Bone marrow mesenchymal stem cell osteogenesis induction enhancing liquid

Abstract

The invention discloses a bone marrow mesenchymal stem cell osteogenesis induction enhancing solution, which is prepared from the following components: 6.5 to 9.5 mg / ml of sodium hydrosulfite, 8.5 to 13.5 mg / ml of nicotinamide ribose, 1.5 to 2.1 mg / ml of deoxycholic acid, 45.0 to 55.0 mu g / ml of estradiol, 0.2 to 0.6 mg / ml of calcitriol, and 90.0 to 110.0 mu g / ml of rrBMP-2. Traditional osteogenic induction and 1% osteogenic induction enhancing liquid have high cell activity on BMSC cell proliferation, and compared with traditional osteogenic induction (containing 0.2 mmol / L of ascorbic acid, 10 mmol / L of beta-sodium glycerophosphate and 100 nmol / L of dexamethasone), the alkaline phosphatase staining color is deeper, the positive area is larger, the alkaline phosphatase activity is higher, more calcium nodules are formed, and the BMSC cell proliferation effect is better. It is shown that the osteogenic induction enhancing liquid provided by the invention enhances the osteogenic ability of the rat BMSC.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to an osteogenic induction enhancing solution for bone marrow mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) have attracted extensive attention in the field of regenerative medicine due to their multi-directional differentiation ability. Currently, bone marrow tissue is the most important source for obtaining MSCs. Bone marrow mesenchymal stem cells (BMSCs) are regarded as ideal seed cells for tissue engineering and have been widely used in the research and treatment of various traumatic, inflammatory, autoimmune, and degenerative diseases with good effects.

[0003] Bone marrow mesenchymal stem cells can differentiate into osteoblasts, adipocytes, chondrocytes, and fibroblasts under specific induction conditions. How to directionally induce bone marrow mesenchymal stem cells into osteoblasts is very important, which can provide treatment methods for fracture repair, bone tissue engineering, osteoporosis, etc. The osteogenic induction and differentiation of bone marrow mesenchymal stem cells mainly rely on osteogenic induction media. The composition of traditional osteogenic induction media includes dexamethasone, vitamin C, and β-glycerophosphate sodium, and there is a dose and time correlation among the three. However, the induction efficiency of traditional osteogenic induction media is relatively low and the induction time is relatively long. Currently, there are also commercially available osteogenic induction media for bone marrow mesenchymal stem cells, but their induction effects are unstable, and the components and ratios of commercially available media are not transparent, which may affect subsequent in-depth research. Summary of the Invention

[0004] The purpose of the present invention is to provide an osteogenic induction enhancing solution for bone marrow mesenchymal stem cells to improve the osteogenic efficiency of bone marrow mesenchymal stem cells. The purpose of the present invention is achieved as follows. An osteogenic induction enhancing solution for bone marrow mesenchymal stem cells, comprising 6.5 - 9.5 mg / ml of sodium dithionite, 8.5 - 13.5 mg / ml of nicotinamide riboside, 1.5 - 2.1 mg / ml of deoxycholic acid, 45.0 - 55.0 μg / ml of estradiol, 0.4 mg / ml of calcitriol, and 90.0 - 110.0 μg / ml of rrBMP-2 (rrBMP-2 is recombinant rat BMP-2 protein, with the full English name: Recombinant Rat BMP-2 Protein).

[0005] Sodium dithionite can regulate the growth microenvironment of stem cells and promote osteogenesis of stem cells; nicotinamide riboside can improve mitochondrial function and promote osteogenesis of stem cells; deoxycholic acid can activate the osteogenic-related signaling pathway of stem cells and promote osteogenesis of stem cells; estradiol and calcitriol can improve cell activity, promote the expression of osteogenesis-related genes, and at the same time inhibit the activity of osteoclasts; rrBMP-2 can directly act on stem cells and promote osteogenesis of stem cells.

