| Objective: To investigate the differentiation of bone marrow mesenchymal stem/stroma cells(BMSCs)in the bone tissue of defect parts under the intervention of exogenous drugs at the micro-level by observing the expression changes of vascular endothelial growth factor(VEGF)and bone morphogenetic protein 2(BMP2)during the osteoblasts differentiation process after introducing the isolated Eupolyphaga extract into the bone defect repair experiments,thereby explore the effect of Eupolyphaga pharmaceutical ingredient on the BMSCs,study the feasibility and applicability of Eupolyphaga pharmaceutical ingredient in the treatment of related disease in orthopedics,as well as offer the theoretical basis for clinical treatment.Methods: This research design is divided into two aspects: Experiment I: The effect of Eupolyphaga extract on the subculture and induced differentiation in vitro of BMSCs in SD rats: the proliferation assay was conducted in partial BMSCs of SD rats cultured for 3 generations by methyl thiazolyl tetrazolium(MTT)colorimetric assay to screen the drug concentration in subsequent experiments.The screening of the concentration of drug used in the preliminary test derived from the previous experiments.The recovery of BMSCs was observed,and the proliferation of BMSCs was evaluated by MTT assay.Experiment II: Quantification and identification of the expression of VEGF and BMP2 in BMSCs differentiation process under the effect of isolated Eupolyphaga extract: the BMSCs cultured for 3 generations,in accordance with the random number table,were divided into five groups on the basis of the concentration of added isolated Eupolyphaga extract.the concentration of isolated Eupolyphaga extract added in each group was 1.25mg/m L,2.5mg/m L,5mg/m L and 7.5mg/m L,in sequence,and the isolated Eupolyphaga extract was replaced by culture medium in the blank control group.The alkaline phosphatase staining and calcium nodules staining were carried out 14 days later to detect the expression of type I collagen and osteocalcin;in addition,PCR and 7 blot were conducted at day 14 and day 18 after culture to determine the expression of BMP2 and VEGF.Results 1.All of the BMSCs were induction differentiated successfully after adding different concentrations of isolated Eupolyphaga extract,and its differentiation degree was better with the increase of concentration;2.After culturing for 24 h,no obvious changes were observed in the number of BMSCs among different drug concentration groups,but subsequently,the number of BMSCs among groups other than blank control group and 0mg/m L group were increased sharply,the higher the concentration,the faster the cell growth rate,which was consistent between the 7.5mg/m L group and 10mg/m L group,and the similar outcome were seen between the 7.5mg/m L group and 10mg/m L group.The growth rate of BMSCs were further accelerated in the 1.25mg/m L group,2.5mg/m L group and 5mg/m L group,as demonstrated by the results of cell culture at 48 h,and which were exceeded that of the other higher drug concentration groups.3.At 14 days and 28 days of proliferation,despite the proliferation multiple of BMP2 in different Eupolyphaga extract concentration groups were rised to some extent with the increasing of drug concentration,which were not significant,even descended compared with that of blank control group.Compared with the blank control group,the proliferation multiple of VEGF in the 5mg/m L group and 7.5mg/m L group were increased remarkably,however,no dramatic increase,even decrease were observed in the other two groups.Moreover,in the above two concentration groups,the amplification times at day 28 was decreased markedly compared with that at day 14 under the same concentration.In addition,at the same point in time,the amplification times of VEGF was also rised continuously with the increase of drug concentration.4.Compared with the blank control group,the trend to decline was observed in the expression of BMP2 protein along with the increase of drug concentration at day 14,whereas the expression of VEGF protein was prominently increased.At day 28,the expression of BMP2 protein was markedly decreased with the increasing of drug concentration,and the expression of VEGF protein was still notably increased,which was significantly greater than that at day 14.Conclusion 1.The osteogenic differentiation in SD rats can be induced to a certain extent by Eupolyphaga extract,some interaction probably exists in the purity and mix ingredients of Eupolyphaga extract,and the effectivity of single ingredient extract is still worthy of further study.2.The Eupolyphaga extract has better effect,which has certain influence on the osteogenic differentiation of BMSCs.In addition,the expression of VEGF is active during the induction of osteogenic differentiation.3.The 7.5mg/m L might be the optimum concentration of Eupolyphaga extract in promoting the differentiation of BMSCs of SD rats to microangiogenesis.The 7.5mg/m L can be chosen for the next in depth research of the action mechanism of VEGF in osteogenic differentiation in future. |
PDF Full Text Request