hTERT启动子调控的HSV-tk基因逆转录病毒载体的构建及SKOV3转染细胞的生物学特性观察

Changes of biological character of SKOV3 cells transfected with HSV-tk gene under the control of hTERT promoter

目的 :观察转染有人端粒酶逆转录酶基因 (humantelomerasereversetranscriptase,hTERT)启动子调控的单纯疱疹病毒Ⅰ型胸苷激酶 (herpessimplexvirusthymidinekinase ,HSV tk)基因的人卵巢癌细胞的部分生物学特性。方法 :构建hTERT启动子调控的HSV tk基因重组逆转录病毒载体 ,筛选携带该载体的包装细胞 ,测定包装细胞产生的逆转录病毒滴度 ,并用病毒颗粒感染人卵巢癌SKOV3细胞 (SKOV3/tk细胞 )并经RT PCR方法鉴定。然后倒置显微镜下观察SKOV3/tk细胞形态 ,MTT法研究细胞生长特性 ,并观察转染细胞裸鼠体内的成瘤能力。结果 :包装细胞产生的病毒滴度可达 2× 1 0 3 cfu·ml-1 。SKOV3/tk细胞扩增出 770bp的tk基因片段 ,形态与SKOV3细胞无明显差异 ;倍增时间 72h ,无明显变化 ;裸鼠体内的成瘤能力较SKOV3细胞下降。结论 :hTERT调控的HSV tk基因逆转录病毒载体构建成功 ;

Aim: To identify the difference between transfected human ovarian cancer cells with HSV tk gene under the control of hTERT promoter and non transfected cells. Methods: The first step was to select packaging cells with high retrovirus titer by G418 . The human ovarian cancer cell line SKOV3 was transfected with the recombinant retrovirus containing HSV tk gene. Positive clones were identified by RT PCR and then the growth rate, phenotype, and heterotransplantative tumourigenecity were observed, respectively. Results: The positive packaging cells were obtained with the titer of 2×10 3 cfu·ml -1 . 770 bp fragment was amplified by RT PCR from the selected cells. The growth rate and phenotype of transfected cells were almost the same as the wild type cells. The heterotransplantative tumourigenecity and the growth rate of the tumour in nude mice decreased compared with non transfected cells. Conclusion: The heterotransplantative tumourigenecity of transfected human ovarian cancer cells with HSV tk gene under the control of hTERT promoter decreased.