Anti-inflammatory mesenchymal stem cell as well as preparation method and application thereof

Abstract

The invention relates to the technical field of mesenchymal stem cell culture. The invention provides anti-inflammatory mesenchymal stem cells as well as a preparation method and application thereof. The anti-inflammatory mesenchymal stem cells are obtained by inducing mesenchymal stem cells through IFN-gamma (interferon-gamma), TNF-alpha (tumor necrosis factor-alpha) and IOX4. According to the present invention, by adjusting the induction factor type and the induction process of the mesenchymal stem cells, the efficiency of the mesenchymal stem cells is effectively improved, and the anti-inflammatory ability is improved. The induction effect is guaranteed, and meanwhile the defects caused by induction through a traditional IFN-gamma single factor are effectively overcome. Compared with common mesenchymal stem cells, the anti-inflammatory mesenchymal stem cells obtained through induction have the advantages that the inflammation index is reduced more quickly after reinfusion, and more anti-inflammatory factors are secreted. The traditional Chinese medicine composition has remarkable treatment effects on diseases such as colitis, diabetes mellitus, psoriasis, chronic obstructive pulmonary disease, asthma and atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cell culture, and in particular to an anti-inflammatory mesenchymal stem cell and a preparation method and application thereof. Background Art

[0002] Mesenchymal Stem Cells (MSCs) are an ideal tool for repairing and treating diseases related to tissue damage. MSCs are a type of precursor cell with the potential for self-renewal and multidirectional differentiation, which are widely distributed in adult bone marrow, fat, dental pulp, placenta, umbilical cord and other tissues. MSCs not only have strong self-renewal ability and multidirectional differentiation potential to mesoderm, ectoderm and endoderm, but also can effectively prevent apoptosis of cells in situ in damaged tissues, promote angiogenesis, and activate stem cells in situ in tissues, and participate in the regeneration and repair of damaged tissues.

[0003] The immunosuppressive and anti-inflammatory functions of MSCs mainly depend on the immunomodulatory factors they secrete. In order to exert the anti-inflammatory function of MSCs, it is very important to direct the induction culture of MSCs. The traditional induction method uses IFN-γ as the induced cytokine. However, MSCs induced by IFN-γ alone have great problems in immunosuppression. In clinical practice, when MSCs pretreated with IFN-γ are used to treat Crohn's disease, the MSCs injected into the body do not play the expected anti-inflammatory role, and when IFN-γ is combined with other cytokines to induce MSCs, it sometimes fails to inhibit T cell proliferation but promotes its activation.

[0004] Therefore, how to improve the efficacy of MSCs is an urgent problem to be solved in the field of stem cells. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-inflammatory mesenchymal stem cell and a preparation method and application thereof, so as to effectively improve the efficacy of mesenchymal stem cells and enhance their anti-inflammatory ability.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an anti-inflammatory mesenchymal stem cell, wherein the anti-inflammatory mesenchymal stem cell is obtained by inducing mesenchymal stem cells with IFN-γ, TNF-α and IOX4.

[0008] Preferably, the mesenchymal stem cells are derived from human fat, bone marrow, dental pulp, umbilical cord, placenta or umbilical cord blood.

[0009] The present invention also provides a method for preparing the anti-inflammatory mesenchymal stem cells, comprising the following steps:

[0010] (1) inoculating mesenchymal stem cells into a culture medium for pre-culturing to obtain pre-cultured mesenchymal stem cells;

[0011] (2) IFN-γ, TNF-α and IOX4 are introduced into the culture medium and the culture is continued. After the culture is completed, the anti-inflammatory mesenchymal stem cells are collected.

[0012] Preferably, the culture medium in step (1) is based on DMEM / F12 culture medium, and further contains 0.5-0.9 mg / L reduced glutathione, 50-60 μg / L recombinant human epidermal growth factor, and 50-60 μg / L gentamicin.

[0013] Preferably, the pre-culture time in step (1) is 12 to 18 hours.

[0014] Preferably, in step (2), the concentration of IFN-γ is 5-7 ng / mL, the concentration of TNF-α is 7-10 ng / mL, and the concentration of IOX4 is 30-35 μmol / L.

[0015] Preferably, the time for continued cultivation in step (2) is 20 to 22 hours.

[0016] The present invention also provides the use of the anti-inflammatory mesenchymal stem cells in the preparation of anti-inflammatory products.

