摘要
目的 :构建NOEY2基因真核表达载体 ,研究其对人乳腺癌细胞系MDA MB 2 31生长的影响。方法 :RT PCR获得目的基因编码区 ,经T A策略克隆后 ,再亚克隆至pcDNA3 ,获得真核表达载体。用lipofectAMINE辅助转染MDA MB 2 31细胞 ,经G418长期筛选而获得稳定转染细胞克隆。Western印迹检测NOEY2蛋白水平的表达。通过记录生长曲线和流式细胞分析术观察转染细胞的生长和细胞周期变化。结果 :NOEY2真核表达载体pcDNA3 NOEY2 CR构建成功。被稳定转染该载体的MDA MB 2 31细胞有明确NOEY2蛋白表达 ,而对照细胞无表达。NOEY2转染细胞的生长抑制率可达 46 .3 % ,在细胞周期改变上可见明显的S期分数和G2 M期分数下降 ,而G0 G1期比例上升。结论 :NOEY2基因转染对体外培养的人乳腺癌细胞生长具有一定的抑制作用 ,支持其作为一个抑癌基因的推测 。
Purpose:To clone and construct an eukaryotic expression vector of NOEY2 gene, and to observe the effects of NOEY2 transfection on the growth of human breast cancer cell line MDA MB 231. Methods:The coding region of NOEY2 was obtained with reverse transcription PCR, and then the PCR product was first cloned into pGEM T vector and further directionally subcloned into pcDNA3. Transfection reagent and selective antibiotic were lipofect AMINE and G418 respectively. The expression level of NOEY2 protein was detected by Western Blotting. The growth cures of the transfected and non transfected cells were recorded. The changes in cell cycle were analyzed by flow cytometry. Results:An eukaryotic expression vector of NOEY2 gene was constructed successfully. The cell transfected with NOEY2 showed definite expression of NOEY2, and the controls were negative. The growth of NOEY2 transfected cells was inhibited by 46.3%, compared with the parental cells on the seventh day after seeding. An obvious decrease in S phase and G2/M phase fraction and an increase in the percentage of G0/G1 phase cells were found in NOEY2 transfected cells. Conclusions:NOEY2 Transfection can inhibit the growth rate of MDA MB 231 cells in vitro, probably via the mechanism of G1 arrest. These data support the suggestion that NOEY2 is a tumor suppressive gene, which merits further investigation for its value as a therapy gene.
出处
《中国癌症杂志》
CAS
CSCD
2001年第1期29-32,共4页
China Oncology
