核心结合因子α1对骨髓间充质干细胞成骨细胞标志基因表达的影响

EFFECT OF CORE-BINDING FACTOR α1 ON THE EXPRESSION OF OSTEOBLAST GENE MARKER MESENCHYMAL STEM CELLS

目的探讨核心结合因子α1(core-binding factor α1,Cbfa1)对骨髓间充质干细胞(mesenchymal stem cells,MSCs)成骨定向分化的作用. 方法抽取3月龄日本大白兔股骨骨髓,密度梯度离心获得MSCs,以Cbfa1重组腺病毒转染第3代MSCs,3 d后免疫荧光法观察目的基因的表达,并于1～7 d MTT法检测其对细胞增殖的影响(Cbfa1腺病毒处理组).以正常培养组、单纯诱导组和对照腺病毒处理组作为对照,各组设置7、14 d两个时间点进行观察.RT-PCR法半定量分析Cbfa1腺病毒处理对MSCs碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OC)、骨桥蛋白(osteopontin,OPN)和I型胶原等多种成骨细胞标志基因的影响,同时观察Cbfa1对MSCs成脂、成肌分化指标过氧化物酶体增殖物激活受体γ2和生肌决定因子的影响. 结果①免疫荧光显示,转染Cbfa1重组腺病毒后MSCs可以表达Cbfa1;②各组MSCs增殖差异无统计学意义(P>0.05);③RT-PCR显示7、14 d,Cbfa1腺病毒处理组ALP、OC和OPN的表达均明显上调,Ⅰ型胶原的表达基本不变;④Cofa1使PPARγ、MyoD的表达受到抑制. 结论 Cbfa1能明显促进MSCs成骨方向的分化.

Objective To study the effect of core binding factor α1 （Cbfal） on the mesenchymal stem cells （MSCs） osteohlastic differentiation. Methods The MSCs were isolated from Japan white rabbits and cultured in vitro. The ;3rd generation MSCs were infected with Cbfal recombinant adenovirus. The expression of Cbfal was detected by immunofluoresccnce after being infected for 3 days and the proliferation was estimated by MTT method from the 1st day to the 7th day. Then the MSCs were divided into four groups： the commonly cultured group, the simply induced group, the control adenovirus treatment group, and the Cbfal adenovirus treatment group. The expressions of mRNA for a various of osteohlasl genc markers such as alkaline phosphatase, osteocalcin, osteopontin and type Ⅰ collagen were analyzed based on reverse transcriptase polymerase chain reaction （RT PCR）. The change of adipose and myoblastic differentiation gent marker PPARγ2 and MyoD expression were detected by RT PCR respectively. Results Positive staining of Cbfal was found in the MSCs infected with Cbfal adenovirus, and there was no significant difference in cell proliferation among the experimental groups（P〉0. 05）. The RT-PCR indicated that all the osteoblast gene markers exccpt type Ⅰ collagen were up- regublted in the Cbfal adenovirus treatment group. In contrast, the expressions of PPARγ2 and MyoI） were restrained. Conclusion Cbfal can directly promote the differentiation of MSCs into ostcoblasts.