[0017] The present invention provides an anti-inflammatory mesenchymal stem cell and a preparation method and application thereof, wherein the anti-inflammatory mesenchymal stem cell is obtained by inducing mesenchymal stem cells through IFN-γ, TNF-α and IOX4. The present invention effectively improves the efficacy of mesenchymal stem cells and improves their anti-inflammatory ability by adjusting the types of induction factors and induction processes of mesenchymal stem cells. While ensuring the induction effect, the disadvantages caused by the traditional induction using a single factor of IFN-γ are effectively avoided. Compared with ordinary mesenchymal stem cells, the induced anti-inflammatory mesenchymal stem cells have a faster decrease in inflammatory indicators and more anti-inflammatory factors after reinfusion. It has significant therapeutic effects on diseases such as colitis, diabetes, psoriasis, chronic obstructive pulmonary disease, asthma, and atherosclerosis. DETAILED DESCRIPTION

[0018] The present invention provides an anti-inflammatory mesenchymal stem cell, wherein the anti-inflammatory mesenchymal stem cell is obtained by inducing mesenchymal stem cells with IFN-γ, TNF-α and IOX4.

[0019] In the present invention, the mesenchymal stem cells are preferably derived from human fat, bone marrow, dental pulp, umbilical cord, placenta or umbilical cord blood.

[0020] The present invention also provides a method for preparing the anti-inflammatory mesenchymal stem cells, comprising the following steps:

[0021] (1) inoculating mesenchymal stem cells into a culture medium for pre-culturing to obtain pre-cultured mesenchymal stem cells;

[0022] (2) IFN-γ, TNF-α and IOX4 are introduced into the culture medium and the culture is continued. After the culture is completed, the anti-inflammatory mesenchymal stem cells are collected.

[0023] In the present invention, the culture medium in step (1) is preferably based on DMEM / F12 culture medium, and further contains 0.5-0.9 mg / L reduced glutathione, 50-60 μg / L recombinant human epidermal growth factor, and 50-60 μg / L gentamicin. It is further preferably based on DMEM / F12 culture medium, and further contains 0.7 mg / L reduced glutathione, 55 μg / L recombinant human epidermal growth factor, and 55 μg / L gentamicin.

[0024] In the present invention, the pre-culture time in step (1) is preferably 12 to 18 hours, more preferably 15 hours.

[0025] In the present invention, the concentration of IFN-γ in step (2) is preferably 5-7 ng / mL, more preferably 6 ng / mL, the concentration of TNF-α is preferably 7-10 ng / mL, more preferably 8-9 ng / mL, and the concentration of IOX4 is preferably 30-35 μmol / L, more preferably 32-33 μmol / L.

[0026] In the present invention, the time for continued culturing in step (2) is preferably 20 to 22 hours, more preferably 21 hours.

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] A method for preparing anti-inflammatory mesenchymal stem cells comprises the following steps:

[0030] (1) Reduced glutathione, recombinant human epidermal growth factor and gentamicin were added to DMEM / F12 medium (Thermo Fisher Scientific) at a ratio of 0.9 mg / L reduced glutathione, 50 μg / L recombinant human epidermal growth factor and 60 μg / L gentamicin, and the mixture was mixed to obtain a culture medium;

[0031] (2) inoculating mesenchymal stem cells (derived from bone marrow) into the culture medium prepared in step (1) and pre-culturing for 18 hours to obtain pre-cultured mesenchymal stem cells;

[0032] (3) IFN-γ, TNF-α, and IOX4 were added to the culture medium at a ratio of IFN-γ 5 ng / mL, TNF-α 10 ng / mL, and IOX4 30 μmol / L, and the culture was continued for 22 h. After the culture was completed, the anti-inflammatory mesenchymal stem cells were collected.

[0033] Example 2

[0034] A method for preparing anti-inflammatory mesenchymal stem cells comprises the following steps:

[0035] (1) reducing glutathione, recombinant human epidermal growth factor and gentamicin were added to DMEM / F12 medium (Thermo Fisher Scientific) at a ratio of 0.5 mg / L reduced glutathione, 60 μg / L recombinant human epidermal growth factor and 50 μg / L gentamicin, and the mixture was mixed to obtain a culture medium;

[0036] (2) inoculating mesenchymal stem cells (derived from the umbilical cord) into the culture medium prepared in step (1) and pre-culturing for 12 hours to obtain pre-cultured mesenchymal stem cells;

[0037] (3) IFN-γ, TNF-α, and IOX4 were added to the culture medium at a ratio of IFN-γ 7 ng / mL, TNF-α 7 ng / mL, and IOX4 35 μmol / L, and the culture was continued for 20 h. After the culture was completed, the anti-inflammatory mesenchymal stem cells were collected.

