补肾健脾活血方干预沉默DKK1、Sost骨细胞对细胞活性和相关蛋白的影响研究

The effect of Bushen Jianpi Huoxue Recipe on cell activity and related proteins in DKK1 or Sost silencing osteoblasts

目的用补肾健脾活血方干预沉默Dickkopf(DKK)1、骨硬化蛋白Sclerostin(Sost)转染的成骨细胞,观察其对细胞活性和相关蛋白的影响。方法将培养好的细胞分为空白对照组(NC组)、沉默DKK1(Ad-sh DKK1)组和沉默Sost(Ad-sh Sost)组,空载腺病毒转染NC组,沉默DKK1和Sost重组腺病毒转染其余两组,再分别给予空白血清和含药血清干预,观察细胞增殖、碱性磷酸酶(alkaline phosphatase,ALP)活性以及骨相关蛋白变化情况。结果 (1)三组含药血清干预后的细胞活性强于空白血清干预后的细胞活性。与空白血清干预的三组比较,含药血清干预后的三组细胞活性在24、48、72 h均增加,尤其在72 h时含药血清对Ad-sh DKK1组的细胞活性作用更强,与同组空白血清比较,差异有统计学意义(P<0.05);(2)三组含药血清干预与空白血清干预比较ALP活性明显升高,差异有统计学意义(P<0.01)。含药血清干预后,与NC组比较,其余两组ALP活性显著升高,差异有统计学意义(P<0.01),Ad-sh DKK1组和Ad-sh Sost组比较,ALP活性升高不明显;(3)与空白血清干预比较,含药血清干预后三组的结缔组织生长因子(connective tissue growth factor,CTGF)、骨保护素(osteoprotegerin,OPG)、骨桥蛋白(osteopontin,OPN)均升高,与NC组比较,Ad-sh DKK1组OPG、OPN差异有统计学意义(P<0.05和P<0.01),Ad-sh Sost组CTGF、OPG、OPN差异有统计学意义(P<0.05)。结论补肾健脾活血方含药血清可显著促进沉默DKK1、Sost干预后细胞增殖和提高ALP活性,上调沉默DKK1、Sost腺病毒转染细胞CTGF、OPG、OPN的表达。

Objective To observe the effect of Bushen Jianpi Huoxue Recipe on cell activity and related proteins expression in Dickkopf（DKK1） or Sclerostin（Sost） silencing osteoblasts. Methods Cultured cells were divided into blank control group（NC group）,DKK1 silencing（Ad-shDKK1） group and Sost silencing（Ad-shSost） group. NC group was transfected with empty adenovirus,Ad-shDKK1 group and Ad-shSost group were transfected with silencing DKK1 and silencing Sost recombinant adenovirus vector,respectively. The three groups were cultured with blank serum and medicated serum,respectively. Cell proliferation,alkaline phosphatase（ALP） activity,and the expression of related proteins were observed. Results（1）Cell activity of the three groups of medicated serum was higher than that of blank serum. Compared with blank serum,cell activity of the three groups of medicated serum increased at 24,48 and 72 hours,respectively,especially the cell activity in the Ad-shDKK1 group was strongest at 72 hour compared with blank serum in the same group,and the difference was statistically significant（P〈0. 05）.（2） ALP activity of the three groups of medicated serum was significantly higher than blank serum,and the differences werestatistically significant（P〈0. 01）. Compared with the NC group,medicated serum could significantly increase ALP activity in the Ad-shDKK1 group and Ad-shSost group（P〈0. 01）. Comparing between the Ad-shDKK1 group and Ad-shSost group,ALP activity was not significantly different.（3） Compared with the blank serum,medicated serum could up-regulate CTGF,OPG and OPN expression. Compared with the NC group,the expression of OPG and OPN in the Ad-shDKK1 group was significantly increased（P〈0. 05 and P〈0. 01）,and the expression of CTGF,OPG and OPN in the Ad-shSost group was significantly increased（P〈0. 05 and P〈0. 01）. Conclusion The serum containing Bushen Jianpi Huoxue Recipe could improve the cell proliferation and ALP activity,and up-regulate the expression of CTGF,OPG and OPN in DKK1 and Sost silencing osteoblasts.