Interleukin-10 (IL-10) high-expression mesenchymal stem cell secretion component, preparation method thereof and application of IL-10 high-expression mesenchymal stem cell secretion component in prevention and treatment of cerebral arterial thrombosis

Abstract

The invention relates to an IL-10 high-expression mesenchymal stem cell secretion component, a preparation method thereof and application thereof in prevention and treatment of cerebral arterial thrombosis, and belongs to the technical field of cerebral apoplexy treatment. In order to solve the problems of current clinical medication and administration methods of stem cells in cerebral arterial thrombosis, the invention provides a specially treated mesenchymal stem cell secretion component (hMSC-M), and the secretion component is obtained by inducing amplified human mesenchymal cells by using a basal culture medium containing human IFN-gamma protein and TNF-alpha mimic peptide. And harvesting, culturing and repeatedly freezing and thawing to obtain the IL-10 high-expression stem cell secretion component. Animal experiments find that the hMSC-M has a remarkable effect on prevention and treatment of cerebral arterial thrombosis, so that a new medicine application is developed for stem cell application, a foundation is laid for developing related medicines for efficiently preventing and treating cerebral arterial thrombosis injury, and a new application scheme and thought are provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of cerebral stroke treatment, and specifically relates to a mesenchymal stem cell secretory component with high expression of IL-10, a preparation method thereof, and application thereof in preventing and treating ischemic cerebral stroke. Background Art

[0002] Stroke is the second leading cause of death and disability in the world. Over time, the morbidity and mortality of this type of disease continue to rise, placing a huge burden on patients' families and society. According to the latest epidemiological studies, ischemic stroke accounts for as much as 69.6% of all stroke cases in my country. Ischemic stroke has diverse pathological types and complex pathogenesis, involving the release of inflammatory mediators, infiltration of inflammatory cells, damage to the blood-brain barrier, and the generation of inflammatory factors. Although the treatment time window for intravascular mechanical thrombectomy or thrombolysis has been expanded in the treatment of ischemic stroke, its narrow treatment time and surgical-induced hemorrhagic complications remain important challenges for clinicians. Therefore, the development of effective prevention and treatment measures is crucial to reducing the incidence of cerebral hemorrhage and improving patient prognosis.

[0003] Stem cell transplantation therapy is considered a new treatment method to promote brain repair after stroke. Among them, human umbilical cord mesenchymal stem cells have received widespread attention because they are easy to obtain and have fewer ethical issues. However, the homing ability and vitality of transplanted stem cells after homing are poor, which reduces their therapeutic effect. Stem cell derivatives - exosomes and vesicles can promote neurogenesis and reduce inflammatory responses. Although they can circumvent the above problems to a certain extent, their clinical application as cell-free therapies still requires more exploration. Among the exosome administration methods, intravenous injection is the most widely used route of administration, but intravenous injection may have the risk of retention in the pulmonary capillary network causing infusion problems. How to effectively avoid the above dilemma is the key issue that needs to be solved in the current use of stem cells for the treatment of ischemic stroke. Summary of the invention

[0004] In view of the shortcomings of current stem cells in clinical medication and administration methods for ischemic stroke, the present invention provides a specially treated mesenchymal stem cell secretory component (hMSC-M), which is a stem cell secretory component with high IL-10 expression obtained by inducing amplified human mesenchymal cells using a basic culture medium containing human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide, and then harvesting, culturing and repeatedly freezing and thawing. The present invention uses hMSC-M to pretreat mice, and finds that pre-administration of hMSC-M can increase the proportion and number of B cells in the lymph nodes of peripheral immune organs, and proves through a cerebral ischemia model that pre-use of stem cell secretory components can significantly reduce the volume of cerebral ischemia and improve the spleen weight loss caused by cerebral ischemia; in addition, it is also found that hMSC-M has a more significant therapeutic effect on cerebral ischemia than mesenchymal stem cell secretion supernatant (hMSC-L), can improve the pathological damage and blood-brain barrier leakage of cerebral ischemia, inhibit the expression of inflammatory factors, and at the same time promote the expression of downstream SOCS1 / STAT6 / p-STAT6 pathway by activating the nuclear translocation of PPAR-γ / p-PPAR-γ, and induce neutrophil N2 type reprogramming, so it has important clinical application value.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide a method for preparing a secretory component of mesenchymal stem cells with high expression of IL-10, the preparation method comprising the following steps:

[0007] S1. Expansion culture: obtain sufficient human mesenchymal stem cells through expansion culture as the first mesenchymal stem cells;

[0008] S2, induction culture: The first mesenchymal stem cells obtained in S1 were placed in an induction medium at 2-5% O 2 , 5% CO 2 , culturing at 37° C. for 12-24 hours to obtain a second mesenchymal stem cell; the induction medium is a basal medium supplemented with cytokines, the cytokines are human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide, the amino acid sequence of the human IFN-γ mimetic peptide is as shown in SEQ ID NO.1, and the amino acid sequence corresponding to the TNF-ɑ mimetic peptide is NKHNRKI;

[0009] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with saline, harvest the cells after trypsin digestion, and add 1×10 6 The inoculum was inoculated into an ultra-low adsorption 96U bottom plate at a volume of 100 μg / mL, centrifuged, and α-MEM medium containing 5-10% human AB serum was added. 2 , 5% CO2 , cultured at 37°C for 12h;

[0010] S4. Repeated freezing and thawing: Place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high expression of IL-10.

[0011] In one embodiment of the present invention, the expansion culture in S1 is to place the human mesenchymal stem cells in a 37°C water bath for recovery, and then inoculate them in a basic culture medium at 5% O 2 , 5% CO 2 , cultured at 37°C until the cell confluence reached 70% to 90%.