[0038] Example 3

[0039] A method for preparing anti-inflammatory mesenchymal stem cells comprises the following steps:

[0040] (1) reducing glutathione, recombinant human epidermal growth factor and gentamicin were added to DMEM / F12 medium (Thermo Fisher Scientific) at a ratio of 0.7 mg / L reduced glutathione, 55 μg / L recombinant human epidermal growth factor and 55 μg / L gentamicin, and the mixture was mixed to obtain a culture medium;

[0041] (2) inoculating mesenchymal stem cells (derived from the umbilical cord) into the culture medium prepared in step (1) and pre-culturing for 15 hours to obtain pre-cultured mesenchymal stem cells;

[0042] (3) IFN-γ, TNF-α, and IOX4 were added to the culture medium at a ratio of IFN-γ 6 ng / mL, TNF-α 8 ng / mL, and IOX4 33 μmol / L, and the culture was continued for 21 h. After the culture was completed, the anti-inflammatory mesenchymal stem cells were collected.

[0043] Test Example 1 Colitis Treatment Test

[0044] Fifty SD male rats weighing 200±10 g were purchased from Shanghai Slake Experimental Animal Co., Ltd. Before the experiment, the rats were randomly divided into 6 groups, 10 in each group, numbered as experimental groups 1 to 3, control group and blank control group.

[0045] The rats in the experimental groups 1 to 3 and the control group were modeled with dextran sulfate sodium DSS (average molecular weight 4W) to construct a rat colitis model. During modeling, the rats were gavaged with 4% DSS solution. After 4 days, the rats in the experimental groups 1 to 3 and the control group all showed symptoms such as weight loss, anorexia, increased bowel movement, soft or loose stools, etc. After the model was successfully established, gavage with DSS solution was stopped.

[0046] On the second day after successful modeling, the anti-inflammatory mesenchymal stem cells prepared in Example 1 were injected into the tail vein of the rats in the experimental group 1. Each rat was injected with 1×10 6 According to the same method and standard, the rats in test group 2 were treated with the anti-inflammatory mesenchymal stem cells prepared in Example 2, and the rats in test group 3 were treated with the anti-inflammatory mesenchymal stem cells prepared in Example 3. An equal volume of normal saline was injected into the tail vein of the rats in the control group and the blank control.

[0047] Test results:

[0048] On the third day after the injection of anti-inflammatory mesenchymal stem cells, the colitis symptoms of rats in test groups 1 to 3 were reduced, and there was no significant difference with the rats in the blank control group after 7 days. The colitis symptoms of rats in the control group did not change significantly on the third day after the injection of normal saline. After 7 days, the symptoms of 4 rats were alleviated, and the symptoms of the remaining rats did not show significant improvement. The analysis showed that the stimulation factor disappeared after the intragastric administration of DSS solution was stopped, and the rats' own immunity took effect, but they could not completely heal themselves in a short period of time.

[0049] Test Example 2 Diabetes Treatment Test

[0050] 70 SD male rats weighing 200±10 g were selected and purchased from Shanghai Slake Experimental Animal Co., Ltd. They were divided into a modeling group and a blank control group, with 60 rats in the modeling group and 10 rats in the blank control group. The rats in the modeling group were fed a high-fat, high-sugar diet for 35 consecutive days, and then supplemented with 40 mg / kg injection of streptozotocin (STZ). The blood glucose concentration of the rat tail was measured with a blood glucose meter, and rats with blood glucose concentrations higher than 16.7 mmol / L for two consecutive times were selected as diabetic models.

[0051] After the modeling was completed, a total of 51 diabetic model rats were obtained, and 40 rats with similar blood glucose concentrations were selected and randomly divided into 4 groups, with 10 rats in each group, numbered as experimental groups 4 to 6 and the control group.

[0052] On the second day after successful modeling, the anti-inflammatory mesenchymal stem cells prepared in Example 1 were injected into the tail vein of rats in the experimental group 4. Each rat was injected with 1×10 6 According to the same method and standard, the rats in test group 5 were treated with the anti-inflammatory mesenchymal stem cells prepared in Example 2, and the rats in test group 6 were treated with the anti-inflammatory mesenchymal stem cells prepared in Example 3. The same volume of normal saline was injected into the tail vein of the control group and the blank control rats that had not undergone modeling. The injection was performed once a week for 4 consecutive times.

[0053] Test results:

[0054] After the injection, the blood glucose concentration of the blank control rats was 6.1mmol / L, the blood glucose concentration of the rats in test group 4 was 7.3mmol / L, the blood glucose concentration of the rats in test group 5 was 6.8mmol / L, the blood glucose concentration of the rats in test group 4 was 7.7mmol / L, and the blood glucose concentration of the rats in the control group was 17.1mmol / L.

[0055] Test Example 3 Asthma Treatment Test

[0056] 70 SD male rats weighing 200±10 g were purchased from Shanghai Slake Experimental Animal Co., Ltd. and divided into a modeling group and a blank control group, including 60 rats in the modeling group and 10 rats in the blank control group.

