miR-21诱导犬BMSCs骨向分化的体外实验研究

摘要

目的 体外研究检测miR-21诱导拉布拉多犬骨髓间充质干细胞(BMSCs)成骨分化的作用.方法 构建LentimiR-21-Luciferase与Lenti-LacZ-Luciferase慢病毒载体,分别转染犬BMSCs,将研究分为miR-21组与LacZ组.利用体外细胞划痕实验来比较转染后BMSCs的爬行迁移能力.在转染后的第0、1、4、7、14天分别提取miR-21组与LacZ组BM-SCs的mRNAs和蛋白.通过Real time-PCR技术和Westernblot检测成骨分化关键因子骨钙素(OCN)和骨桥蛋白(OPN)的表达.结果 Lenti-miR-21-Luciferase与Lenti-LacZ-Luciferase慢病毒载体成功转入BMSCs后,miR-21组的BMSCs细胞爬行迁移能力显著增强(P＜0.001);Real time-PCR和Western blot结果显示,miR-21可以显著上调BMSCs成骨分化关键因子OCN和OPN的表达(P＜0.05).结论 miR-21可以显著促进BMSCs的成骨分化,为进一步的体内实验奠定基础.%Objective To explore the effect of miR-21 on the osteogenic differentiation of Labrador dog bone marrow mesenchymal stem cells (BMSCs) in vitro.Methods Lenti-miR-21-Luciferase and Lenti-LacZ-Luciferase lentiviral vector were constructed and then were transfected into canine BMSCs.The study was divided into miR-21 group and LacZ group.The cell scratch test was used to compare the migration ability of BMSCs after lentiviral vector transfection.The mRNA and protein of BMSCs were extracted on 0,1,4,7,and 14 days.The expression of osteocalcin (OCN) and osteopontin (OPN),the key factors of osteogenic differentiation of BMSCs,were detected by Real time-PCR and Western blot.Results After Lenti-miR-21-Luciferase and Lenti-LacZ-Luciferase were successfully transfected into BMSCs,the migration of BMSCs in miR-21 group was enhanced significantly (P ＜ 0.001).Expression of OCN and OPN at the mRNA and protein levels were significantly increased (P ＜ 0.05).Conclusion MiR-21 can significantly promote the osteogenic differentiation of BMSCs,lay the foundation for further in vivo experiments.