[0012] In one embodiment of the present invention, the amount of human IFN-γ mimetic peptide added in S2 is 600 U / mL, and the amount of TNF-ɑ mimetic peptide added is 10 ng / mL.

[0013] In one embodiment of the present invention, the basic culture medium in S2 is α-MEM culture medium containing double antibody and 10-20% FBS or double antibody and 10-20% human AB serum.

[0014] The second object of the present invention is to provide a secretory component of mesenchymal stem cells with high expression of IL-10 obtained by the above preparation method.

[0015] The third object of the present invention is to provide the use of the above-mentioned mesenchymal stem cell secretory components in increasing the proportion and number of B cells in the lymph nodes of peripheral immune organs.

[0016] The fourth object of the present invention is to provide the use of the above-mentioned mesenchymal stem cell secretory components in the preparation of a drug for preventing ischemic stroke.

[0017] The fifth object of the present invention is to provide the use of the above-mentioned mesenchymal stem cell secretory components in the preparation of a drug for treating ischemic stroke.

[0018] In one embodiment of the present invention, the application includes at least any one of the following:

[0019] (1) Reduce the volume of cerebral ischemia;

[0020] (2) Promote motor function recovery;

[0021] (3) Improve pathological damage of ischemic brain tissue;

[0022] (4) Inhibit the expression of inflammatory factors in the brain;

[0023] (5) Promote the expression of immunoregulatory N2 neutrophils;

[0024] (6) Improve blood-brain barrier leakage;

[0025] (7) Promote angiogenesis;

[0026] (8) Activation of PPAR-γ nuclear translocation;

[0027] (9) Promote the reprogramming of N2 neutrophils.

[0028] Beneficial effects of the present invention:

[0029] The present invention proposes to induce stem cell secretory components with high anti-inflammatory ability by combining human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide with hypoxia. Specifically, amplified human mesenchymal cells are induced using a basic culture medium containing human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide, and then the IL-10 highly expressed stem cell secretory components are obtained after harvesting, culture and repeated freezing and thawing.

[0030] The present invention uses hMSC-M to pretreat mice, and finds that pre-administration of hMSC-M can increase the proportion and number of B cells in the lymph nodes of peripheral immune organs, while the number of innate immune cells and T lymphocytes has no significant change. It is further found that the main effector cells of hMSC-M in preventing ischemic stroke are B cells in the lymph nodes, and the cerebral ischemia model proves that pre-use of hMSC-M can significantly reduce the volume of cerebral ischemia and improve the spleen weight loss caused by cerebral ischemia. In addition, it was found in the prevention experiment that subcutaneous administration is more advantageous than intravenous administration and the effect is more lasting. Specifically, a cerebral ischemia model was constructed 7 days after stopping the administration of stem cell secretory components, and the subcutaneous administration method can still reduce the volume of cerebral ischemia. The present invention prevents ischemic stroke by using stem cell secretory components, which not only avoids the risk of stem cell tumorigenesis and immune rejection, but also can exert the preventive effect of various factors in stem cells on the disease to a greater extent. The present invention lays a theoretical foundation for the preventive application of stem cell secretory components, proposes the main effector cells of stem cell secretory components in the preventive health care function, and proposes a more effective medication route. At the same time, it provides new solutions and ideas for the preventive application of stem cells for various diseases including ischemic stroke.

[0031] The present invention finds and confirms that stem cell secretory components have a therapeutic effect on ischemic stroke. Specifically, studies using the middle cerebral artery infarction (MCAO) model have shown that nasal mucosal administration of stem cell secretory components can reduce the volume of cerebral ischemia in mice, improve ischemia-induced brain tissue pathological damage, promote motor function recovery in stroke mice, improve blood-brain barrier leakage, and promote immunoregulatory N2 neutrophil expression. Specifically, stem cell secretory components can activate PPAR-γ nuclear translocation and promote N2 type neutrophil reprogramming, and the effect of stem cell secretory components on neutrophils is better than that of stem cell supernatant. In addition, the method of nasal mucosal administration can not only achieve the same effect as other modes of administration, but also has high compliance.

[0032] The present invention opens up new drug uses for stem cell applications, and at the same time, lays a foundation for the development of highly effective drugs for preventing and treating ischemic stroke damage, and provides new application solutions and ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 ELISA test results of the anti-inflammatory factor IL-10 concentration in hMSCs-M obtained in Examples 1-3 and hMSCs-M obtained in various comparative examples; wherein, hypoxia-, means 20% O 2 normoxic culture environment, hypoxia+, means 5% O 2 The hypoxic culture environment, ①②③ respectively represent the addition of human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide according to the addition amounts in Example 1, Example 2 and Example 3;

[0034] Figure 2 The figure shows the effect of pre-administering hMSCs-M obtained in Example 2 by iv or ih on the proportion and number of B cells and T cells in the lymph nodes and bone marrow of mice; wherein LN represents lymph nodes, BM represents bone marrow; ns represents no statistical significance, **P<0.01, ***P<0.001, ****P<0.0001;

[0035] Figure 3 The figure shows the effect of pre-administering hMSCs-M obtained in Example 2 on the proportion and number of innate immune cells in the lymph nodes (LN) and bone marrow (BM) of mice by iv or ih; wherein, Figure 3 A in the figure represents NK1.1 in lymph nodes and bone marrow + The results of the effects of the proportion and number of NK cells are shown in the figure. Figure 3 B in the figure is for Ly6C in bone marrow and peripheral blood (PB) + The effect of the proportion of monocytes on the results of Figure 3 C in the figure is for Ly6G in bone marrow and peripheral blood +The effect of the proportion of neutrophils on the results of Figure 3 D in the figure is for F4 / 80 in bone marrow and peripheral blood + The results of the effect of the proportion of macrophages; ns means no statistical significance, **P<0.01, ***P<0.001;