[0006] The beneficial effects of the present invention are as follows: Traditional osteogenic induction + 1% osteogenic induction enhancer has a relatively high cell activity on the proliferation of BMSC cells. Compared with traditional osteogenic induction (containing 0.2 mmol / L ascorbic acid, 10 mmol / L β-glycerophosphate, and 100 nmol / L dexamethasone), its alkaline phosphatase staining has a deeper color, a larger positive area, stronger alkaline phosphatase activity, and more calcium nodule formation, demonstrating that the osteogenic induction enhancer described in this application enhances the osteogenic ability of rat BMSC. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Cell morphology of rat bone marrow mesenchymal stem cells (BMSC) at different time points (inverted microscope, 100×); Figure 2 Flow cytometry identification results of rat bone marrow mesenchymal stem cells (BMSC); Figure 3 Effect of osteogenic induction enhancer on the activity of rat bone marrow mesenchymal stem cells (BMSC); Figure 4 Alkaline phosphatase staining results (upright microscope, 200×); Figure 5 Alizarin red staining results (inverted microscope, 100×). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0008] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments fall within the scope of protection of the present invention.

[0009] The object of the present invention is achieved in this way. An osteogenic induction enhancer for bone marrow mesenchymal stem cells includes 6.5 - 9.5 mg / ml sodium dithionite, 8.5 - 13.5 mg / ml nicotinamide riboside, 1.5 - 2.1 mg / ml deoxycholic acid, 45.0 - 55.0 μg / ml estradiol, 0.2 - 0.6 mg / ml calcitriol, and 90.0 - 110.0 μg / ml rrBMP-2.

[0010] Preferably: including 8.5 mg / ml sodium dithionite, 13 mg / ml nicotinamide riboside, 2 mg / ml deoxycholic acid, 50 μg / ml estradiol, 0.5 mg / ml calcitriol, 100 μg / ml rrBMP-2.

[0011] Example 1 An osteogenic induction enhancing solution for bone marrow mesenchymal stem cells, including 8.0 mg / ml sodium dithionite, 11.0 mg / ml nicotinamide riboside, 1.8 mg / ml deoxycholic acid, 50.0 μg / ml estradiol, 0.4 mg / ml calcitriol, 100.0 μg / ml rrBMP-2.

[0012] Example 2 An osteogenic induction enhancing solution for bone marrow mesenchymal stem cells, including 6.5 mg / ml sodium dithionite, 8.5 mg / ml nicotinamide riboside, 1.5 mg / ml deoxycholic acid, 45.0 μg / ml estradiol, 0.2 mg / ml calcitriol, 90.0 μg / ml rrBMP-2.

[0013] Example 3 An osteogenic induction enhancing solution for bone marrow mesenchymal stem cells, including 9.5 mg / ml sodium dithionite, 13.5 mg / ml nicotinamide riboside, 2.1 mg / ml deoxycholic acid, 55.0 μg / ml estradiol, 0.6 mg / ml calcitriol, 110.0 μg / ml rrBMP-2.

[0014] Example 4 An osteogenic induction enhancing solution for bone marrow mesenchymal stem cells, including 8.5 mg / ml sodium dithionite, 13 mg / ml nicotinamide riboside, 2 mg / ml deoxycholic acid, 50 μg / ml estradiol, 0.5 mg / ml calcitriol, 100 μg / ml rrBMP-2.

[0015] Select the enhancing solution described in Example 4 for the following experiment: 1. Experimental animals 3-4 week-old SD rats, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. [SCXK (Xiang) 2021-0002], and raised in the SPF animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Dian) K2021-0003].

[0016] 2. Main reagents, consumables Reagent Name Company DMEM / F-12 Basal Medium Gibco L-Glutamine Gibco Penicillin-Streptomycin Gibco 0.25% Trypsin Gibco APC Mouse Anti-Rat CD90 BD Biosciences FITC Mouse Anti-Rat CD45 BD Biosciences Phosphate Buffered Saline (PBS) Gibco Vitamin C Sigma β-Glycerophosphate Sodium Mce Dexamethasone Sigma Sodium Dithionite Sigma Nicotinamide Riboside (NR) Mce Deoxycholic Acid Mce Estradiol Mce rrBMP-2 (Recombinant Rat BMP-2 Protein) Beijing Protein Innovation Calcitriol Mce CCK-8 Kit Beyotime Alizarin Red Staining Solution Cyagen Alkaline Phosphatase Staining Kit RayGen 3. Main instruments Instrument Name Company Cell Culture Incubator Thermo Fisher Scientific 3141 Laminar Flow Hood Haier HCB-1300V Inverted Microscope OLYMPUS BX53 Flow Cytometer ACEA Novocyte 2060R Multifunctional Microplate Reader Aosheng Feyond-A300 Microscope OLYMPUS BX53 4. Experimental methods 4.1 Primary culture of rat BMSCs 1. Decapitate and sacrifice an SD rat, and soak it in 75% ethanol for disinfection for 10 min; 2. Under sterile conditions, cut off the bilateral hindlimbs of the rat, and strip off the external skin and muscles. Cut the femur intact and place it in a culture dish containing DMEM / F12 basal medium; 3. Cut off the femoral heads on both sides of the femur, aspirate DMEM / F12 medium with a 2.5 ml syringe, and flush the bone marrow out of the bone marrow cavity until the femur turns white; 4. Pipette and blow several times to blow the bone marrow into a single-cell suspension, centrifuge at 1000 rmp / min for 3 min to collect the cells; 5. Resuspend the cells with 5 ml of DMEM / F12 complete medium and inoculate them into a T25 flask, and culture them in an incubator at 37 °C and 5% CO2. After culturing for 48 h, perform the first cell medium change, and then change the medium every 72 h. When the cell confluence reaches 80%, perform cell passage.

