中文摘要：

目的：利用原代人胚肾上皮细胞转化模型阐明端粒酶在细胞癌变过程中的作用。方法：用逆转录病毒介导的基因导入法，使人端粒酶催化亚基（human telomerase catalytic subunit，hTERT）、H—Ras癌基因、猿猴病毒40（simian virus 40，SV40）编码的早期抗原在原代人胚肾上皮细胞中稳定地表达，观察细胞的生长特性、染色体畸变率以及细胞转化特征。结果：端粒酶的激活虽然能延长细胞的寿命，但不能使细胞永生化。如果细胞同时表达端粒酶和SV40编码的大T抗原（large Tantigen，LT），细胞就能获得永生化。在此永生化细胞株中导入H—Ras癌基因以及SV40编码的小T抗原（small Tantigen，ST），细胞发生恶性转化，表现为在软琼脂上形成克隆并在裸鼠皮下形成肿瘤。与原代细胞相比，永生化细胞株的染色体畸变率无改变，而恶性转化细胞的畸变率明显增加。此外在转化细胞和几种肿瘤细胞中表达阻抑人端粒酶的特异性siRNA，能显著地抑制肿瘤细胞的生长、诱导细胞凋亡并减少软琼脂上形成的克隆数目。结论：细胞永生化是癌变过程的必要阶段，端粒酶的激活不仅是细胞永生化的重要环节，而且在维持肿瘤细胞生长中起重要的作用。

英文摘要：

Objective：To elucidate the role of telomerase in the process of tumorigenesis using the transformation model of human primary embryonic kidney epithelial （HEK）cells. Methods：Primary HEK cells were transfected with recombinant retrovirus containing cDNA encoding human telomerase reverse transcriptase enzyme （hTERT）, H-ras or Simian virus 40 （SV40） early antigens, respectively. Cell clones stably expressing hTERT, H-ras,SV40 T（LT） or T（ST） antigen were screened and expanded. Cell proliferation, chromosomal aberration, and morphological phenotypes in cell transformation were observed. Results：Activation of telomerase prolonged cell life span but could not immortalize HEK cells. Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HEK cells in vitro. Introduction of oncogenic H-ras and SV40-ST into immortal HEK cells induced malignant cell transformation, as shown by increased colony number in soft agar and subcutaneous tumor formation in nude mice. Compared with primary cells,chromosomal aberration rate had no significant change in immortal cells but significantly increased in malignantly transformed cells. Moreover, expression of a small hairpin RNAi （shRNA） targeted hTERT in transformed cells or tumor cell lines significantly inhibited cell growth, induced apoptosis, and reduced the colony number formed in soft agar. Conclusion：Cell immortalization is a critical stage in tumorigenesis. Activation of telomerase is not only a crucial step in cell immortalization, but also plays an important role in maintaining the growth of tumor cells.