[0036] Figure 4 The results of the effect of pre-administering hMSCs-M obtained in Example 2 on the cerebral ischemic volume in the mouse MCAO model by iv or ih; wherein, pre7+0, pre7+3, and pre7+7 respectively represent modeling 7 days after pre-administration, modeling on the 3rd day after 7 days of pre-administration, and modeling on the 7th day after 7 days of pre-administration; *P<0.05;

[0037] Figure 5 The figure shows the effect of pre-administering hMSCs-M obtained in Example 2 on the spleen weight in the mouse MCAO model by iv or ih; wherein, pre7+0, pre7+3, and pre7+7 respectively represent modeling 7 days after pre-administration, modeling on the 3rd day after 7 days of pre-administration, and modeling on the 7th day after 7 days of pre-administration; **P<0.01, ##P<0.01;

[0038] Figure 6 The results of the effects of hMSC-L and hMSC-M treatment on brain ischemia volume and neurological dysfunction in mice are shown in Figure 2. Figure 6 A in the figure is the result of TTC staining to detect the brain ischemic volume after ischemic stroke in mice treated with hMSC-L or hMSC-M in different ways. Figure 6 Figure B is the statistical result of cerebral ischemic volume after ischemic stroke in mice in the hMSCs-L group. Figure 6 C in the figure is the result of neurological damage score of mice in the hMSCs-L group. Figure 6 D in the figure is the statistical result of cerebral ischemic volume after ischemic stroke in mice in the hMSCs-M group. Figure 6 E in the figure is the neurological dysfunction score result of mice in the hMSCs-M group; ns means no statistical significance, *P<0.05, **P<0.01, ***P<0.001;

[0039] Figure 7 This is a result diagram of the effect of hMSC-L and hMSC-M treatment on the recovery of motor function in stroke mice using corner turning test and body lifting test; Figure 7 A in the figure is the result of the corner turning experiment of mice in the hMSCs-L group. Figure 7 B is the result of the body lifting experiment of mice in the hMSCs-L group. Figure 7C in the figure is the result of the corner turning experiment of mice in the hMSCs-M group. Figure 7 D in the figure is the result of the body lifting experiment of mice in the hMSCs-M group; ns indicates no statistical significance, *P<0.05, **P<0.01, ***P<0.001;

[0040] Figure 8 This is the result of using HE staining and Nissl staining to detect the effect of hMSCs-M treatment on the pathological damage of ischemic mouse brain tissue; Figure 8 A is a representative image of HE staining of the cortex (magnification 200 times, scale bar 50 μm). Figure 8 B is a representative image of Nissl staining in the cortical area (magnification 200 times, scale bar 50 μm). Figure 8 Figure C is the statistical result of HE staining. Figure 8 D in the figure is the statistical result of Nissl staining; ***P<0.001, ##P<0.01, ###P<0.001;

[0041] Fig. 9 The figure shows the detection results of the gene levels of major pro-inflammatory cytokines in the brain of mice after ischemic stroke; Fig. 9 A in the figure is the result of detecting the expression level of IFN-γ mRNA in the brain after cerebral ischemia by qPCR. Fig. 9 B is the result of qPCR detection of TNF-α mRNA expression level in the brain after cerebral ischemia. Fig. 9 C in the figure is the result of qPCR detection of IL-1β mRNA expression level in the brain after cerebral ischemia; *P<0.05, **P<0.01, ***P<0.001;

[0042] Fig.10 hMSC-L and hMSC-M treatments for immunoregulatory N2 neutrophils (CD45 high CD11b + 6G + CD206 + ) expression; ##P<0.01, ***P<0.001;

[0043] Fig.11 The results of hMSC-L and hMSC-M treatment on blood-brain barrier leakage after stroke in mice are shown in Figure 2. Fig.11 A in the figure is the result of immunofluorescence staining to detect occludin expression (magnification 400 times, scale bar 50 μm), Fig.11 B in the figure is the result of EB method to detect the integrity of blood-brain barrier. Fig.11 C in the figure is the result of statistical analysis of the integrity of the blood-brain barrier. Fig.11 D in the figure is the result of statistical analysis of immunofluorescence staining; *P<0.05, **P<0.01, ***P<0.001;

[0044] Fig.12 The results of the effects of hMSC-L and hMSC-M treatment on the permeability of the blood-brain barrier are shown in Figure 2. Fig.12 A in the figure is the result of immunofluorescence staining (400×) to detect the expression of blood-brain barrier tight junction-related protein ZO-1, the scale bar is 50 μm, Fig.12 B in Fig.12 Statistical results of A in Figure 1, **P<0.01;

[0045] Fig.13 The results of the effects of hMSC-L and hMSC-M treatment on PPAR-γ nuclear translocation are shown in Figure 1. Fig.13 A in the figure is the result of WB detection of the expression level of PPAR-γ protein in the nucleus. Fig.13 B in the figure is the statistical result of the expression level of PPAR-γ protein in the nucleus. Fig.13 C in the figure is the result of WB detection of cytoplasmic PPAR-γ, total PPAR-γ and p-PPAR-γ protein expression levels. Fig.13 D in the figure is the statistical result of the expression levels of cytoplasmic PPAR-γ, total PPAR-γ and P-PPAR-γ proteins; vs PMA, **P<0.01, ***P<0.001; PMA+L+inhibitior vs PMA+L, ##P<0.01, ###P<0.001; PMA+M+inhibitior vs PMA+M, $P<0.01, $$P<0.001;

[0046] Fig.14 The results of the effects of hMSC-L and hMSC-M treatment on N2 neutrophil reprogramming are shown in Figure 2. Fig.14 A in the figure is the detection of Ly6G in neutrophils by flow cytometry + CD206 + and Ly6G + iNOS + Expression level results graph, Fig.14 B in the figure is for Ly6G in neutrophils + CD206 + and Ly6G + iNOS +The result of statistical analysis of the expression levels; compared with PMA *P<0.05, **P<0.01, ***P<0.001; PMA+L+inhibition vs PMA+L, ###P<0.001, PMA+M+inhibition vs PMA+M, $$P<0.001. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the field unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0048] The amino acid sequence of the human IFN-γ mimetic peptide used in the present invention is LTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQM (SEQ ID NO.1); the amino acid sequence corresponding to the TNF-ɑ mimetic peptide used in the present invention is NKHNRKI.

