GPI-PLD过表达对肺癌细胞株A549生物学特征的影响  

Effect of Overexpression of Glycosylphosphatidylinositol Specific Phospholipase D on Biological Characters of Lung Carcinoma Cell Line A549

作　　者：石新云[1] 黄明清[1] 黄洁[1] 

机构地区：[1]浙江省绍兴市第六人民医院呼吸内科,绍兴312000

出　　处：《华中科技大学学报（医学版）》2009年第2期228-231,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

摘　　要：目的研究糖基化磷脂酰肌醇特异性磷酯酶D(GPI-PLD)基因转染并过度表达对肺癌细胞株A549生物学特征的影响。方法用脂质体转染法将GPI-PLD真核表达载体pcDNA3.1(+)/GPI-PLD转入A549细胞,以未转染的A549细胞及转染空载体pcDNA3.1(+)的细胞为对照,筛选出阳性细胞克隆。以胎盘碱性磷酸酶(PLAP)为底物,定量检测GPI-PLD活性,RT-PCR法确定GPI-PLD mRNA表达水平,细胞计数绘制生长曲线,锥虫蓝排斥法观察补体介导的细胞毒(CDC)杀伤率,分光光度法测定PLAP活性,ELISA检测癌胚抗原(CEA)的表达情况。结果阳性细胞克隆GPI-PLD酶活性水平比对照组升高约3倍,GPI-PLD mRNA表达水平显著增加;GPI-PLD基因过度表达可促进GPI锚定的蛋白质如CEA和PLAP等被大量水解释放入培养液,导致细胞增殖能力减弱,对补体杀伤的敏感性增强。结论成功将GPI-PLD基因转染入A549细胞,并在细胞中稳定表达。GPI-PLD过表达能抑制肿瘤细胞增殖,增强肿瘤细胞对补体杀伤的敏感性。Objective To investigate the effect of overexpression of glycosylphosphatidylinositol specific phospholipase D （GPI-PLD） on the biological characters of lung carcinoma cell line A549. Methods The GPI-PLD gene eukaryotic expression vector pcDNA3. 1（＋）/GPI-PLD was transiently transfeeted into A549 cells by lipid media transfection. The un-transfected A549 cells and A549 cells transfected with pcDNA3.1（＋） were used as controls. After screening with G418,the single clone was obtained. The expression level of GPI-PLD mRNA in A549 cells was detected by using reverse transcription polymerase chain reaction（RT-PCR）. GPI-PLD activities were analyzed quantitatively by TX-114 partition with GPI anchored placental alkaline phosphatase（PLAP） as a substrate. Cell counting was used to measure the proliferation of the 3 groups,and complement dependent cytotoxicity（CDC） effects were observed by the staining of trypan blue. Carcinoembryonic antigen（CEA） was determined by enzyme linked immunosorbent assay（ELISA）. Results Compared with A549 and pcDNA3. 1（＋）/A549 cells, the levels of GPI-PLD activities and its mRNA from pcDNA3.1 （＋）/GPI-PLD/A549 were increased by almost 3 to 6 times, respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD,and the rate of CDC killing on the cells was significantly increased. However,the proliferative capacity was obviously decreased. Conclusion The stable cell line with the overexpression of GPI-PLD has been constructed successfully. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of eells.

关 键 词：糖基化磷脂酰肌醇特异性磷酯酶D 肺癌 A549细胞 基因转染 

分 类 号：R734.2[医药卫生—肿瘤]