刺头复叶耳蕨总黄酮促进脐带间充质干细胞成骨分化

摘要

目的 研究刺头复叶耳蕨总黄酮(total flavonoids from arachniodes exilis,TFAE)在人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)成骨分化中的作用.方法 采用组织块贴壁法分离培养hUCMSCs,取P3代细胞进行实验,不同浓度的TFAE处理hUCMSCs,CCK-8法检测细胞活性;AMP法测定碱性磷酸酶(ALP)活力,茜素红染色检测钙结节形成;RT-PCR检测成骨相关基因I型胶原酶a1(collagen type I alpha1, Col1a1)、骨桥蛋白(osteopotin, OPN)、Runx2、Osterix(Osx)mRNA表达水平,Western blot检测Col1a1、OPN蛋白表达水平.结果 在一定浓度范围内,TFAE(1、5 mg·L-1)促进细胞增殖;钙结节数量增多;ALP活力增强,成骨相关基因Col1a1、OPN、Runx2、OsxmRNA及Col1a1、OPN蛋白表达水平上调.结论 一定浓度的TFAE促进hUCMSCs增殖及成骨分化.%Aim To study the role of the total flavonoids from arachniodes exilis(TFAE) in osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCMSCs).Methods hUCMSCs were isolated and cultured by tissue explants adherent method, hUCMSCs at passage 3 were used to do the experiment, hUCMSCs were treated by different concentrations of TFAE, and cellular viability was measured by CCK 8 assay;alkaline phosphatase(ALP) activity was tested by AMP method;calcium nodule formation was detected by alizarin red staining;expression level of osteogenesis-related gene type I collagen enzyme a1(collagen type Ia1, Col1a1), osteopontin(OPN), Runx2, Osterix(Osx) mRNA was measured by RT-PCR;expression level of Col1a1,OPN protein was measured by Western blot.Results Certain concentration of TFAE(1 mg·L-1, 5 mg·L-1) promoted cellular proliferation, calcium nodules were more, ALP activity was enhanced;expression level of osteogenesis-related gene Col1a1, OPN, Runx2, OsxmRNA and Col1a1, OPN protein was up-regulated.Conculsion A certain concentration of TFAE promotes proliferation and osteogenic differentiation of hUCMSCs.