[0057] The rats in the modeling group were sensitized twice on day 0 and day 14. A mixture of 1% ovalbumin saline solution and 160 mg aluminum hydroxide powder (prepared at 4°C overnight and mixed well) was injected subcutaneously at three points on the chest. At the same time, 1 ml of inactivated Bordetella pertussis vaccine (6 × 10 9 / ml). Starting from the 21st day, the rats were placed in a sealed container and challenged with 5% OVA aerosol inhalation, 3 min / time, for 14 consecutive days. After the modeling was completed, rats with rapid breathing, forelimb retraction, nodding or abdominal breathing, irregular rhythm, slow movement, and asthma-like performance were selected as asthma models.

[0058] After the modeling was completed, a total of 47 asthma model rats were obtained, and 40 rats with similar symptoms were selected and randomly divided into 4 groups, with 10 rats in each group, numbered as experimental groups 7 to 9 and the control group.

[0059] On the second day after successful modeling, the anti-inflammatory mesenchymal stem cells prepared in Example 1 were injected into the tail vein of rats in test group 7. Each rat was injected with 1×10 6According to the same method and standard, the rats in test group 8 were treated with the anti-inflammatory mesenchymal stem cells prepared in Example 2, and the rats in test group 9 were treated with the anti-inflammatory mesenchymal stem cells prepared in Example 3. The same volume of normal saline was injected into the tail vein of the control group and the blank control rats that had not undergone modeling. The injection was performed once a week for 4 consecutive times.

[0060] Test results:

[0061] The condition of rats in each group was observed on the third day after the last injection. The results showed that the asthma-like symptoms of rats in test groups 7 to 9 were significantly relieved or disappeared, and none of them showed aggravated symptoms, which was not significantly different from the rats in the blank control group. The symptoms of rats in the control group remained basically unchanged, and one rat showed aggravated symptoms.

[0062] As can be seen from the above embodiments, the present invention provides an anti-inflammatory mesenchymal stem cell and a preparation method and application thereof, wherein the anti-inflammatory mesenchymal stem cell is obtained by inducing mesenchymal stem cells through IFN-γ, TNF-α and IOX4. The present invention effectively improves the efficacy of mesenchymal stem cells and improves their anti-inflammatory ability by adjusting the types of induction factors and induction processes of mesenchymal stem cells. While ensuring the induction effect, the disadvantages caused by the traditional induction using a single factor of IFN-γ are effectively avoided. Compared with ordinary mesenchymal stem cells, the induced anti-inflammatory mesenchymal stem cells have a faster decrease in inflammatory indicators and more anti-inflammatory factors after reinfusion. It has significant therapeutic effects on diseases such as colitis, diabetes, psoriasis, chronic obstructive pulmonary disease, asthma, and atherosclerosis.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-inflammatory mesenchymal stem cell, characterized in that: The anti-inflammatory mesenchymal stem cells are obtained by inducing mesenchymal stem cells with IFN-γ, TNF-α and IOX4.

2. The anti-inflammatory mesenchymal stem cell according to claim 1, characterized in that The mesenchymal stem cells are derived from human fat, bone marrow, dental pulp, umbilical cord, placenta or umbilical cord blood.

3. The method for preparing the anti-inflammatory mesenchymal stem cells according to claim 1 or 2, characterized in that: The steps include: (1) inoculating mesenchymal stem cells into a culture medium for pre-culturing to obtain pre-cultured mesenchymal stem cells; (2) IFN-γ, TNF-α and IOX4 are introduced into the culture medium and the culture is continued. After the culture is completed, the anti-inflammatory mesenchymal stem cells are collected.

4. The preparation method according to claim 3, characterized in that: The culture medium in step (1) is based on DMEM / F12 culture medium, and also contains 0.5-0.9 mg / L reduced glutathione, 50-60 μg / L recombinant human epidermal growth factor, and 50-60 μg / L gentamicin.

5. The preparation method according to claim 4, characterized in that: The pre-culture time in step (1) is 12 to 18 hours.

6. The preparation method according to claim 5, characterized in that: In step (2), the concentration of IFN-γ is 5-7 ng / mL, the concentration of TNF-α is 7-10 ng / mL, and the concentration of IOX4 is 30-35 μmol / L.

7. The preparation method according to any one of claims 3 to 6, characterized in that: The time for continued cultivation in step (2) is 20 to 22 hours.

8. Use of the anti-inflammatory mesenchymal stem cells according to claim 1 or 2 or the anti-inflammatory mesenchymal stem cells prepared according to any one of claims 3 to 7 in the preparation of anti-inflammatory products.