Antagomir-30d对大鼠骨髓基质干细胞成骨分化的影响

目的：探讨miR-30d拮抗剂（antagomir-30d）正性调控大鼠骨髓基质干细胞（BMSCs）的成骨分化作用并确认转染的最佳浓度。方法：BMSCs培养并分组，分为不同浓度的antagomir-30d（antagomir-30d组）、阴性对照（NC组）和未做转染的BMSCs（空白组）；将不同浓度的antagomir-30d及其NC转染至BMSCs后进行成骨诱导。通过RT-PCR技术进行比较，测定成骨基因碱性磷酸酶（ALP）、骨钙素（OC）、Runt相关转录因子2（RUNX2）的mRNA的表达情况，并确定最佳转染浓度及其转染效率。通过ALP染色验证antagomir-30d对成骨分化的调控作用。结果：RT-PCR结果表明，antagomir-30d体外可以促进BMSCs的成骨分化。不同浓度antagomir-30d组的成骨基因表达量随浓度变化而改变，当antagomir-30d转染浓度为150 nmol/L时，ALP、OC、RUNX2的mRNA水平均显著高于空白组和NC组（P＜0.05）。Antagomir-30d的转染最佳浓度为150 nmol/L，此时ALP染色结果显示其活性最高，成骨分化作用好。结论：Antagomir-30d体外正性调控BMSCs的成骨分化，其转染至BMSCs的最佳浓度为150 nmol/L。

The effect of antagomir-30d on osteogenic differentiation of bone mesenchymal stem cells

Objective: To verify the upregulating effect of antagomir-30d (miR-30d) on osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and to confirm the optimum transfection concentration. Methods: Antagomir-30d of different concentrations was divided into antagomir-30d (antagomir-30d group), negative control (NC group) and untransfected BMSCs (blank group). Transfecting different concentrations of antagomir-30d/NC/blank to BMSCs, gene expression of osteoblast marker genes (ALP, OC and RUNX2) was analyzed using RT-PCR and then optimum transfection concentration and transfection efficiency were confirmed. The role of antagomir-30d in regulating osteogenesis was verified by ALP staining. Results: The results of RT-PCR showed that antagomir-30d enhanced osteogenic differentiation of BMSCs in vitro. The transfection effect changed according to transfection concentration. When transfected with 150 nmol/L, the expression of ALP, OC and RUNX2 in the antagomir-30d group was significantly higher than the other groups. Meanwhile, there was no significant change in the proliferation and apoptosis of BMSCs when transfected with 150 nmol/L. Therefore, 150 nmol/L proved to be optimum ALP staining showed ALP activity of antagomir-30d group was significantly upregulated. Conclusion: The antagonist of miR-30d (antagomir-30d) promotes osteogenic differentiation of BMSCs in vitro and 150 nmol/L is the optimum transfected concentration.