A mesenchymal stem cell-based composition for treating chronic diseases and its preparation method

Abstract

The present invention relates to the field of biomedicine technology, and more specifically, to a chronic disease treatment composition based on mesenchymal stem cells and a preparation method thereof, wherein the chronic disease treatment composition comprises mesenchymal stem cells; the mesenchymal stem cells are adipose-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. 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, experimental tests have shown that 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.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition for treating chronic diseases 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 that have an insidious onset, a long course of illness, a persistent condition, 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 mesenchymal stem cells (DPSCs) possess immunomodulatory, self-renewal, and multidirectional differentiation capabilities, along with advantages such as abundant sources and low immunogenicity. A "human dental pulp mesenchymal stem cell injection," independently developed by a team led by Academician Wang Songling of Capital Medical University, has received clinical trial approval from the Center for Drug Evaluation of the National Medical Products Administration. Current clinical trial results demonstrate that the injection is safe, effective, and quality-controlled in humans. It exerts therapeutic effects by altering the local immune microenvironment and has broad clinical application prospects. It has been shown to treat chronic inflammation caused by betel nut chewing, which disrupts the homeostasis of the oral mucosal epithelial cell barrier.

[0007] The biological behaviors of BPH-1 cells, including proliferation and apoptosis, are crucial for the study of BPH. Numerous studies have shown that epithelial and stromal cells in BPH have greater proliferative capacity than those in normal prostate glands, and that BPH epithelial cells have lower apoptosis rates than normal prostate epithelial cells. Studies have also shown that expression of the anti-apoptotic gene Bcl2 is significantly higher in BPH epithelial cells than in normal prostate epithelial cells, confirming that BPH epithelial cells have a stronger anti-apoptotic capacity than normal prostate epithelial cells.

[0008] Huang Ming et al. discovered that adipose-derived stem cells inhibit the proliferation of benign prostatic hyperplasia epithelial BPH-1 cells by inhibiting the Wnt / β-catenin pathway (see Adipose-derived stem cells regulate the proliferation and apoptosis of benign prostatic 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 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 a composition for treating chronic diseases based on mesenchymal stem cells in order to achieve the above-mentioned purpose. The composition for treating chronic diseases includes 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 further comprise other drugs for treating benign prostatic 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 comprises 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 passage 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, which is used for preparing medicines for treating chronic diseases.

[0029] Preferably, the chronic disease is benign prostatic 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 for adipose-derived mesenchymal stem cells, resulting in adipose-derived mesenchymal stem cells with enhanced proliferation activity. Furthermore, experimental testing has shown that the adipose-derived mesenchymal stem cells described in this application can inhibit the proliferation of prostate epithelial prostatic hyperplasia (BPH-1) cells and significantly promote apoptosis in these cells, providing a new approach for the treatment of prostate hyperplasia and possessing significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A diagram showing the effect of adipose-derived stem cells on the proliferation of benign prostatic hyperplasia epithelial BPH-1 cells;

[0033] Figure 2 Effects of adipose-derived stem cells on collagen synthesis in benign prostatic hyperplasia epithelial BPH-1 cells;

[0034] Figure 3 Figure 3. Effect of adipose-derived stem cells on apoptosis of benign prostatic 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 clearer, 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 apoptosis detection kit, Transwell chamber, hydroxyproline detection kit; HRP-labeled goat anti-rabbit IgG, and CCK8 kit were all purchased from commercial companies.

[0039] Example 1 Preparation of Adipose-Derived Mesenchymal Stem Cells

[0040] 1) Under sterile conditions, human adipose tissue was obtained from liposuction, rinsed with physiological saline, minced 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 the fat, seal it, and shake it upside down to mix thoroughly. Transfer it to a 37°C constant temperature air bath shaker and digest for 60 minutes.

[0042] 3) First, ultrasonicate the tube at 100 W for 30 seconds. Place the tube in a 37°C constant temperature shaker at 100 rpm and digest for 30 minutes until the tube becomes a uniform suspension.

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

[0044] 5) Add 10 mL of saline to the centrifuge tube containing the adipose-derived stem cell cluster. Resuspend the cells by pipetting repeatedly to form a uniform single-cell suspension. Wash thoroughly, filter the cells through a 40 μm diameter filter, collect the single cells, and centrifuge at 2000 rpm for 5 minutes. Discard the supernatant and retain the adipose-derived stem cell cluster at the bottom. Repeat this washing step two more times.

[0045] 6) After thorough washing, add physiological saline and repeatedly pipette the cells to form a single-cell suspension of adipose-derived mesenchymal stem cells. Add the suspension to a culture flask containing DMEM + 30 μmol / L icariin + 0.5% insulin-transferrin-selenium medium and culture in a 5% CO2 incubator at 37°C. Change the medium on days 3 and 6 of cell culture. On days 7-10, if five randomly observed cell colonies reach over 80%, the cells can be passaged.