[0049] The experimental data of the present invention were statistically analyzed using GraphPad Prism 9.0 software, and the comparison between two groups of samples was performed using the unpaired t test; the comparison between three or more groups of samples was performed using one-way analysis of variance. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

[0050] Example 1: A method for preparing a secretory component of mesenchymal stem cells with high expression of IL-10

[0051] The induction medium used in this embodiment is a basal medium supplemented with human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, human IFN-γ mimetic peptide 300U / mL, and TNF-ɑ mimetic peptide 10ng / mL.

[0052] The method for preparing the secretory component of mesenchymal stem cells with high expression of IL-10 using the above-mentioned induction medium is as follows:

[0053] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then inoculated in α-MEM medium containing 10% FBS and double antibodies in 5% O 2, 5% CO 2 , cultured at 37°C until the cell confluence reached 70% to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;

[0054] S2, induction culture: The first mesenchymal stem cells obtained in S1 were placed in an induction medium at 5% O 2 , 5% CO 2 , cultured at 37°C for 24 hours to obtain the second mesenchymal stem cells;

[0055] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6 The inoculum was inoculated into an ultra-low adsorption 96U bottom plate at a volume of 100 μL / mL, centrifuged at 300 g for 3 min, and 100 μL of α-MEM medium containing 10% human AB serum was added to each well. 2 , 5% CO 2 , cultured at 37°C for 12h;

[0056] S4. Repeated freezing and thawing: Place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high expression of IL-10 (hMSC-M).

[0057] The preparation method of the mesenchymal stem cell supernatant (hMSCs-L) is as follows: after step S3 is completed, a portion of cells are taken and the cell supernatant is centrifuged to obtain the mesenchymal stem cell supernatant.

[0058] Example 2: A method for preparing a secretory component of mesenchymal stem cells with high expression of IL-10

[0059] The induction medium used in this embodiment is a basal medium supplemented with human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, human IFN-γ mimetic peptide 1000U / mL, and TNF-ɑ mimetic peptide 5ng / mL.

[0060] The method for preparing the secretory component of mesenchymal stem cells with high expression of IL-10 using the above-mentioned induction medium is as follows:

[0061] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then inoculated in α-MEM medium containing 10% FBS and double antibodies in 5% O 2 , 5% CO 2 , cultured at 37°C until the cell confluence reached 70% to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;

[0062] S2, induction culture: The first mesenchymal stem cells obtained in S1 were placed in an induction medium at 5% O 2 , 5% CO 2 , cultured at 37°C for 24 hours to obtain the second mesenchymal stem cells;

[0063] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6 The inoculum was inoculated into an ultra-low adsorption 96U bottom plate at a volume of 100 μL / mL, centrifuged at 300 g for 3 min, and 100 μL of α-MEM medium containing 10% human AB serum was added to each well. 2 , 5% CO 2 , cultured at 37°C for 12h;

[0064] S4. Repeated freezing and thawing: Place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high expression of IL-10 (hMSC-M).

[0065] The preparation method of the mesenchymal stem cell supernatant (hMSCs-L) is as follows: after step S3 is completed, a portion of cells are taken and the cell supernatant is centrifuged to obtain the mesenchymal stem cell supernatant.

[0066] Example 3: A method for preparing a secretory component of mesenchymal stem cells with high expression of IL-10

[0067] The induction medium used in this embodiment is a basal medium supplemented with human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, human IFN-γ mimetic peptide 600U / mL, and TNF-ɑ mimetic peptide 10ng / mL.

[0068] The method for preparing the secretory component of mesenchymal stem cells with high expression of IL-10 using the above-mentioned induction medium is as follows:

[0069] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then inoculated in α-MEM medium containing 10% FBS and double antibodies in 5% O 2 , 5% CO 2 , cultured at 37°C until the cell confluence reached 70% to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;

[0070] S2, induction culture: The first mesenchymal stem cells obtained in S1 were placed in an induction medium at 5% O 2 , 5% CO 2, cultured at 37°C for 24 hours to obtain the second mesenchymal stem cells;

[0071] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6 The inoculum was inoculated into an ultra-low adsorption 96U bottom plate at a volume of 100 μL / mL, centrifuged at 300 g for 3 min, and 100 μL of α-MEM medium containing 10% human AB serum was added to each well. 2 , 5% CO 2 , cultured at 37°C for 12h;

[0072] S4. Repeated freezing and thawing: Place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high expression of IL-10 (hMSC-M).

[0073] The preparation method of the mesenchymal stem cell supernatant (hMSCs-L) is as follows: after step S3 is completed, a portion of cells are taken and the cell supernatant is centrifuged to obtain the mesenchymal stem cell supernatant.

[0074] Comparative Example 1: Preparation method of secretory components of mesenchymal stem cells

[0075] The induction medium used in this comparative example is a basal medium supplemented with commercially available IFN-γ and TNF-ɑ. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, IFN-γ 300U / mL, TNF-ɑ 10ng / mL.

