一种高效细胞永生化载体的构建及其功能分析

摘要

细胞永生化在基因功能验证、转基因动物制作、药物研发等领域发挥关键作用,然而由于细胞永生化难度大,所以到目前为止仅有少数种类的细胞被永生化.为了解决这一技术问题,本研究拟通过构建人端粒酶逆转录酶基因(human telomerase reverse transcriptase,hTERT)的慢病毒表达载体来提高原代细胞永生化效率,从而最终建立细胞系.本研究先用PCR的方法从载体pBABE-neo-hTERT上扩增获得hTERT基因片段,然后将其连接到慢病毒骨架载体pLV-puro上,最终获得目标载体pLV-puro-hTERT.为了验证这一载体的有效性,先将其包装成慢病毒颗粒,再对猪原代前体脂肪细胞进行侵染,然后用嘌呤霉素筛选获得阳性克隆,之后检测了阳性克隆的端粒酶的活性、细胞增殖和分化能力.研究结果表明,与对照组相比,pLV-puro-hTERT慢病毒侵染猪前体脂肪细胞,可使细胞端粒酶mRNA的表达水平上调12倍,并极显著的促进了猪前体脂肪细胞的增殖,同时不影响猪前体脂肪细胞的分化.本研究所构建的慢病毒表达载体能够有效的提高原代细胞的增殖能力,同时保持了原代细胞的生物学特性,这将为进一步永生化原代细胞提供便利.%Cell lines as a immortalized cell materials play an important role in exploring gene function,producing transgenic animals and discovering new drugs.However,because of difficulties in immortalizing primary cells,until now only a few types of cells were reported to be immortalized.In order to overcome this problem,we aimed to construct a recombination lentivirus expressing human telomerase reverse transcriptase to improve the efficiency of immortalization so as to establish cell lines.So,we cloned ORF of hTERT gene from the retrovirus vector of pBABE-neo-hTERT and inserted into pLV-puro vector to construct pLV-puro-hTERT.After that,lentivirus particals were successfully packaged and used to infect pig preadipocytes.Then,positive cell clones were selected by puromycin.Finally,we examined the expression of telomerase reverse transcriptase by Real-time PCR,and evaluated the ability of proliferation and differetiation by DAPI and Oil red O staining.The results showed that porcine preadipocytes infected with pLV-puro-hTERT lentivirus displayed a 12-fold increase in telomerase mRNA level compared to control(P＜0.01) with significant enhancement in cell proliferative activity,whilst no effects of lentivirus on porcine preadipocyte differentiation were observed.Taken together,these results indicate that the lentivirus vector can be used to promote primary cells proliferation,meanwhile it doesn't change primary cells' original biological characters,so it provides us a powerful tool to immortalize more primary cells in the future.