNA was extracted by TRIzol method to detect the mRNA expression levels of TGF-β1, IL-10 and TSG6 secreted by MSCs.

[0028] PCR reaction system: TB Premix Ex TaqTM II 5μl, upstream primer 0.4μl, downstream primer 0.4μl, cDNA template 1μl, ddH2O 3.2μl; reaction conditions: 95℃10s, 60℃30s, 40 cycles.

[0029] Table 1: PCR primer sequences

[0030]

[0031] After the reaction is completed, the computer automatically analyzes the fluorescence signal, converts it into a Ct value, and calculates the relative expression level. The results are as follows Figure 1 shown.

[0032] 1.3 Expression levels of TGF-β1, IL-10 and TSG6 in culture supernatant: MSCs of the third generation in logarithmic growth phase qualified by flow cytometry were selected and prepared into cell suspension using DMEM culture medium. 5 The cells were inoculated at a density of 1:1 / ml in a 6-well plate, and then different concentrations of CBD were added. An equal volume of PBS was added to the control group. After incubation for 48 h, the supernatant was collected by centrifugation and tested using an ELISA kit according to the instructions. The test results are shown in Table 4.

[0033] 2 Results: SPSS software was used to perform statistical analysis on the experimental data. Indicates that t test was used.

[0034] 2.1 Effect of CBD on MSC proliferation: As can be seen from Table 2 below, compared with the control group, CBD had no significant effect on MSC activity when it was in the concentration range of 0.1-10 nmol / L, and there was no statistical difference compared with the control group. However, when it was ≥100 nmol / L, it showed the effect of promoting MSC proliferation, and this effect increased with the increase of concentration, and the difference was statistically significant.

[0035] Table 2 Effects of CBD on MSC activity ( , n=6)

[0036]

[0037] Note: * P<0.05, ** P<0.01, compared with the control group.

[0038] 2.2 Effect of CBD on MSC cell cycle: The results are shown in Table 3 below. After incubation with different concentrations of CBD for 48 hours, compared with the control group, the G1 phase cells in the 100 and 1000 nmol / L concentration groups were significantly reduced, while the S phase cells were significantly increased. However, there was no statistical difference in the S phase cells and G1 phase cells in the 1 and 10 nmol / L concentration groups compared with the control group.

[0039] Table 3 Effects of CBD on MSC cell cycle ( , %)

[0040]

[0041] Note: * P<0.05, ** P<0.01, *** P<0.001, compared with the control group.

[0042] 2.3 Effects on cytokine mRNA expression levels: Figure 1 The results showed that the mRNA expression levels of TGF-β1, IL-10 and TSG6 in the CBD 10nmol / L and 100nmol / L groups were significantly higher than those in the control group, especially in the 10nmol / L group.

[0043] 2.4 Expression levels of TGF-β1, IL-10, and TSG6 in culture supernatant

[0044] Table 4 ELISA test results

[0045]

[0046] Note: * P<0.05, ** P<0.01, *** P<0.001, compared with the control group.

[0047] The above experimental results show that CBD has a proliferation-promoting effect on MSC cells, and CBD within a specific concentration range can significantly increase the levels of TGF-β1, IL-10 and TSG6 factors secreted by MSC. However, the mechanism by which CBD acts on MSC to produce the above effects still needs further experimental proof.

[0048] Finally, it should be noted that the above embodiments are used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of cannabidiol in the preparation of bone marrow mesenchymal stem cells that highly express anti-inflammatory factors; the anti-inflammatory factors are TGF-β1, IL-10 and TSG6; the concentration of cannabidiol is 10nmol / L.

2. A method for preparing bone marrow mesenchymal stem cells that highly express anti-inflammatory factors, characterized in that: The method comprises the steps of culturing bone marrow mesenchymal stem cells in a culture medium containing cannabidiol; the anti-inflammatory factors are TGF-β1, IL-10 and TSG6; and the concentration of cannabidiol is 10 nmol / L.

3. The method according to claim 2, characterized in that The culture medium is DMEM, F12, DMEM / F-12 or RPMI1640 culture medium.

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