[0017] 4.2 Flow cytometry identification of BMSC cells 1. Select passage 3 BMSC cells. When the cell confluence reaches 80%, aspirate the medium, add 1 ml of PBS, gently shake and rinse, aspirate all the PBS, add 500 μl of 0.25% trypsin, and digest in a cell incubator at 37 °C for 2 min. When it is found that the cells shrink into spheres or show a flowing sand-like state, immediately add 500 μl of DMEM / F12 complete medium to terminate the digestion; 2. Collect the cell suspension into a 1.5 ml centrifuge tube, centrifuge at 1300 rpm at room temperature for 3 minutes, and discard the supernatant; add 1 ml of PBS to resuspend the cells, centrifuge at 1300 rpm at room temperature for 3 minutes, and discard the supernatant to collect the cell pellet; 3. Add 100 μl of PBS to resuspend the cells, add 0.5 μl of the corresponding flow antibody to the centrifuge tube, blow and mix well, incubate at 4 °C in the dark for 30 min; 4. Take out the centrifuge tube, add 1 ml of PBS, centrifuge at 1300 rpm at room temperature for 3 minutes, discard the supernatant to collect the cell pellet, add 200 μl of PBS to resuspend the cells, and immediately perform flow cytometry detection.

[0018] 4.3 CCK8 assay Experimental grouping ①. BMSC normal culture group ②. BMSC + osteogenic induction enhancer group (containing 0.1%, 0.5%, 1.0%, 2.0%, 5.0% osteogenic induction enhancer) After digestion and counting of passage 3 bone marrow mesenchymal stem cells, they were seeded in a 96-well plate at a density of 5000 cells per well and cultured in DMEM / F12 complete medium containing osteogenic induction enhancer at different concentrations (with the volume ratio of DMEM / F12 complete medium to osteogenic induction enhancer being 1000:1, 500:1, 200:1, 100:1, 50:1, 20:1). At 24 h, 48 h, and 72 h, the original medium was removed, and then 100 μl of DMEM / F12 basal medium containing 10% CCK8 solution was added. The plate was placed in an incubator and incubated for 2 h in the dark. The absorbance at 450 nm was measured using a full-wavelength microplate reader.

[0019] 4.4 Osteogenic differentiation induction of BMSC Experimental grouping ① Control group: BMSC was cultured normally. ② Osteogenic induction group: Traditional osteogenic induction group of BMSC (containing 0.2 mmol / L ascorbic acid, 10 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone). ③ Osteogenic induction enhancer group: Traditional osteogenic induction + osteogenic induction enhancer group of BMSC (containing 0.2 mmol / L ascorbic acid, 10 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, 1% osteogenic induction enhancer).

[0020] After digestion and counting of passage 3 bone marrow mesenchymal stem cells, they were seeded in a 12-well plate coated with 0.1% gelatin at a density of 3×10 4 cells. When the cell confluence reached 80%, the medium in the wells was discarded. In groups ② and ③, the corresponding osteogenic induction media were changed according to the grouping, and the medium was changed every 48 h. ALP staining was performed after 7 days of culture; alizarin red staining was performed after 14 days of culture to verify the osteogenic effect of the cells.