[0076] The method for preparing the secretory components of mesenchymal stem cells using the above-mentioned induction medium is as follows:

[0077] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then inoculated in α-MEM medium containing 10% FBS and double antibodies in 5% O 2 , 5% CO 2 , cultured at 37°C until the cell confluence reached 70% to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;

[0078] S2, induction culture: The first mesenchymal stem cells obtained in S1 were placed in an induction medium at 5% O 2 , 5% CO 2 , cultured at 37°C for 24 hours to obtain the second mesenchymal stem cells;

[0079] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6The inoculum was inoculated into an ultra-low adsorption 96U bottom plate at a volume of 100 μL / mL, centrifuged at 300 g for 3 min, and 100 μL of α-MEM medium containing 10% human AB serum was added to each well. 2 , 5% CO 2 , cultured at 37°C for 12h;

[0080] S4. Repeatedly freeze and thaw the cells: place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells, hMSCs-M.

[0081] The present invention detects the IL-10 content in the hMSCs-M obtained in Examples 1-3 and Comparative Example 1, and uses hMSCs-M cultured under normoxic conditions without adding any cytokines, hMSCs-M cultured under hypoxic conditions without adding any cytokines, and hMSCs-M cultured under normoxic conditions with the addition of IFN-γ and TNF-ɑ (the addition amount is the same as that in Example 1) as controls. The IL-10 content detection results are shown in Figure 1 ,Depend on Figure 1 It can be seen that the IL-10 concentration in hMSCs-M obtained in Examples 1-3 is significantly increased compared with various controls. The above results show that the induction medium and the method for preparing the secretory component of mesenchymal stem cells provided by the present invention can significantly increase the IL-10 secretion of mesenchymal stem cells.

[0082] Example 4: Application of secretory components of mesenchymal stem cells with high expression of IL-10 in increasing the proportion and number of B cells in lymph nodes of peripheral immune organs

[0083] C57B / 6 male mice weighing 20-22g were divided into sham group, intravenous (iv) administration group, and subcutaneous (ih) administration group, with 5 mice in each group. The sham group was not administered, and the other groups were pretreated with the IL-10 highly expressed mesenchymal stem cell secretory component (hMSCs-M) obtained in Example 2 for 7 consecutive days, with a concentration of 2mg / mL and a dosage of 100μg per mouse. After the administration, the lymph nodes and bone marrow tissues of the mice were collected for flow cytometry detection; the proportion of different immune cell subsets and changes in cell counts were evaluated.

[0084] The results are as follows Figure 2 After hMSCs-M obtained in Example 2 were administered to mice by iv or ih for 7 consecutive days, the proportion and number of B cells in the lymph nodes of mice in the ih administration group increased. + T and CD8 + The proportion of T cells decreased but the number did not change significantly. B cells and CD4 + T and CD8 +There was no significant change in the proportion and number of T cells; B cells and CD4 + T and CD8 + There was no significant change in the proportion and number of T cells. Only the proportion of B cells in the bone marrow decreased, and there was no significant difference in the number.

[0085] hMSCs-M obtained in Example 2 were administered to mice by iv or ih for 7 consecutive days in advance, and then the innate immune cells (NK1.1 + NK cells, Ly6C + Monocytes, Ly6G + Neutrophils, F4 / 80 + The results showed that the proportion of NK cells decreased, but the number did not change significantly. The proportion of innate immune cells did not change significantly regardless of whether the administration method was iv or ih (see Figure 3 ).

[0086] It can be seen that subcutaneous administration of secretory components of mesenchymal stem cells with high IL-10 expression in mice can increase the proportion and number of B cells in the lymph nodes of peripheral immune organs, but there is no significant change in the number of innate immune cells and T lymphocytes.

[0087] Example 5: Application of secretory components of mesenchymal stem cells with high expression of IL-10 in preventing ischemic stroke

[0088] The mice were divided into a sham group (i.e., no drug administration group), an intravenous (iv) drug administration group, and a subcutaneous (ih) drug administration group, with 5 mice in each group. They were pretreated with the IL-10 highly expressed mesenchymal stem cell secretory component (hMSCs-M) obtained in Example 2 for 7 consecutive days at a concentration of 2 mg / mL, with a dosage of 100 μg per mouse.

[0089] Construction of ischemic stroke model (MCAO): On the 1st, 3rd and 7th day after pretreatment, 2% sodium pentobarbital solution was prepared and intraperitoneally injected into C57 / BL6 male mice weighing 20-22g. The anesthetized mice were fixed in supine position, the neck skin was prepared and disinfected, and the skin was cut along the middle of the neck with ophthalmic scissors. The neck muscles were bluntly separated, and the right common carotid artery, external carotid artery and internal carotid artery were carefully separated. The common carotid artery and external carotid artery were ligated with sutures, and a slipknot was tied when the internal carotid artery was ligated. Sutures were inserted at the ligation site of the external carotid artery and the common carotid artery branch, and reserved as ligature line plugs. An incision was made between the two lines of the external carotid artery, and the line plug was slowly inserted into the common carotid artery, and the reserved suture was ligated. The internal carotid artery ligature was removed, the line plug was withdrawn to the bifurcation, the line plug was gently reversed and inserted into the internal carotid artery, and the line plug was slowly pushed in until the initial segment of the cerebral artery was blocked, causing local ischemia of brain tissue. Finally, the neck incision is sutured.

[0090] TTC staining:

[0091] On the third day after MCAO, mice were killed, brain tissue was quickly removed, placed in a pre-cooled brain slice mold, and cut into slices of about 2 mm using a matching blade. The slices were placed in a 2% TTC solution and incubated at 37°C for 30 min. The specific incubation time can be adjusted according to the degree of staining. After the staining is uniform, the brain slices were removed for photographing and statistics.

