人端粒酶基因(hTERT)对牛耳成纤维细胞凋亡的影响  

Effect of hTERT gene transfection on bovine ear fibroblast apoptosis

作　　者：隋丽娜[1] 彭新荣[1] 王亚刚[1] 张涌[1] 

机构地区：[1]西北农林科技大学生物工程研究所,陕西杨凌712100

出　　处：《黑龙江畜牧兽医》2007年第10期17-19,共3页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家"863"高技术研究发展计划项目(2004AA213072)

摘　　要：为了提高体细胞核移植的效率,试验探讨了转染外源性hTERT基因对核移植供体细胞———牛耳成纤维细胞的影响。试验应用脂质体介导hTERT基因转染,用相差显微镜观察转染前后的细胞状态,用RT-PCR、W estern blot法检测hTERT基因是否整合并表达,用流式细胞仪检测转染前后细胞周期的变化和凋亡的情况,并进行核型分析。结果表明:转染48 h后能看到明显的荧光,经600μg/mLG418筛选后,获得阳性克隆,转染后的细胞形态上基本无变化,hTERT基因整合并表达;经流式细胞仪检测,转染前后生长周期无明显变化,凋亡率极显著降低(P<0.01),核型正常;转染hTERT基因后的牛耳成纤维细胞的凋亡率显著低于未转染的细胞,并且形态正常,表明转染hTERT后,使体外培养的牛耳成纤维细胞生命力得到延长,为核移植提供了数量更多的高质量的核供体细胞。To explore the effect of exogenous human telomerase reverse transcriptase （hTERT） on the donor cells of nuclear transfer bovine ear fibroblast, the plasmid of pEGFP - hTERT was transfeeted into bovine fibroblast with Lipofeetime 2 000. Integration and Expression of gene were detected by RT - PCR and Western blot methods. The cell apoptosis rate was detected by/low cytometer, doing karyotype analysis. The results showed that the positive clones were subcultored and expressed the hTERT protein after screening of 600 μg/mL G418. The morphological phenotype of the transfeeted cells was similar to that of the non - transfected cells. There was no difference about cell cycle between transfeeted and non - transfeeted fibroblasts, but the apoptosis rate of transfeeted fibroblasts were decreased compared with that of pre - transfection. The fibroblasts transfeeted by pEGFP - hTERT maintained the nomal diploid karyotype. The results suggested that the bovine ear fibroblasts transfected by pEGFP - hTERT can express the hTERT and prolong life span of culture, and the cell apoptosis rate significantly decreased. The transfected fibroblasts can provide a large number of nomal donor cells for nuclear transfer.

关 键 词：HTERT基因 牛耳成纤维细胞 转染 细胞凋亡 

分 类 号：S858.23[农业科学—临床兽医学]