新伤续断汤对成骨细胞增殖及骨形态发生蛋白的影响

【目的】探讨新伤续断汤(由骨碎补、续断、地鳖虫等组成)对大鼠成骨细胞增殖以及骨形态发生蛋白2(BMP-2)表达的影响。【方法】采用血清药理学方法制备新伤续断汤含药血清,与α-MEM培养基配成细胞培养液。按酶序列消化法从SD大鼠乳鼠颅骨获取、培养成骨细胞,以碱性磷酸酯酶染色和钙结节染色鉴定细胞。取第3~4代细胞用细胞增殖—毒性(CCK-8)法检测经体积分数为5%、10%、20%新伤续断汤含药血清处理24、48、72 h后的细胞增殖情况。用含体积分数10%的新伤续断汤、续断、骨碎补、空白对照含药血清分别培养细胞,采用微量酶标法检测碱性磷酸酶的活性,酶联免疫吸附法检测BMP-2的表达。【结果】新伤续断汤对成骨细胞的影响呈时间、浓度上的依赖性,其中浓度为10%时效果明显。体积分数10%的新伤续断汤、骨碎补、续断含药血清的成骨细胞碱性磷酸酶活性较空白对照组显著升高,差异有统计学意义(P0.01);7 d时,新伤续断汤组、骨碎补组与空白对照组比较,BMP-2表达显著升高,差异有统计学意义(P0.05)。【结论】新伤续断汤能促进大鼠成骨细胞碱性磷酸酶活性及BMP-2的表达,其中单味药骨碎补可能发挥重要作用,其促进骨折愈合机制可能与提高细胞的成骨功能有关。

Effects of Xinshang Xuduan Decoction on Osteoblast Proliferation and Bone Morphogenetic Protein in Vitro

Objective To investigate the effects of Xinshang Xuduan Decoction(XXD, mainly composed of Rhizoma Drynariae, Radix Dipsaci, and Eupolyphaga seu Steleophaga) on the proliferation and differentiation of rat osteoblasts and bone morphogenetic protein 2(BMP-2)expression in the osteoblasts . Methods The animal serum containing XXD was prepared by serum pharmacological method and then was mixed together with α-MEM for the cell culture. Osteoblasts were isolated from the skull bone of SD neonatal rats by collagenase digestion and were identified by alkaline phosphatase(ALP) staining and alizarin red staining. The third and fourth generations of osteoblasts were treated with XXD at the volume fraction of 5%, 10%, 20% for 24, 48 , 72 hours respectively, and then the proliferation of the cells was evaluated by Cell Counting Kit-8(CCK-8). In the other test, osteoblasts were cultured with blank control serum, and 10% serum containing XXD, Radix Dipsaci, and Rhizoma Drynariae, respectively, and then the ALP activity was examined by using ALP assay kit and the expression of BMP-2 was investigated by enzyme-linked immunosorbent assay(ELISA). Results XXD had a dose- and time-dependent effect on the proliferation of rat osteoblasts in a suitable volume fraction range from 5% to 20%, and the effect of XXD at 10% was the best. Compared with the blank control serum group, ALP activity was increased in the cells treated with 10% serum containing XXD, Radix Dipsaci, and Rhizoma Drynariae(P<0.01). On culturing day 7, the expression of BMP-2 was increased in 10% XXD group and Rhizoma Drynariae group(P<0.05). Conclusion XXD can increase the ALP activity and BMP-2 expression in the osteoblasts in vitro, so does the single herb of Rhizoma Drynariae. And their therapeutic mechanism in promoting the healing of fractures may be related with the enhancement of osteogenesis of osteoblasts.