补肾活血汤石油醚提取物对BMSCs迁移过程中Wnt5a/PKC通路的影响

摘要

目的 探讨补肾活血汤(熟地黄、菟丝子、补骨脂,等)石油醚提取物对大鼠骨髓间充质干细胞(BMSCs)的迁移作用及相关机制.方法 全骨髓贴壁法培养BMSCs,取第3代细胞用于实验,采用流式细胞仪检测细胞表面抗原,细胞划痕、Transwell实验观察补肾活血汤石油醚提取物0、25、50、100、200μg/mL剂量组的细胞迁移能力,Western blot和RT-PCR检测Wnt5a、蛋白激酶C(PKC)表达情况.结果 第3代BMSCs表面抗原CD29、CD90、CD44表达阳性,而CD45表达阴性;细胞划痕6h及12h后,药物各剂量组细胞迁移速度明显高于空白组(P＜0.05,P ＜0.01);Transwell实验中100 μg/mL补肾活血汤石油醚提取物为促细胞迁移最佳剂量,差异有统计学意义(P＜0.01);RT-PCR结果显示与空白对照组比较,药物各剂量组的Wnt5a mRNA水平均升高(P＜O.05,P＜0.01),而与空白对照组的PKC mRNA水平比较,药物只有100μg/mL剂量mRNA表达水平升高(P＜0.01);Western blot结果表明药物组各剂量的Wnt5a、PKC蛋白表达较空白对照组都明显升高(P＜0.01),均且以100 μg/mL剂量最高(P＜0.01).结论 补肾活血汤石油醚提取物可以促进BMSCs体外迁移,其作用机制可能与Wnt5a/PKC信号通路有关.%AIM To investigate the effect and mechanism of petroleum ether extract from Bushen Huoxue Decoction (BSHXD,Rehmanniae Radix Praeparata,Cuscutae Semen,Psoraleae Fructus,etc.) on rat BMSCs migration.METHODS Bone marrow mesenchymal stem cells isolated from whole bone marrow of rats were amplified by adherent culture in vitro.Flow cytometry was employed to detect the cell surface antigens of the third generation cells.Migration examination of cells treated with petroleum ether extract from BSHXD at concentrations of 25,50,100 and 200 μg/mL was accomplished by cell scratch test and Transwell assay.Expressions of WntSa and PKC protein were traced by Western blot,and so were the mRNA expressions of Wnt5a and PKC by RT-PCR.RESULTS Positive expressions of CD29,CD44 and CD90 in the passage 3 of BMSCs but negative expression of CD45 were observed.The cells given different concentrations of petroleum ether extract from BSHXD groups displayed a significantly higher wound healing speed than the control group after 6 h and 12 h cell scratch (P ＜ 0.05).100 μg/mL BSHXD was shown to be the most active concentration for promoting cell migration by Transwell migration experiments,with a statistically significant difference (P ＜ O.O1).Compared with the control group,the levels of Wnt5a mRNA in the BSHXD groups were significantly higher (P ＜ 0.05),and the PKC mRNA level in 100 μg/mL petroleum ether extract from BSHXD group was significantly increased as well (P ＜0.01)by RT-PCR.Although BSHXD groups brought forth significant improvement in both the expressions of Wnt5a and PKC protein (P ＜ 0.01),the 100 μg/mL concentration conferred the highest (P ＜ 0.01).CONCLUSION Petroleum ether extract from BSHXD can promote BMSCs migration in vitro,and the mechanism may lie in its association with WntSa signaling pathway.