论文部分内容阅读
目的 构建稳定表达由60SE2启动子驱动的增强型绿色荧光蛋白(EGFP)的MG63细胞株,并筛选出能反映Cbfal活性的细胞株.方法 合成60SE2启动子的序列,并将其构建到T载体中:然后通过双酶切得到启动子片段;与去除CMV启动子的报告基因载体pEGFP-Nl的酶切片段连接,构建真核表达载体;并转染到MG63细胞系,经G418筛选得到了稳定转染60SE2-EGFP基因的MG63细胞株.用不同浓度的IGF-I和VD,处理,通过EGFP荧光强度分析,ALP活性测定,筛选出能够响应IGF-I和VD3的细胞株.结果 构建了pUC-60SE2扩增载体和p60SE2-EGFP真核表达载体,通过稳定转染以及IGF-I、VD_3筛选获得一株荧光强度随IGF-I和VD_3处理而增强的细胞株.OSE-MG63.结论 构建了能反应Cbfal活性的成骨细胞模型,为进一步研究微重力对Cbfal的转录活性及信号传导途径的影响奠定了基础.“,”Objective To obtain MG63 cell lines which are stably transfected with enhanced green fluorescent protein(EGFP) reporter gene drived by 6OSE2 promoter and select the cell strain which can reflect Cbfal activity by EGFP fluorescence. Methods 6OSE2 promoter was synthesized and cloned into pUC57-T vector. The 140 bp promoter fragment inserted into deleted CMV-promoter pEGFP-Nl vector to construct eukaryotic expression vector. MG63 cell was transfected with this vector by Lipofectamine 2000 and stably selected by G418. Analysed the EGFP fluorescence intensity of the stably transfected MG63 cell stain and detection of ALP activity after treatment with different concentration of IGF-I or VD_3 ,then selected one which could reflecting Cbfal activity. Results The pUC-6OSE2 vector and p6OSE2-EGFP expression vector were constructed. Through stably transfection and selection, one OSE-MG63 cell strain was gained,which fluorescence intensity could reflect the treatment of IGF-I or VD_3. Conclusion The osteoblastic model which can reflect Cbfal activity is established successfully. This cell line will be useful for studying the effects of microgravity on the activity of Cbfal and the signal pathways related with bone form.