小鼠11β-HSD1基因过表达的前成骨细胞系的建立
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国家自然科学基金青年基金项目(30900505)


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

    Abstract:

    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 293FT 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 osteogenic differentiation was detected using Alizarin red staining. Results:The lentiviral vector of mouse 11β-HSD1 overexpression was successfully constructed,and the MC3T3-E1 preosteoblast cell lines were infected efficiently by the lentivirus. The mRNA and protein levels of 11β-HSD1 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.

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祁寒梅,吴 琳,王 龙,毕建华,丁国宪.小鼠11β-HSD1基因过表达的前成骨细胞系的建立[J].南京医科大学学报(自然科学版),2012,(5):626-630

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  • 收稿日期:2011-12-02
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