Chronic disease treatment composition based on mesenchymal stem cells and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a chronic disease treatment composition based on mesenchymal stem cells and a preparation method thereof, and the chronic disease treatment composition comprises the mesenchymal stem cells; the mesenchymal stem cells are adipose mesenchymal stem cells, dental pulp mesenchymal stem cells and bone marrow mesenchymal stem cells. According to the invention, the culture medium of the adipose-derived mesenchymal stem cells is improved, and the prepared adipose-derived mesenchymal stem cells have higher proliferation activity. Meanwhile, through experimental tests, the adipose-derived stem cells can inhibit proliferation of the prostatic hyperplasia epithelium BPH-1 cells and remarkably promote apoptosis of the prostatic hyperplasia epithelium BPH-1 cells, a new direction is provided for treatment of prostatic hyperplasia, and the adipose-derived stem cells have huge application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chronic disease treatment composition based on mesenchymal stem cells and a preparation method thereof. Background Art

[0002] The full name of chronic disease is chronic non-communicable diseases. It does not refer to a specific disease, but is a general term for a class of diseases with hidden onset, long course and protracted illness, lack of definite evidence of infectious biological causes, complex causes, and some of which have not yet been fully confirmed.

[0003] Common chronic diseases include cardiovascular and cerebrovascular diseases, cancer, diabetes, chronic respiratory diseases, prostate hyperplasia, etc.

[0004] Currently, stem cell therapy for chronic diseases has become a research hotspot.

[0005] For example, bone marrow mesenchymal stem cells have been shown to be able to treat chronic diseases including atherosclerosis, heart failure, osteoporosis, psoriasis, and steroid-induced femoral head necrosis.

[0006] Dental pulp stem cells (DPSCs) have the ability of immunoregulation, self-renewal and multidirectional differentiation, and have the advantages of rich sources and low immunogenicity. The "human dental pulp mesenchymal stem cell injection" independently developed by the team of Academician Wang Songling of Capital Medical University has obtained the clinical trial license of the Drug Review Center of the State Food and Drug Administration. The current clinical trial results show that the injection is safe, effective and quality-controlled for human administration. It can play a therapeutic role by changing the local immune microenvironment and has broad clinical application prospects. It has been proven to be able to treat chronic inflammation caused by the imbalance of oral mucosal epithelial cell barrier homeostasis caused by chewing betel nut.

[0007] The biological behaviors of proliferation and apoptosis of BPH-1 cells in prostate hyperplasia are very important for the study of prostate hyperplasia. A large number of studies have shown that the epithelial and stromal cells of prostate hyperplasia have stronger proliferation ability than normal prostate, and the apoptosis rate of prostate hyperplasia epithelial cells is lower than that of normal prostate epithelial cells. Studies have shown that the expression of the anti-apoptotic gene Bcl2 in prostate hyperplasia epithelial cells is significantly higher than that in normal prostate epithelial cells, confirming that the anti-apoptotic ability of prostate hyperplasia epithelial cells is stronger than that of normal prostate epithelial cells.

[0008] Huang Ming et al. discovered that adipose-derived stem cells inhibit the proliferation of prostate hyperplasia epithelial BPH-1 cells by inhibiting the Wnt / β-catenin pathway (see Adipose-derived stem cells regulate the proliferation and apoptosis of prostate hyperplasia epithelial BPH-1 cells through the Wnt / β-catenin pathway, Genomics and Applied Biology, 2019, Vol. 38, No. 8, pp. 3857-3862).

[0009] The isolation and culture of adipose-derived mesenchymal stem cells will greatly affect their activity. Therefore, this application will optimize the isolation and culture methods of adipose-derived mesenchymal stem cells to obtain adipose-derived mesenchymal stem cells with higher activity, and thereby develop a chronic disease treatment composition based on mesenchymal stem cells. Summary of the invention

[0010] The present invention first provides, in order to achieve the above-mentioned purpose, a chronic disease treatment composition based on mesenchymal stem cells, wherein the chronic disease treatment composition comprises mesenchymal stem cells.

[0011] Preferably, the mesenchymal stem cells are adipose-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

[0012] Preferably, the composition may also contain other drugs for treating prostate hyperplasia.

[0013] Preferably, the raw material used in the method for preparing adipose-derived mesenchymal stem cells is adipose tissue.

[0014] Preferably, the method for preparing adipose-derived mesenchymal stem cells comprises the following steps:

[0015] (1) Obtaining adipose tissue;

[0016] (2) enzymatically hydrolyzing the adipose tissue of step (1) to obtain an enzymatic hydrolysis product;

[0017] (3) ultrasonically treating the enzymatic hydrolysis product of step (2) to obtain an ultrasonically treated product;

[0018] (4) centrifuging and washing the ultrasonic treatment product of step (3) to obtain a single cell suspension of adipose-derived mesenchymal stem cells;

[0019] (5) Cultivating and passage the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4).

