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利用磷酸钙沉淀法将重组的质粒 p CMV5 -pemt2与 p SV-neo共转染 ,经 G418筛选 ,免疫细胞化学和免疫印迹法检测蛋白表达 ,[3 H] -SAM掺入测定酶的活性 ,获得稳定表达 PEMT2的阳性克隆 .经台盼蓝排阻法测生长曲线 ,流式细胞仪测定细胞周期 ,MTT 法检测细胞生长抑制率 .观察了磷脂酰乙醇胺 N-甲基转移酶 2( phosphatidylethanolam ine N -m ethyltransferase 2 ,PEMT2 )的表达对大鼠甲胎蛋白阳性 ( AFP+ )肝癌细胞系 CBRH-7919增殖的影响 .结果表明 :PEMT2在 CBRH-7919肝癌细胞中已成功表达 ,细胞的增殖能力下降 ,S期明显减少 ,AFP含量减少 .说明 PEMT2的表达可明显抑制大鼠 CBRH -7919肝癌细胞的生长 .
The recombinant plasmids p CMV5 -pemt2 and p SV-neo were cotransfected by calcium phosphate precipitation method, and then screened by G418. The protein expression was detected by immunocytochemistry and immunoblotting. The activity of [3 H] -SAM incorporation was measured, The positive clones stably expressing PEMT2 were obtained.The growth curve was determined by trypan blue exclusion method, the cell cycle was determined by flow cytometry, and the cell growth inhibition rate was measured by MTT assay.Phosphatidylethanolamine 2 N-m ethyltransferase 2 and PEMT2 on the proliferation of rat AFP-positive hepatocellular carcinoma cell line CBRH-7919.The results showed that PEMT2 was successfully expressed in CBRH-7919 hepatocarcinoma cells and the proliferation of cells was decreased , S phase decreased significantly, AFP decreased, indicating that PEMT2 expression can significantly inhibit the growth of rat CBRH-7919 hepatoma cells.