[0046] 7) Adipose-derived mesenchymal stem cells of passage 3 and passage 8 were used for experiments.

[0047] 8) Cell identification: Adipose-derived stromal stem cells at passages 3 and 8 efficiently expressed CD49, CD73, and CD105, but did not express CD45 or HLA-DR. The differentiation potential of passage 8 adipose-derived stromal stem cells was also assessed. Oil red O staining and silver nitrate staining confirmed that adipose-derived stromal stem cells possessed significant adipogenic and osteogenic abilities.

[0048] Example 2

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

[0050] Example 3

[0051] Except that the culture medium in step 6) is DMEM+0.5% by mass of 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 collected at a concentration of 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL into T25 cell culture flasks. After 10 days, the number of clones with more than 10 cells was observed under a microscope and recorded. Based on the cell number in Example 4, the relative multiple of the cell number in each group was calculated. 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 cell number multiples Example 1 8.7 Example 2 2.5 Example 3 2.3 Example 4 1

[0058] Example 6 Experiment on the treatment of benign 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 benign prostatic hyperplasia epithelial BPH-1 cells through the Wnt / β-catenin pathway, Genomics & Applied Biology, 2019, Vol. 38, No. 8, pp. 3857-3862) for the method to detect the effects of adipose-derived stem cells on the proliferation, collagen synthesis, and apoptosis of benign prostatic 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: benign prostatic hyperplasia epithelial BPH-1 cells were 1:1), and the benign prostatic 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 seeded 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 the Transwell (the upper chamber of the control group only contained cell culture medium without adipose-derived stem cells). Human adipose tissue-derived stem cells were co-cultured with benign prostatic hyperplasia epithelial BPH-1 cells. After 24 hours of culture, the benign prostatic hyperplasia epithelial BPH-1 cells in the lower chamber were removed for examination.

[0068] CCK8 assay for cell viability: The third generation of human adipose-derived stem cells were collected and incubated at 0 and 6 × 10 4 100 cells / well were seeded in the upper chamber of the Transwell chamber (0 was the control group, containing only cell culture medium), and the 4th generation of prostate hyperplasia epithelial BPH-1 cells in the logarithmic growth phase were taken and plated with 6×10 4 Cells were seeded into the lower chamber of a 6-well plate. 1% FBSDMEM culture medium was used in both the upper and lower chambers of the Transwell. Three replicate wells were set up in each group and cultured in an incubator for 24 h. 10 μL of 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. The cell culture fluid from the lower chamber (6-well plate) was collected and assayed according to the instructions of the hydroxyproline detection kit. To this end, 0.25 mL of cell culture fluid (or standard application solution) was added to 0.05 mL of digestion buffer. The mixture was incubated at 37°C in a water bath for 3 hours. Then, 0.5 mL of reagent 1 was added, mixed, and allowed to stand at room temperature for 10 minutes. Then, 0.5 mL of reagent 2 was added, mixed, and allowed to stand for 5 minutes. Finally, 1 mL of reagent 3 was added, and the mixture was incubated at 60°C in a water bath for 15 minutes. After centrifugation, the supernatant was collected and the absorbance was measured at 550 nm.

[0071] Flow cytometry was used to assess cell apoptosis: The effect of adipose-derived stem cells on apoptosis in prostate epithelial hyperplasia (BPH-1) cells was examined using the Annexin V-APC / 7-AAD double staining assay. Cell seeding and treatment were performed as described in the "Cell Co-culture" section. Following the instructions for the apoptosis detection kit, cells were resuspended in Annexin V binding buffer, and 5 μL of diluted Annexin V-APC working solution was added. The cells were incubated on ice for 15-20 minutes. The cells were then counterstained with 5 μL of diluted 7-AAD working solution. After incubation on ice for 5 minutes, the cells were immediately analyzed by flow cytometry.

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

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

[0074] The results of the effect of adipose-derived stem cells on apoptosis of benign prostatic 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 them, 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 variations and improvements can be made without departing from the creative concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An application of a chronic disease treatment composition, characterized in that: The application is to prepare a drug for treating benign prostatic hyperplasia; The chronic disease treatment composition includes adipose-derived mesenchymal stem cells; The method for preparing adipose-derived mesenchymal stem cells comprises the following steps: (1) Obtaining adipose tissue; (2) enzymatically hydrolyzing the adipose tissue in 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) culturing and passage the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4); Wherein, in step (5), the culture medium used is DMEM culture medium, icariin and insulin-transferrin-selenium.

2. The use according to claim 1, characterized in that The chronic disease treating composition further comprises other drugs for treating benign prostatic hyperplasia.

3. The use according to claim 1 or 2, characterized in that In the culture medium used in the culture in step (5), the concentration of icariin is 30 μmol / L, and the mass fraction of the insulin-transferrin-selenium is 0.5%.

Images