[0020] Preferably, the culture medium used for the culture contains DMEM, icariin and insulin-transferrin-selenium.

[0021] Preferably, the concentration of icariin is 30 μmol / L, and the mass fraction of insulin-transferrin-selenium is 0.5%.

[0022] The present invention also provides a method for preparing the above-mentioned chronic disease treatment composition, wherein the method for preparing adipose-derived mesenchymal stem cells comprises the following steps:

[0023] (1) Obtaining adipose tissue;

[0024] (2) enzymatically hydrolyzing the adipose tissue of step (1) to obtain an enzymatic hydrolysis product;

[0025] (3) ultrasonically treating the enzymatic hydrolysis product of step (2) to obtain an ultrasonically treated product;

[0026] (4) centrifuging and washing the ultrasonic treatment product of step (3) to obtain a single cell suspension of adipose-derived mesenchymal stem cells;

[0027] (5) culturing and passaging the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4);

[0028] The present invention also provides the use of the above-mentioned chronic disease treatment composition, wherein the use is for preparing medicines for treating chronic diseases.

[0029] Preferably, the chronic disease is prostate hyperplasia.

[0030] Compared with the prior art, the above technical solution of the present invention has the following beneficial effects:

[0031] The present invention improves the culture medium of adipose-derived mesenchymal stem cells, and the prepared adipose-derived mesenchymal stem cells have higher proliferation activity. At the same time, through experimental tests, the adipose-derived mesenchymal stem cells of the present application can inhibit the proliferation of prostate hyperplasia epithelial BPH-1 cells and can significantly promote the apoptosis of prostate hyperplasia epithelial BPH-1 cells, providing a new direction for the treatment of prostate hyperplasia and having great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Diagram showing the effect of adipose-derived stem cells on the proliferation of prostate hyperplasia epithelial BPH-1 cells;

[0033] Figure 2 A diagram showing the effect of adipose-derived stem cells on collagen synthesis in prostate hyperplasia epithelial BPH-1 cells;

[0034] Figure 3 Effect of adipose-derived stem cells on apoptosis of prostate hyperplasia epithelial BPH-1 cells. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0036] Icariin was purchased from Beijing Suocaibao Technology Co., Ltd. (China) with a purity of ≥98.0%;

[0037] Insulin-transferrin-selenium and type I collagenase were purchased from Gibco;

[0038] Prostatic hyperplasia epithelial BPH-1 cells, Annexin V-APC / 7-AAD cell apoptosis detection kit, Transwell chamber, hydroxyproline detection kit; HRP-labeled goat anti-rabbit IgG, CCK8 kit were all purchased from commercial companies.

[0039] Example 1 Preparation of adipose-derived mesenchymal stem cells

[0040] 1) Under sterile conditions, adipose tissue obtained from human liposuction was obtained, rinsed with physiological saline, chopped with scissors, and dispensed into 50 mL centrifuge tubes at a rate of 5 mL / tube.

[0041] 2) Add type I collagenase (purchased from Gibco) to the centrifuge tube containing fat, seal it, shake it upside down, and mix it thoroughly. Transfer it to a 37°C constant temperature air bath shaker and digest it for 60 minutes.

[0042] 3) First, ultrasonically treat the tube at 100 W for 30 seconds, then place the centrifuge tube in a 37°C constant temperature shaker and shake at a constant speed of 100 rpm for 30 minutes until the tube is digested into a uniform suspension.

[0043] 4) The obtained suspension was centrifuged at 2000 rpm for 20 min, the upper layer of fat cells and the middle liquid were discarded, the cell precipitate was collected, and the fat stem cell clusters at the bottom layer were retained.

[0044] 5) Add 10 mL of physiological saline to the centrifuge tube containing the adipose stem cell mass, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a filter with a diameter of 40 μm, collect single cells, and then centrifuge at 2000 rpm for 5 minutes, discard the upper layer of liquid, and retain the adipose stem cell mass at the bottom; repeat this washing step twice.

[0045] 6) After thorough washing, add physiological saline, repeatedly blow and beat the cells to make a single cell suspension of adipose-derived mesenchymal stem cells, add them to a culture bottle containing DMEM + 30 μmol / L icariin + 0.5% insulin-transferrin-selenium medium, and culture them in a 5% CO2 concentration 37°C incubator. When the cells are in culture on the 3rd and 6th days, the medium should be changed in time; on the 7th to 10th day, if 5 randomly observed cell colonies reach more than 80%, the cells can be passaged.

[0046] 7) The 3rd and 8th passage adipose-derived mesenchymal stem cells were used for the experiment.

