h1-calponin基因对五指山小型猪骨髓间充质干细胞体外成骨分化的影响

Effect of h1-Calponin Gene on in Vitro Osteogenesis Differentiation of Bone Marrow Mesenchymal Stem Cells of Wuzhishan Minipig

【目的】通过碱性调宁蛋白(h1-calponin)基因与五指山小型猪骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM MSCs)成骨分化关系的研究,探讨成骨分化机制。【方法】添加成骨分化液对BM MSCs进行成骨分化诱导,采用茜素红染色的方法鉴定细胞成骨分化的程度;通过定量PCR分析h1-calponin基因及成骨相关基因骨桥蛋白(osteopotin,OPN)、骨钙蛋白(osteocalcin,OCN)、核心结合因子α1(core binding factor a1,Cbfα1)及核心结合因子β(core binding factor beta,Cbfβ)表达谱。【结果】①随着诱导时间的延长,茜素红染色矿化结节逐渐增多;②成骨标志基因OPN和OCN表达趋势一致:诱导0至14 d,基因表达量呈增长趋势,诱导14 d表达量最高,而后下降;成骨相关基因Cbfα1和Cbfβ表达量于第7天达到最高峰,之后逐渐下降,但仍高于诱导前;③h1-calponin基因表达量随着诱导的延续逐渐下降;诱导14—21 d时,该基因基本不表达。【结论】h1-calponin基因的表达与五指山小型猪骨髓间充质干细胞体外成骨分化存在着负相关,可能是成骨分化的负调控基因。

[Objective] The objective of the present study is to explore the relationship between hl-calponin gene and osteoblast differentiation of Wuzhishan minipig＇s bone marrow mesenchymal stem cells （BM MSCs） and further understand the mechanism of osteoblast differentiation. [Method] BM MSCs were induced to osteoblast differentiation by adding osteogenesis differentiation medium. Alizarin Red staining was used to indentify the degree of osteoblast differentiation of BM MSCs. The expression profile of hl-calponin gene and the osteo-related genes osteopontin （OPN）, osteocalcin （OCN）, core binding factor al （Cbfal） and core binding factor 13 （Cbfβ） were analyzed by quantitative PCR. [Result] Using Alizarin Red staining, it was found that the nodules of cells were increasing as the induction time went on. Q-PCR results showed that the expression patterns of OPN and OCN （two marker genes of osteogesis） were the same. Both OPN and OCN genes increased from day 0 to day 14, peaked at day 14 and finally declined. The osteo-related genes, Cbfal and Cbfβ, were highestly expressed at day 7 and then decreased gradually, but still higher than pre-induction. The expression of hl-calponin gene was gradually decreased during the induction and was nearly undetected from day 14 to 21. [Conclusion] Results of the experiment suggested there is a negative correlation between the expression of hl-calponin gene and osteoblast differentiation of Wuzhishan minipig＇s BM MSCs is in vitro. The hl-calponin genemay be a negative-regulated gene during osteoblast differentiation.