[0092] Mouse spleen weight detection:

[0093] On the third day after MCAO, the mice were killed and their spleens were quickly removed. The changes in spleen length and weight were detected twice, and then weighed.

[0094] TTC staining results showed that the hMSCs-M obtained in Example 2 was administered to mice by iv or ih, which could reduce the volume of cerebral ischemia in mice. The MCAO model was established 7 days after stopping administration. The ih administration method was more able to reduce the volume of cerebral ischemia than the iv administration method, indicating that the immunomodulatory effect of hMSCs-M administered in advance by ih on the body lasted longer (see Figure 4 ).

[0095] The results of mouse spleen weight test showed that the hMSCs-M obtained in Example 2 was administered to mice by iv or ih to construct MCAO model. Compared with MCAO model mice, pretreatment significantly increased the weight of mouse spleen, indicating that hMSCs-M pretreatment can improve the phenomenon of spleen function inhibition caused by cerebral ischemia (see Figure 5 ).

[0096] Example 6: Application of secretory components of mesenchymal stem cells with high expression of IL-10 in the treatment of ischemic stroke

[0097] Construction of the middle cerebral artery occlusion (MCAO) model:

[0098] Prepare 2% sodium pentobarbital solution and inject it intraperitoneally to anesthetize C57 / BL6 male mice weighing 20-22g. After anesthesia, fix the mouse in supine position, prepare the neck skin and disinfect it, and use ophthalmic scissors to cut the skin along the middle of the neck. Bluntly separate the neck muscles and carefully separate the right common carotid artery, external carotid artery, and internal carotid artery. Use sutures to ligate the common carotid artery and external carotid artery, and tie a slipknot when ligating the internal carotid artery. Insert sutures at the ligation site of the external carotid artery and the branch of the common carotid artery, leaving them as ligatures. Make an incision between the two lines of the external carotid artery, slowly insert the suture into the common carotid artery, and ligate the reserved suture. Remove the internal carotid artery ligature, withdraw the suture to the bifurcation, gently reverse the suture and insert it into the internal carotid artery, and slowly push the suture until the starting segment of the cerebral artery is blocked, causing local ischemia of the brain tissue. Finally, suture the neck incision.

[0099] Drug administration:

[0100] After the mice woke up, they were treated with hMSC-L (mesenchymal stem cell supernatant) and hMSC-M (IL-10 highly expressed mesenchymal stem cell secretory component) obtained in Example 2 by intraperitoneal injection, tail vein injection and nasal mucosal administration, respectively. The concentrations were 1 mg / mL and 2 mg / mL, and the administration volume was 100 μL per mouse for 3 consecutive days.

[0101] The therapeutic effects of hMSC-L and hMSC-M were evaluated using the following experiments:

[0102] TTC staining:

[0103] On the third day after MCAO, mice were killed, brain tissue was quickly removed, placed in a pre-cooled brain slice mold, and cut into slices of about 2 mm using a matching blade. The slices were placed in a 2% TTC solution and incubated at 37°C for 30 min. The specific incubation time can be adjusted according to the degree of staining. After the staining is uniform, the brain slices were removed for photographing and statistics.

[0104] Neurological function score:

[0105] Longa's five-point method is often used to evaluate the degree of neurological deficit in mouse ischemic stroke models. The specific evaluation criteria are as follows:

[0106] 0 points: no nerve damage, indicating that the experimental animals have no obvious abnormalities in nerve function;

[0107] 1 point: The contralateral forepaw cannot be fully extended: one of the forepaws of the experimental animal shows limited movement and cannot be fully extended;

[0108] 2 points: Turning in circles to the opposite side: This means that the experimental animal will lean towards the paralyzed side during the movement;

[0109] 3 points: Leaning to the contralateral side: The experimental animal loses balance and leans toward the paralyzed side while walking;

[0110] 4 points: Unable to walk spontaneously and loss of consciousness.

[0111] Generally speaking, a higher score indicates more severe neurological damage in the experimental animal.

[0112] Brain edema detection:

[0113] On the third day of MCAO, mice were killed, brain tissue was removed, and the weight of the brain was recorded. The brain tissue was placed in a drying oven at 60°C-80°C and dried for 24 hours. The specific drying time was adjusted according to the drying condition of the brain tissue. After drying, the brain tissue was reweighed and the weight was recorded. The percentage of water content of the brain tissue was calculated as: (wet weight-dry weight) / wet weight×100%.

[0114] Mouse behavioral experiments:

[0115] (1) Corner test: The experimental device consists of two flat plates forming a 30° angle. The mouse is placed in the middle of the device and allowed to move forward to the depth of the angle. When the mouse touches the flat plate wall, it will stand up and then turn left or right. Only when the mouse stands up completely on its hind legs and then turns around is considered an experiment. Each mouse is tested 10 times, with a 1-minute interval between each test. The score is calculated as: number of turns on the healthy side / total number of turns × 100%.

[0116] (2) Body lifting test: The tail of the mouse is lifted so that the tail is about 5 cm away from the plane. At this time, the mouse's head will rotate to the left or right, and the counting standard is that the unilateral rotation angle is greater than 100°. Each mouse is repeated 20 times, with an interval of 1 minute each time, and the calculation is: the number of rotations on the affected side / the total number of rotations × 100%.

[0117] Evans Blue staining:

[0118] 200 μL of 2% Evans blue dye solution was injected into the mouse via tail vein injection. The mouse was killed 2 hours after the injection, the mouse chest cavity was opened, and the heart was perfused with PBS until the outflowing liquid became clear, and the perfusion was stopped. The brain tissue was removed, photographed, and statistically analyzed.

