辛伐他汀对大鼠BMSCs成骨诱导Wnt信号传导通路相关因子mRNA表达的影响

EFFECT OF SIMVASTATIN ON mRNA EXPRESSIONS OF SOME COMPONENTS OF Wnt SIGNALING PATHWAY IN DIFFERENTIATION PROCESS OF OSTEOBLASTS DERIVED FROM BMSCs OF RATS

目的探讨辛伐他汀(simvastatin,SIM)对大鼠BMSCs成骨分化过程中Wnt信号传导通路相关因子mRNA表达的影响,研究SIM促进BMSCs成骨分化的作用机制。方法取6周龄雌性SD大鼠双侧股骨、胫骨骨髓细胞,全骨髓培养法进行原代和传代培养。取第2代细胞换用成骨诱导培养基培养,并将细胞分为2组,实验组加入1×10-7mol/LSIM,对照组加入等量无水乙醇和PBS。诱导培养7d倒置相差显微镜观察两组细胞形态,并行ALP染色观察;培养28d行vonKossa染色观察ECM矿化;培养7、14d采用实时定量PCR检测Wnt信号传导通路相关因子Axin2、β-catenin、骨钙素(osteocalcin,OC)、frizzled-2、Lef-1、Wnt5amRNA的表达。结果诱导培养7d倒置相差显微镜观察显示,实验组以多边形和三角形细胞居多;对照组细胞多呈长梭形,少数为小圆形或三角形。诱导培养7dALP染色观察显示,实验组细胞质中出现蓝染的ALP,阳性染色细胞和区域明显多于对照组;培养28dvonKossa染色观察显示,两组细胞均可见点状及片状钙沉积,与对照组相比,实验组钙沉积明显增多。实时定量PCR检测显示,诱导培养7d,实验组Axin2mRNA表达水平显著低于对照组,frizzled-2、Lef-lmRNA表达水平显著高于对照组,差异均有统计学意义(P<0.05);其余各因子mRNA表达两组差异无统计学意义(P>0.05);培养14d,实验组除β-catenin外,其余各因子mRNA表达水平均显著高于对照组(P<0.05)。结论SIM可促进BMSCs向成骨细胞分化,并伴Wnt信号途径相关因子mRNA表达水平的改变。

Objective To confirm the stimulating effect of simvastatin on BMSCs of SD rats osteogenic differentiation, and to further study the role of Wnt signaling pathway in this process. Methods BMSCs derived from the tibia and femur of 6-week-old female SD rats were cultured in vitro. Two groups were established： control group and experimental group. After the 2nd passage, the cells of experimental group were treated with simvastatin （1 × 10^-7mol/L） and the cells of control group with absolute ethyl alcohol and PBS. ALP staining was used at 7 days and von Kossa staining was applied at 28 days to assess osteoblastic differentiation and mineralization. Real-time quantitative PCR was performed to evaluate the expressions of Axin2, β-catenin, osteocalcin （OC）, frizzled-2, Lef-1, and WntSa mRNA at 7 days and 14 days after simvastatin treatment. Results The observation of inverted phase contrast microscope showed that the majority of cells were polygonal and triangular in the experimental group, and were spindle-shaped in the control group at 7 days. The ALP staining showed blue cytoplasm, the positive cells for ALP staining in the experimental group were more than those in the control group at 7 days. The yon Kossa staining showed that mineralization of extracelluar matrix at 28 days in two groups, but the mineralization in the experimental group was more obvious than that in the control group. The expression of Axin2 mRNA was significantly lower, and frizzled-2, Lef-1 mRNA were significantly higher in the experimental group than in the control group （P 〈 0.05） at 7 days, while the mRNA expressions of Axin2, OC, frizzled-2, Lef-1, and Wnt5a were significantly higher in the experimental group than in the control group at 14 days （P 〈 0.05）. Conclusion Simvastatin can promote the osteogenic differentiation of BMSCs and change the expression of mRNA of some components of Wnt signaling pathway.