转hTERT基因的山羊胎儿成纤维细胞的生物学特性及其表达分析

用脂质体介导法将hTERT基因转染到山羊胎儿成纤维细胞中,以期获得永生化的山羊胎儿成纤维细胞系。对转染后的阳性细胞进行细胞学和分子学相关检测。结果显示,导入hTERT的阳性细胞形态正常,并已传至第58代;未转染组细胞经长期传代后（30代）,增殖缓慢,部分细胞表现衰老和凋亡的迹象。RT-PCR检测转染组细胞中有hTERT基因表达。生长曲线绘制结果显示转染组（30代和50代）山羊胎儿成纤维细胞生长速度稍快于未转染组（5代）细胞,但差异不显著（P〉0.05）。转染组细胞生长旺盛,细胞接种后第6天,基本覆盖培养孔。而未转染组（30代）细胞一直生长缓慢,差异显著（P〈0.05）。未转染组（30代）山羊胎儿成纤维细胞处于S期细胞和G1期细胞分别为28.9%和60.8%,而转染组（30代）分别为45.2%和45.0%。说明外源性hTERT基因可促进山羊胎儿成纤维细胞由G1期向S期转变,并提高细胞增殖能力。转染组（50代）山羊胎儿成纤维细胞染色体核型正常,未发生变化。未转染组（30代）细胞凋亡率和死亡率分别为39.7%和29.4%,而转染组（30代）分别为11.0%和12.7%,差异极显著（P〈0.01）。hTERT蛋白在转染组（30代）山羊胎儿成纤维细胞中表达,蛋白相对分子质量为127 000。说明外源性hTERT基因可以延长胎儿成纤维细胞在体外培养的寿命,降低细胞凋亡率。

Biological specificity and expression analysis of goat fetal fibroblast cell transfected hTERT gene

In this study,hTERT gene was transfected into goat fetal fibroblast cell by Lipofection 2000,and we obtained the immortalized goat fetal fibroblast cell lines.The experimental results obtained were as follows： transfected cells maintain to be normal shape,and they have been cultured to passage 58 by now.Negative cells cultured after 30th generation grew slowly.and displayed apoptosis phenomena.RT-PCR shows that expression of hTERT gene can be detected in positive cells.The cell cycles indicate positive cells（30Th generation and 50Th generation） grew a little faster than negative cells（5Th generation）,but there was not significant difference（P〉0.05）.However,negative cells（30Th generation） show a slow growth tendency and there is a significant differences with positive cells（P〈0.05）.For negative cells group（30Th generation）,the percentage of cells in S phase is 28.9%,in G1 phase is 60.8%,while for positive cells group（30Th generation）,the percentage is 45.2% and 45.0%,respectively.It indicates that hTERT gene promoted the goat fetal fibroblast cells transforming from G1 phase to S phase and improved the ability of cell proliferation.Positive cells（50Th generations） have normal karyotype.The apoptosis rate and death rate of negative cells group（30Th generations） are 39.7% and 29.4%.While the apoptosis rate and death rate for positive cells group（30Th generations） are 11.0% and 12.7%,respectively（P〈0.01）.hTERT protein is expressed in transfected cells（30 generations）,protein size 127 000.The results remind that hTERT gene can extend the life span of fetal fibroblast cells in vitro and reduce cell apoptosis.