骨特异性转录因子Runx2对抗空间骨丢失效应的初步研究

Bone-specific Transcription Factor Runx2 on the Role of Antagonistic Bone Loss in Space

为了构建稳定过表达Runx2(骨特异性转录因子)的C2C12(小鼠成肌细胞)和MG63(前成骨细胞)细胞株,并用于研究Runx2在对抗空间骨丢失效应中的作用.利用实时定量聚合酶链式反应鉴定Runx2下游基因I型胶原、碱性磷酸酶表达情况,在二维回转器中培养稳定细胞株,通过定量聚合酶链式反应,观察在模拟失重效应下Runx2基因对其下游基因表达的影响.结果表明,通过筛选获得稳定转染的C2C12-Runx2和MG63-Runx2细胞株,经鉴定都能过表达Runx2.转染后的细胞I型胶原和碱性磷酸酶mRNA表达增高.回转组与对照组相比,MG63,C2C12-Runx2,MG63-Runx2细胞的I型胶原和碱性磷酸酶mRNA表达降低,但在模拟失重效应下,转染细胞中I型胶原和碱性磷酸酶的mRNA表达下降程度明显低于末转染细胞株.所构建的C2C12-Runx2和MG63-Runx2细胞株比较稳定,并证实Runx2能部分对抗失重引起的成骨特异性分子的降低.

To construct a stable expression of Runx2 in C2C12, MG63 cells, and primarily investigate the role of Runx2 against bone loss induced by spaceflight. Myoblast C2C12 and pre-osteoblast MG63 was transfected with Runx2 and selected overexpressing cell clone with G418. The stably transfected C2C12, MG63 cell lines with Runx2 were identified by westernblot and named C2C12-Runx2 and MG63-Runx2 respectively. The expression of genes regulated by Runx2 were determined using real-time quantitative PCR. The stable cell lines and untransfected cell lines were cultured in clinostat to simulate mierogravity condition and detected the expression of Runx2 and its downstream genes by real-time quantitative PCR. C2Cl2-Runx2, MG63-Runx2 cell lines were gained which were over- expressing Runx2. The mRNA expression level of Alkaline Phosphatase （ALP） and Collal increased in C2Cl2-Runx2 and MG63-Runx2 cells. The mRNA expression of ALP and Collal decreased in the clinorotation group compared with the control group in MG63, C2Cl2-Runx2 and MG63-Runx2 cells. The decreased degree of ALP, Collal mRNA expression in both transfected cell lines was lower than in untransfected cell lines under simulated microgravity condition. C2Cl2-Runx2, MG63-Runx2 cell lines were successfully constructed. The data from clinorotation and real-time quantitative PCR suggest that Runx2 can partly antagonize the decreased expression of osteogenie specific molecules induced by simulated microgravity.