[0119] HE staining:

[0120] (1) Take out the frozen sections and place them at room temperature for 5 minutes;

[0121] (2) Fix the sections in cold acetone for 15-30 minutes;

[0122] (3) Wash the sections with PBS for 3 times, 3 minutes each time;

[0123] (4) Shake off the surface liquid and circle the tissue using a tissue pen;

[0124] (5) Immerse the sections in hematoxylin stain for 3 minutes;

[0125] (6) Use pathological PBS to wash away excess stain;

[0126] (7) Drop the differentiation solution on the tissue surface and wash it with pathological PBS after 1 minute;

[0127] (8) Immerse the sections in eosin staining solution for 2 minutes;

[0128] (9) Use pathological PBS to wash away excess staining solution;

[0129] (10) The sections were sequentially immersed in 80%, 90%, and 100% ethanol for dehydration, then immersed in xylene for transparency, sealed with neutral resin, and photographed under a microscope.

[0130] Nissl staining:

[0131] (1) Take out the frozen sections and place them at room temperature for 5 minutes;

[0132] (2) Fix the sections in cold acetone for 15-30 minutes;

[0133] (3) Wash the sections with PBS for 3 times, 3 minutes each time;

[0134] (4) Shake off the surface liquid and circle the tissue using a tissue pen;

[0135] (5) Immerse the sections in Nissl stain at 37°C for 10 minutes;

[0136] (6) Use pathological PBS to wash away excess stain;

[0137] (7) 95% ethanol rapidly differentiates, while anhydrous ethanol rapidly dehydrates;

[0138] (8) After the slides are made transparent with xylene, they are sealed with a neutral resin and photographed under a microscope.

[0139] Extract tissue and cell proteins, and measure protein concentration using BCA:

[0140] Extract tissue protein samples: After blood is collected from the mouse eyeball, PBS is used for cardiac perfusion until the liver tissue turns white. The required brain tissue is taken out and placed in 1.5mL EP containing lysis buffer. After the tissue is ultrasonically treated, it is oscillated on a vortex oscillator for 6 times, with an interval of 5 minutes each time. Next, centrifuge at 12000rpm, 4℃, 15 minutes. Pipette the supernatant into a new EP tube and store it for a long time at -80℃. In a 96-well flat-bottom plate, add the standard sample in sequence to draw a standard curve, and add 19.5μL of NaCl and 0.5μL of sample to the measurement well. Prepare the BCA mixture according to the instructions, add 200μL of the mixture to each well, and incubate in a 37℃ incubator away from light for 30 minutes. Use an enzyme reader to detect the absorbance of each well, obtain the standard curve and calculate the sample concentration. Use loading buffer and lysis buffer to prepare the loading system, mix well and place in a metal bath at 100℃ for 9 minutes to denature the protein. Store at -20℃ for short-term and -80℃ for long-term.

[0141] Western Blot:

[0142] (1) Glue preparation: Clean the glass plate with a thickness of 1.00 mm, assemble the glue preparation device, add deionized water to test for leaks for 10 minutes, and proceed to the subsequent steps if there is no obvious leakage. Prepare and add separation glue, flatten it with deionized water, and after solidification for 30 minutes, pour out the upper deionized water, add the prepared concentrated glue, insert the comb, and let it stand for 15 minutes;

[0143] (2) Electrophoresis: Assemble the electrophoresis system, add electrophoresis buffer to the electrophoresis tank, remove the comb and add markers and samples. Run the concentrated gel at a constant voltage of 90V for 15 minutes; after the samples are evenly packed, run the separation gel at a constant voltage of 120V for 60 minutes;

[0144] (3) Transfer: Cut the gel according to the molecular weight of the target protein, cut the PVDF membrane, activate it in methanol, assemble the transfer sandwich, keep the constant current at 300mA, and adjust the time according to the molecular weight of the target protein;

[0145] (4) Blocking: Place the strips in 5% skimmed milk powder and block on a shaker at room temperature for 2 hours;

[0146] (5) Incubation with primary antibody: Prepare primary antibody using antibody diluent according to the instruction manual at 4°C overnight;

[0147] (6) Washing the membrane: Wash the incubated strips twice with TBST and once with TBS, each time for 10 min;

[0148] (7) Incubation with secondary antibody: prepare secondary antibody using TBST according to the instructions and incubate on a shaker at room temperature for 1 hour;

[0149] (8) Wash membrane: TBST twice, TBS once, each time for 10 min;

[0150] (9) Color development: Add Western Blue or ECL color development solution for color development. After the color development is terminated, the bands are scanned and analyzed.

[0151] The results of TTC staining for detecting brain ischemic volume after ischemic stroke and the results of neurological function injury score showed that compared with intravenous administration and intraperitoneal injection (intravenous route iv, intraperitoneal injection ip), nasal mucosal administration (in) has the unique advantages of convenience, non-invasiveness, and high absorption while achieving the same therapeutic effect (see Figure 6 ).

[0152] Corner turning test and body raising test were used to evaluate the effect of 1mg / mL hMSC-L and 1mg / mL hMSC-M treatment on the recovery of motor function in stroke mice. Corner turning test mainly evaluates asymmetric sensory motor disorder in mice and can detect unilateral abnormalities in sensory and motor function in stroke mice. Statistical results showed that hMSC-L administration by in, iv and ip routes could significantly improve the abnormal sensory and motor function in mice (see Figure 7 A and B in Figure 3), hMSC-M treatment also showed the same effect as hMSC-L (see Figure 7 C and D in ).

