The Experimental Research Of Effect Of Kidney Tonifying Recipe On Proliferation And Osteogenic Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells

Objective:To observe the effect of serum containing tonifying recipe of kidney on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in rabbit.Methods:1. BMSCs were isolated and cultured by the whole bone marrow. Then the cells were identified by the effect of osteogenic differentiation and adipogenic, and the surface antigens of BMSCs were used to identify the cells. Finally, the stable system of BMSCs was established.2. The serum containing tonifying recipe of kidney was prepared into different concentrations, and the proliferation of BMSCs cultured by different concentrations (5%、10%、15%、20%、25%)medicated serum was detected by MTT method.3. The different concentrations (5%、10%、15%、20%、25%)medicated serum was used to induce BMSCs into osteogenic differentiation, and the best concentration of medicated serum was detected by the activity of ALP4. BMSCs were divided into control group 1 (the best medicated serum),control group Ⅱ (the best medicated serum and classic induced liquid of osteogenesis), the blank serum group and the classic osteogenic differentiation group the activity of ALP in different groups were detected by ALP Kit at different time (3d,7d,14d).5. The amount of osteocalcin in different groups were detected by ELISA method at different time (7d,14d,21d).6. After 21 days of the BMSCs of different groups induced into osteogenic differentiation, they were dyed with Alizarin Red, and observed in low magnification microscope to count the number of calcified nodule.Results:1. BMSCs isolated from the whole bone marrow were adhered to plastic surface, and they could be induced into osteoblast and adipose cell. Flow cytometry analysis showed that BMSCs were positive for CD44and negative for CD34.2. Compared to the same concentration of the blank serum,5% and 15% medicated serum had better effect on proliferation of BMSCs after 24h incubation(P<0.05). After 24h incubation,15% medicated serum promoted the proliferation of BMSCs(P<0.05).Other concentration medicated serum did not promote the proliferation of BMSCs comparing to the same concentration of the blank serum(P>0.05).3.The activity of ALP in different concentrations (5%、10%、15%、20%、 25%)medicated serum groups were detected to show the best concentration is 5%.4. At the same time point, the activity of ALP in control group Ⅰ was significantly higher than the blank serum group (P<0.05), while the control group Ⅱ ALP activity was significantly higher than the classic osteogenic differentiation group and control group Ⅰ (P<0.05).5. After BMSCs induced into osteogenic differentiation, the osteocalcin of each group was increasesing with the time. The osteocalcin of control group I was significantly higher than the blank serum group (P<0.05), while the control group Ⅱ ALP activity was significantly higher than the classic osteogenic differentiation group (P<0.05).6. After BMSCs induced into osteogenic differentiation 21days, Calcified nodules was not found in the blank serum group, while other group had different number of calcified nodule. The number of calcified nodules of control group Ⅱ was significantly higher than the classic osteogenic differentiation group and control group Ⅰ (P<0.05).Conclusion:The serum containing tonifying recipe of kidney can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. The effect may make bone become stronger, and provide the basis on treatment for bone defect and nonunion. The research indicate common opinion between the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and the theory of Traditional Chinese Medicine on the kidney storing essence, producing marrow and bone, from the angle of taking medicine to prove effect.