论文部分内容阅读
目的人和小鼠正常体细胞端粒酶的活性，存在着种属差异性．肿瘤细胞中端粒酶活性的变化是否亦存在种属差异性尚不清楚．因此，我们在人和小鼠的肿瘤细胞系中，分别转导细胞因子基因ＩＬ２和（或）ＴＮＦα，以观察其对肿瘤细胞增殖活性及端粒酶活性的影响．方法首先将目的基因ＩＬ２和（或）ＴＮＦα构建入ｐＬＸＳＮ或ｐＧＣＥＮ逆转录病毒载体，用脂质体法转染ＰＡ３１７细胞进行病毒包装，再以病毒颗粒分别感染ＭＫＮ２８，ＭＫＮ４５，Ｔｃａ８１１３及Ｈ２２细胞，后者经Ｇ４１８筛选、挑克隆及扩增培养，然后用ＰＣＲ，ＲＴＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ鉴定其外源基因的整合及表达，并制作细胞生长曲线，以观察肿瘤细胞增殖活性变化，同时用ＴＲＡＰＰＣＲ法观察转基因肿瘤细胞端粒酶活性变化．结果人ＭＫＮ２８，ＭＫＮ４５及Ｔｃａ８１１３转导ＩＬ２或ＴＮＦα后，肿瘤细胞增殖受到不同程度的抑制；相应地，其端粒酶活性也受到抑制．而小鼠Ｈ２２细胞转导ＩＬ２和（或）ＴＮＦα后，增殖未受抑制，端粒酶活性亦未受抑制．结论人和小鼠肿瘤细胞系中转细胞因子基因ＩＬ２和（或）ＴＮＦα后，端粒酶活性及肿瘤细胞增殖发生不同的变化，提示肿瘤细胞中端粒酶的作用机制，在人和小?
1. The purpose of human and mouse normal somatic cell telomerase activity, there are species differences. It is still unclear whether the variation of telomerase activity in tumor cells also exists. Therefore, we transduced the cytokine gene IL2 and/or TNFα in human and mouse tumor cell lines to observe its effect on tumor cell proliferation activity and telomerase activity. METHODS: The target genes IL2 and/or TNFα were first constructed into pLXSN or pGCEN retroviral vectors, transfected with PA317 cells by liposome method for viral packaging, and then infected with MKN28, MKN45, Tca8113 and H22 cells by viral particles. After screening by G418, picking out the clones and expanding the culture, PCR, RT-PCR and Southernbloting were used to identify the integration and expression of the foreign genes, and cell growth curves were made to observe the changes in the proliferation activity of tumor cells. Meanwhile, TRAP-PCR was used. Methods To observe the change of telomerase activity in transgenic tumor cells. Results After transfection of IL2 or TNFα by human MKN28, MKN45 and Tca8113, the proliferation of tumor cells was inhibited to varying degrees; accordingly, the telomerase activity was also inhibited. However, after transduction of IL2 and/or TNFα in mouse H22 cells, proliferation was not inhibited and telomerase activity was not inhibited. Conclusion The changes of telomerase activity and tumor cell proliferation in human and mouse tumor cell lines after transduction of cytokine gene IL2 and/or TNFα suggest that the mechanism of telomerase activity in tumor cells is both in human and small?