[0047] 8) Cell identification: Adipose-derived mesenchymal stem cells of the 3rd and 8th passages can efficiently express adipose-derived mesenchymal stem cell markers such as CD49, CD73, and CD105, but do not express CD45 and HLA-DR. At the same time, the differentiation potential of adipose-derived mesenchymal stem cells of the 8th passage was detected. The results of Oil Red O staining and silver nitrate staining confirmed that adipose-derived mesenchymal stem cells have obvious adipogenic and osteogenic abilities.

[0048] Example 2

[0049] Except that the culture medium in step 6) is DMEM+30 μmol / L icariin, the other conditions are the same as those in Example 1.

[0050] Example 3

[0051] Except that the culture medium in step 6) is DMEM+0.5% insulin-transferrin-selenium culture medium, the other conditions are the same as those in Example 1.

[0052] Example 4

[0053] Except that DMEM medium was used as the culture medium in step 6), the other conditions were the same as those in Example 1.

[0054] Example 5

[0055] The adipose-derived mesenchymal stem cells isolated from Examples 1, 2, 3 and 4 were concentrated to 2×10 4 Pieces / cm 2 The cells were inoculated into T25 cell culture flasks at a density of 100 μg / mL. After 10 days, the number of clones with more than 10 cells was observed and recorded under a microscope. The relative multiples of the number of cells in each group were calculated based on the number of cells in Example 4. The results showed that the cells obtained in Example 1 contained more adipose-derived mesenchymal stem cells with proliferation ability, as shown in Table 1.

[0056] Table 1 Proliferation ability of adipose-derived mesenchymal stem cells prepared in different examples

[0057] Group Relative multiple of cell number Example 1 8.7 Example 2 2.5 Example 3 2.3 Example 4 1

[0058] Example 6 Experimental treatment of prostatic hyperplasia

[0059] Grouping:

[0060] Experimental group 1: adipose-derived mesenchymal stem cells prepared in Example 1;

[0061] Experimental group 2: adipose-derived mesenchymal stem cells prepared in Example 2;

[0062] Experimental group 3: adipose-derived mesenchymal stem cells prepared in Example 3;

[0063] Experimental group 4: adipose-derived mesenchymal stem cells prepared in Example 4;

[0064] Control group.

[0065] See (Huang Ming et al., Adipose-derived stem cells regulate the proliferation and apoptosis of prostate hyperplasia epithelial BPH-1 cells through the Wnt / β-catenin pathway, Genomics and Applied Biology, 2019, Vol. 38, No. 8, pp. 3857-3862) method to detect the effects of adipose stem cells on the proliferation, collagen synthesis, and apoptosis of prostate hyperplasia epithelial BPH-1 cells.

[0066] The cell co-culture method is:

[0067] Transwell chambers were used for co-culture. The pore size of the Transwell chamber membrane was 0.4 μm. The third generation of human adipose-derived stem cells were taken and cultured at 0 and 6 × 10 4 100 cells / well were seeded in the upper chamber of the Transwell chamber (adipose stem cells: prostate hyperplasia epithelial BPH-1 cells were 1:1), and prostate hyperplasia epithelial BPH-1 cells in the logarithmic growth phase of the 4th generation were taken and plated with 6×10 4 The cells were inoculated into the lower chamber of a 6-well plate, and 1% FBS DMEM culture medium was used in both the upper and lower chambers of Transwell (only cell culture medium was added to the upper chamber of the control group without adipose-derived stem cells). Human adipose tissue-derived stem cells were co-cultured with prostate hyperplasia epithelial BPH-1 cells. After 24 hours of culture, the prostate hyperplasia epithelial BPH-1 cells in the lower chamber were removed for examination.

[0068] CCK8 method to detect cell viability: The third generation of human adipose-derived stem cells were taken and the concentrations of 0 and 6 × 10 4 100 cells / well were inoculated into the upper chamber of the Transwell chamber (0 was the control group, containing only cell culture medium), and 4 generations of prostate hyperplasia epithelial BPH-1 cells in the logarithmic growth phase were taken and inoculated with 6×10 4 Each well was inoculated with 1% FBSDMEM culture medium in the upper and lower chambers of Transwell. Three replicate wells were set up in each group and cultured in an incubator for 24 h. 10 μL CCK8 solution was added to each well and cultured for 2 h. The absorbance of each well (A450) was measured at 450 nm on a microplate reader. The A450 value represents cell viability.

[0069] Among them, hydroxyproline detection:

[0070] Human adipose tissue-derived stem cells were co-cultured with prostate hyperplasia epithelial BPH-1 cells, and the cell culture fluid in the lower chamber (6-well plate) was collected and tested according to the instructions of the hydroxyproline detection kit. Take 0.25mL of cell culture fluid (or standard application solution), add 0.05mL of digestion solution, incubate in a 37℃ water bath for 3h, add 0.5mL of reagent 1, mix well and let stand at room temperature for 10min, add 0.5mL of reagent 2, mix well and let stand for 5min, add 1mL of reagent 3, incubate in a 60℃ water bath for 15min, centrifuge and take the supernatant to measure the absorbance at 550nm.