[0153] The body raising test was used to evaluate the asymmetry of the motor function of mice. The frequency and intensity of body raising on both sides of the body of healthy mice were similar, but the neurological deficit on the ischemic side of MCAO mice resulted in a relatively low proportion of rotations to the lesion side. Statistical results showed that the administration of hMSC-L via three routes significantly improved the proportion of mice rotating to the right side (see Figure 7 B and D)

[0154] After constructing the MACO model, HE staining results showed that the cells in the MCAO group were disordered, with vacuoles and nuclear atrophy, and other pathological changes. After treatment with hMSC-M at concentrations of 1 mg / mL and 2 mg / mL, these phenomena were alleviated. Statistical results showed that 1 mg / mL and 2 mg / mL of hMSC-M significantly improved the number of damaged cells in the mouse brain tissue, and the 2 mg / mL group more significantly reduced the number of damaged cells in the mouse brain tissue (see Figure 8 A and C in ).

[0155] Nissl staining results showed that the number of Nissl bodies in the brain of mice in the MCAO group decreased and their arrangement was disordered. After treatment with hMSC-M at concentrations of 1 mg / mL and 2 mg / mL, the above phenomenon was alleviated. Statistical results showed that 1 mg / mL and 2 mg / mL significantly increased the number of Nissl bodies in the brain tissue of mice, and the number of Nissl bodies in the brain tissue of mice in the 2 mg / mL group increased significantly (see Figure 8 The above results show that the treatment of hMSC secretory factors and cell secretory components significantly improved the pathological damage of the ischemic mouse brain tissue, and the therapeutic effect of high concentrations of secretory factors and cell secretory components was more significant.

[0156] By detecting the expression of the main pro-inflammatory cytokine gene levels in the brain of mice after ischemic stroke, it was found that the transcription levels of IFN-γ, TNF-α and IL-1β in the brain decreased significantly after hMSC-L and hMSC-M treatment. In particular, the IFN-γ and TNF-α in the brain of the hMSC-M group mice decreased more significantly than those in the hMSC-L group (see Fig. 9 ).

[0157] In addition, hMSC-L and hMSC-M treatment can significantly reduce the blood-brain barrier leakage volume, and the degree of angiogenesis may be more significant after hMSC-M treatment (see Fig.11 and Fig.12 ).

[0158] After treatment with hMSCs, the function of PPAR-γ was further activated, nuclear translocation was significantly enhanced, and phosphorylation levels were reduced. The use of T0070907 to inhibit PPAR-γ significantly inhibited the activation of neutrophils caused by hMSC-L and hMSC-M. It was manifested as down-regulation of nuclear PPAR-γ expression and up-regulation of p-PPAR-γ expression. After stimulation with hMSC-L and hMSC-M, the proportion of N2 neutrophils was significantly increased, and the proportion of N1 neutrophils was significantly decreased. After inhibiting PPAR-γ, the polarization direction of neutrophils in both groups was significantly biased toward the N1 type (see Fig.10 , Fig.13 and Fig.14 ).

[0159] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a secretory component of mesenchymal stem cells with high expression of IL-10, characterized in that: The following steps are involved: S1. Expansion culture: obtain sufficient human mesenchymal stem cells through expansion culture as the first mesenchymal stem cells; S2, induction culture: placing the first mesenchymal stem cells obtained in S1 in an induction medium, and culturing them at 2-5% O2, 5% CO2, and 37°C for 12-24 hours to obtain second mesenchymal stem cells; the induction medium is a basal medium with cytokines added thereto, wherein the cytokines are human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide, the amino acid sequence of the human IFN-γ mimetic peptide is shown in SEQ ID NO.1, and the amino acid sequence corresponding to the TNF-ɑ mimetic peptide is NKHNRKI; S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with saline, harvest the cells after trypsin digestion, and add 1×10 6 Inoculate the plate with an inoculum of 1000 μg / mL into an ultra-low adsorption 96U bottom plate, centrifuge, add α-MEM medium containing 5-10% human AB serum, and culture at 5% O2, 5% CO2, 37°C for 12 h; S4. Repeated freezing and thawing: Place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high expression of IL-10.

2. The preparation method according to claim 1, characterized in that: S1 The expansion culture is to place the human mesenchymal stem cells in a 37°C water bath for recovery, then inoculate them in a basic culture medium, and culture them under the conditions of 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70% to 90%.

3. The preparation method according to claim 1, characterized in that: S2: The amount of human IFN-γ mimetic peptide added is 200-1000 U / mL, and the amount of TNF-ɑ mimetic peptide added is 5-10 ng / mL.

4. The preparation method according to claim 3, characterized in that: The optimal addition amount of the human IFN-γ mimetic peptide in S2 is 600 U / mL, and the optimal addition amount of the TNF-ɑ mimetic peptide is 10 ng / mL.

5. The preparation method according to claim 1, characterized in that: S2 The basic culture medium is α-MEM culture medium containing double antibodies and 10-20% FBS or double antibodies and 10-20% human AB serum.

6. The secretory component of mesenchymal stem cells with high expression of IL-10 obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the secretory component of mesenchymal stem cells according to claim 6 in increasing the proportion and number of B cells in lymph nodes of peripheral immune organs.

8. Use of the mesenchymal stem cell secretory component according to claim 6 in the preparation of a drug for preventing ischemic stroke.

9. Use of the mesenchymal stem cell secretory component according to claim 6 in the preparation of a medicament for treating ischemic stroke.

10. The use according to claim 9, characterized in that: The application includes at least one of the following: (1) Reduce the volume of cerebral ischemia; (2) Promote motor function recovery; (3) Improve pathological damage of ischemic brain tissue; (4) Inhibit the expression of inflammatory factors in the brain; (5) Promote the expression of immunoregulatory N2 neutrophils; (6) Improve blood-brain barrier leakage; (7) Promote angiogenesis; (8) Activation of PPAR-γ nuclear translocation; (9) Promote the reprogramming of N2 neutrophils.

Images