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目的:探讨补肾活血汤石油醚提取物对骨髓间充质干细胞(BMSCs)表达CCR2及其Wnt5a的影响。方法:索氏提取法提取补肾活血汤石油醚提取物;全骨髓贴壁筛选法培养BMSCs,取BMSCs第三代用于实验。石油醚部位按10,50,100μg/mL浓度梯度刺激BMSCs,利用免疫蛋白印迹法(Western blot)及其细胞免疫荧光检测CCR2蛋白表达情况,采用Western blot和qPCR检测Wnt5a蛋白和mRNA的表达。结果:Western blot和细胞免疫荧光结果显示石油醚提取物按照浓度梯度促进CCR2蛋白表达,以100μg/mL浓度最佳(P<0.01)。石油醚提取物同样促进Wnt5a蛋白及mRNA表达,其中Western blot结果亦以100μg/mL浓度最佳(P<0.01)。结论:补肾活血汤石油醚提取物亦能刺激MCP-1/CCR2信号轴,上调Wnt5a蛋白表达,初步发现迁移活性可能与Wnt5a通路相关。
Objective: To investigate the effect of petroleum ether extract from Bushen Huoxue decoction on the expression of CCR2 and Wnt5a in bone marrow mesenchymal stem cells (BMSCs). Methods: Soxhlet extraction method was used to extract petroleum ether extract from Bushen Huoxue Decoction. BMSCs were cultured with whole bone marrow adherent screening method. The third generation of BMSCs was used in the experiment. BMSCs were stimulated with petroleum ether (10,50,100μg / mL), and the expression of CCR2 protein was detected by Western blot and immunofluorescence. The expression of Wnt5a protein and mRNA was detected by Western blot and qPCR. Results: The results of Western blot and immunofluorescence showed that petroleum ether extract promoted the expression of CCR2 protein at a concentration gradient of 100 μg / mL (P <0.01). Petroleum ether extract also promoted the expression of Wnt5a protein and mRNA, and Western blot results also showed the best concentration of 100μg / mL (P <0.01). Conclusion: Bushenhuoxue Tang petroleum ether extract can stimulate MCP-1 / CCR2 signal axis and up-regulate the expression of Wnt5a protein. It is preliminarily found that the migration activity may be related to Wnt5a pathway.