[0021] 4.5 Alkaline phosphatase staining On the 7th day of osteogenic induction and differentiation, the cells were washed twice with PBS, then fixed with pre-cooled ALP fixative at 2 - 8°C for 5 minutes at 4°C, washed three times with distilled water, 20 μl of ALP staining solution was added, and incubated in an incubator at 37°C for 20 min, then washed three times with distilled water; 20 μl of nuclear fast red staining solution was added and incubated at room temperature for 3 min, washed twice with distilled water, and mounted with glycerin jelly. Observation and photography were performed under a microscope.

[0022] 4.6 Alizarin red staining On the 14th day of osteogenic induction and differentiation, the cells were washed twice with PBS, then fixed with 4% paraformaldehyde for 20 min and washed three times with double-distilled water. They were incubated with alizarin red staining solution at room temperature for 30 min, washed three times with double-distilled water, and observed and photographed under an inverted microscope.

[0023] 5. Experimental results 5.1 Primary culture of rat BMSCs After 24 h of inoculation, some cells adhered to the wall. The adherent cells were spherical, and a few were spindle-shaped. There were a large number of non-adherent cells in the culture medium. After changing the medium, the cells were continuously cultured. At 3 d, the cells began to grow in shape but were not fully developed, and the cells grew in a spindle shape. At 5 d, all cells had grown in shape, the cell morphology was extended, and they grew in a spindle shape, with a confluence of about 50%. At 7 d, the cell confluence reached about 70%. At 10 d, the cells were confluent to over 90%, the cell state was good, the cell bodies were translucent, and the refraction was good. The cells grew radially or in a vortex shape.

[0024] 5.2 Flow cytometry identification of rat BMSCs The results of flow cytometry detection and analysis showed that CD90 with stem cell characteristics was positively expressed on BMSCs (positive cells > 99%), and CD45 with hematopoietic and epithelial cell specificity was negatively expressed (positive cells < 1%), which was in line with the characteristics of mesenchymal stem cells.

[0025] 5.3 Effect of osteogenic induction enhancing solution on the proliferation of rat BMSCs The CCK-8 results showed that low concentrations of osteogenic induction enhancing solution (0.5%, 1.0%, 2.0%) could promote the proliferation of bone marrow mesenchymal stem cells. However, as the concentration increased, the inhibitory effect of the osteogenic induction enhancing solution gradually appeared, and the osteogenic induction enhancing solution showed higher cell activity at 1%.

[0026] 5.4 Osteogenic induction enhancing solution enhanced the osteogenic ability of rat BMSCs 5.4.1 Alkaline phosphatase staining Osteoblasts can secrete alkaline phosphatase, synthesize extracellular matrix such as type I collagen and osteocalcin, and then further mineralize to form bone tissue. These characteristics are used as the criteria for identifying the differentiation of bone marrow mesenchymal stem cells into osteoblasts. And alkaline phosphatase (ALP) is an enzyme necessary for bone formation and is an early marker of osteoblast differentiation and functional maturity. Compared with the control group, the staining color in the osteogenic induction group and the osteogenic induction enhancing solution group was darker, and the positive area was larger (the active site of alkaline phosphatase was black cobalt sulfide precipitate), and the positive area in the osteogenic induction enhancing solution group was larger and the color was darker than that in the osteogenic induction group.

[0027] 5.4.2 Alizarin red staining During the differentiation of bone marrow mesenchymal stem cells into osteoblasts, calcium salts are deposited on the cell surface to form calcium nodules. Calcium ions can chelate with alizarin red and be stained red or red-violet. Compared with the control group, calcium nodules were formed in the osteogenic induction group and the osteogenic induction enhancing solution group, and there were more calcium nodules in the osteogenic induction enhancing solution group than in the osteogenic induction group.

Claims

1. A bone marrow mesenchymal stem cell osteogenic induction enhancement solution, characterized in that: Including 6.5-9.5 mg / ml sodium dithionite, 8.5-13.5 mg / ml nicotinamide riboside, 1.5-2.1 mg / ml deoxycholic acid, 45.0-55.0 μg / ml estradiol, 0.2-0.6 mg / ml calcitriol, and 90.0-110.0 μg / ml rrBMP-2.

2. The bone marrow mesenchymal stem cell osteogenic induction enhancement solution according to claim 1, characterized in that: It includes 8.5mg / ml sodium dithionite, 13mg / ml nicotinamide riboside, 2mg / ml deoxycholic acid, 50ug / ml estradiol, 0.5mg / ml calcitriol, and 100μg / ml rrBMP-2.

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