hREV3基因表达被反义阻断的人胚肾细胞系的建立及其某些生物学特性观察

目的:建立hREV3基因表达反义阻断的细胞系并观察其生长特性与对烷化剂N-甲基-N’-硝基-N-亚硝胍(N-methyl-N’-nitro-N-nitrosoguanidine, MNNG)敏感性的改变。方法:利用改良磷酸钙-DNA共沉淀法分别将本室构建的持续和诱导表达反义hREV3 RNA的真核细胞重组表达质粒pBK-RSV-hREV3和pMAMneo-amp^--hREV3^-转染人胚胎肾细胞(HEK-293细胞),经G418筛选,建立相应的细胞系293-B-hREV3^-和293-M-hREV3^-。通过细胞计数方法观察这些细胞系的生长速率和不同浓度MNNG对细胞的细胞毒作用。 结果:持续或诱导阻断hREV3基因的表达对细胞生长速率均无影响;反义阻断细胞系对MNNG的细胞毒作用比母本293细胞较为敏感。结论:hREV3基因的编码产物并非细胞生长所必需而可能在细胞的DNA损伤修复中起作用。

Establishment of cell lines whose hREV3 gene expression was inhibited by transfection of antisense RNA expression plasmids and their biological characteristics

AIM: to establish the cell lines whose hREV3 gene expression was blocked by antisense RNA and observe their characteristis of cell growth rate and N - methyl - N' - nitrosoguanidine (MNNG) sensitivi- ty . METHODS: With modified calcium phosphate - DNA coprecipitation method the eukaryocytic expression plasmid expressing antisense fragment of hREV3,pBK - RSV - hREV3- and pMAMneo - amp hREV3 were transfected into human embryo kidney cell line of HEK - 293. After G418 selection, cell lines of 293 - B - hREV3- and 293 - M hREV3- were established. By cell counting method, the cell growth rate and MNNG sensitivity of these cell lines were characterized. RESULTS: No change of cell growth rates of these cell lines was observed whether hREV3 gene expres- sion was blocked by either the persistent or induced expression of the antisense hREV3 RNA. While the sensitivity of these cell lines to MNNG was somewhat elevated, as compared with their parent cell line 293 and the cell lines trans- fected with vector DNAs. CONCLUSION: The gene product of hREV3 was not essential for the cell growth, but it may play a role in the DNA repair functions of the cells after exposure to DNA damaging agents.