小鼠11β-HSD1基因过表达的前成骨细胞系的建立  

Establishment of mouse 11β-HSD1 gene overexpression model in a MC3T3-E1 preosteoblast cell lines

作　　者：祁寒梅[1] 吴琳[1] 王龙[1] 毕建华[1] 丁国宪[1] 

机构地区：[1]南京医科大学第一附属医院老年医学科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2012年第5期626-630,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金青年基金项目(30900505)

摘　　要：目的:探索小鼠11β-HSD1基因过表达的前成骨细胞系的建立方法。方法:构建过表达小鼠11β-HSD1基因的慢病毒载体,包装、收集、纯化慢病毒,感染小鼠MC3T3-E1前成骨细胞系,用BSD(一种核苷抗生素)筛选出感染成功的细胞,荧光定量PCR及Western blot检测11β-HSD1过表达情况,诱导成骨分化后用茜素红染色法染色矿化结节。结果:成功构建了小鼠11β-HSD1基因过表达慢病毒载体,高效感染MC3T3-E1细胞系。荧光定量PCR及Western blot结果显示,11β-HSD1过表达病毒组细胞11β-HSD1 mRNA水平是空载病毒对照组的17.4倍,蛋白表达量比空载病毒对照组增加。茜素红染色结果表明慢病毒感染的细胞成骨分化良好,细胞正常成骨分化功能未受影响,11β-HSD1过表达细胞成骨分化减少。结论:本研究成功建立了过表达小鼠11β-HSD1的前成骨细胞系,为研究11β-HSD1对成骨细胞的具体作用提供了研究基础。Objective：To establish a mouse preosteoblast cell lines overexpressing 11β-HSD1 gene. Methods：A lentiviral vector overexpressing mouse 11β-HSD1 was constructed. Lentivirus particles were generated in 293Vr cells, followed by concentration and purification. MC3T3-E1 preosteoblast cells were infected by the lentivirus,which were later selected by BSD. The mRNA and protein expression of 11β-HSD1 was analyzed by real-time PCR and Western blot, respectively. MC3T3,E1 0steogenic differentiation was de- tected using Alizarin red staining. Results：The lentiviral vector of mouse 11β-HSD 1 overexpression was successfully constructed, and the MC3T3-E1 preosteoblast cell lines Were infected efficiently by the lentivirus. The mRNA and protein levels of 11βHSD1 were higher in MC3T3-E1 cells infected by 11β-HSD1 overexpression lentivirus than that of cells infected by the negative control lentivirus. Alizarin red staining showed that the differentiation ability toward osteoblasts of MC3T3-E1 cells was not influenced by lentivirus infection. Conclusion： Our findings showed that 11β-HSD1 gene was effectively overexpressed in MC3T3-E1 preosteoblast cells using lentivirus infection,which provides a useful tool for further studies of 11β-HSD1 function in osteoblasts.

关 键 词：慢病毒载体 11β-HSD1 MC3T3-E1前成骨细胞系 

分 类 号：Q786[生物学—分子生物学]