[0071] Flow cytometry was used to detect cell apoptosis: the effect of adipose stem cells on apoptosis of prostate hyperplasia epithelial BPH-1 cells was detected by Annexin V-APC / 7-AAD double staining. Cell inoculation and drug administration were the same as those in the "cell co-culture" item. According to the instructions of the cell apoptosis detection kit, the cells were resuspended with Annexin V binding buffer, 5 μL of diluted Annexin V-APC working solution was added and incubated on ice for 15-20 minutes, and then 5 μL of diluted 7-AAD working solution was added to counterstain the cells. After incubation on ice for 5 minutes, the cells were immediately detected by flow cytometry.

[0072] The results of the effect of adipose-derived stem cells on the proliferation of prostate hyperplasia epithelial BPH-1 cells can be found in Figure 1 As shown, compared with the control group, after the adipose stem cells of the examples were co-cultured with the prostate hyperplasia epithelial BPH-1 cells for 24 hours, the proliferation ability of the prostate hyperplasia epithelial BPH-1 cells was significantly inhibited (p < 0.01), among which Example 1 had the best proliferation ability inhibition effect (p < 0.001).

[0073] The results of the effects of adipose-derived stem cells on collagen synthesis in prostate hyperplasia epithelial BPH-1 cells are shown in Figure 2 As shown, compared with the control group, after the adipose stem cells of the embodiment were co-cultured with the prostate hyperplasia epithelial BPH-1 cells for 24 hours, the collagen synthesis ability of the prostate hyperplasia epithelial BPH-1 cells was significantly inhibited (p < 0.01), among which the collagen synthesis inhibition effect of Example 1 was the best (p < 0.001).

[0074] The results of the effects of adipose-derived stem cells on apoptosis of prostate hyperplasia epithelial BPH-1 cells can be found in Figure 3 As shown, compared with the control group, the apoptosis rate of prostate hyperplasia epithelial BPH-1 cells was significantly increased after adipose-derived stem cells were co-cultured with prostate hyperplasia epithelial BPH-1 cells for 24 hours (p < 0.01), among which Example 1 had the best apoptosis-promoting effect (p < 0.001).

[0075] It should be understood that the above are only some embodiments of the present invention. It should be pointed out that for ordinary technicians in this field, other modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A chronic disease treatment composition based on mesenchymal stem cells, characterized in that: The chronic disease treating composition includes mesenchymal stem cells.

2. The chronic disease treatment composition according to claim 1, characterized in that: The mesenchymal stem cells are adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and bone marrow mesenchymal stem cells.

3. The chronic disease treatment composition according to claim 1, characterized in that: The composition may also contain other drugs for treating prostate hyperplasia.

4. The chronic disease treatment composition according to claim 2, characterized in that: The raw material used in the method for preparing adipose-derived mesenchymal stem cells is adipose tissue.

5. The chronic disease treatment composition according to claim 2, characterized in that: The method for preparing adipose-derived mesenchymal stem cells comprises the following steps: (1) Obtaining adipose tissue; (2) enzymatically hydrolyzing the adipose tissue of step (1) to obtain an enzymatic hydrolysis product; (3) ultrasonically treating the enzymatic hydrolysis product of step (2) to obtain an ultrasonically treated product; (4) centrifuging and washing the ultrasonic treatment product of step (3) to obtain a single cell suspension of adipose-derived mesenchymal stem cells; (5) Cultivating and passage the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4).

6. The chronic disease treatment composition according to claim 5, characterized in that: The culture medium used for the culture comprises DMEM culture medium, icariin and insulin-transferrin-selenium.

7. The chronic disease treatment composition according to claim 6, characterized in that: The concentration of icariin is 30 μmol / L, and the mass fraction of insulin-transferrin-selenium is 0.5%.

8. A method for preparing the chronic disease treatment composition according to any one of claims 1 to 7, characterized in that: The method for preparing adipose-derived mesenchymal stem cells comprises the following steps: (1) Obtaining adipose tissue; (2) enzymatically hydrolyzing the adipose tissue of step (1) to obtain an enzymatic hydrolysis product; (3) ultrasonically treating the enzymatic hydrolysis product of step (2) to obtain an ultrasonically treated product; (4) centrifuging and washing the ultrasonic treatment product of step (3) to obtain a single cell suspension of adipose-derived mesenchymal stem cells; (5) Cultivating and passage the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4).

9. The use of the chronic disease treatment composition according to any one of claims 1 to 7, characterized in that: The application is to prepare medicines for treating chronic diseases.

10. The use of the chronic disease treatment composition according to claim 9, characterized in that: The chronic disease is prostate hyperplasia.

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