摘要
目的 探讨Na+ /H+ 交换蛋白 1(NHE 1)反义表达载体 (pNHE 1)对人肺癌细胞NHE 1基因的抑制作用及生物学效应。方法 实验细胞分为 3组 ,A5 4 9 pNHE 1组 (反义组 )、A5 4 9空载体组(空载体组 )、A5 4 9对照组 (A5 4 9组 )。采用阳离子脂质体使pNHE 1转染人肺腺癌A5 4 9细胞株。采用半定量逆转录 聚合酶链式反应 (RT PCR)法测定 3组细胞的NHE 1mRNA表达水平 ;用荧光法测定 3组细胞内pH(pHi)值 ;了解转染细胞增殖生长状况 ;用细胞凋亡原位检测法检测 3组细胞凋亡率。结果 在转染细胞基因组DNA中扩增出外源真核表达载体的DNA片段 ,证明转染成功。半定量RT PCR法证实反义组细胞的NHE 1mRNA表达水平 (0 4 2± 0 0 6 )显著低于A5 4 9组 (0 71± 0 0 8,P <0 0 1)和空载体组 (0 6 9± 0 16 ,P <0 0 1)。A5 4 9组细胞在培养 12、2 4和 4 8h后pHi值分别为 7 15 0±0 0 0 4、7 14 0± 0 0 0 7和 7 12 0± 0 0 0 8,反义组pHi值分别为 6 990± 0 0 0 5、6 84 0± 0 0 0 5和 6 75 0±0 0 0 5 ,两组差异均有极显著性 (P <0 0 1)。A5 4 9组、空载体组和反义组的细胞倍增时间分别为 3 2 0、3 2 8和 4 97d ,反义组细胞的增殖、生长明显慢于A5 4 9组及空载体组 (P <0 0 1)。反义组细胞凋亡率为 (2
Objective To explore the inhibitory effect of Na +/H + exchanger 1(NHE 1) antisense expression vector on NHE 1 gene in human lung neoplasm cells and to observe its biological effect Methods NHE 1 antisense vector (pNHE 1) was introduced into human lung adenocarcinoma cell line A549 cells with cationic liposome transfection methods Semi quantification RT PCR was performed to analyze the expression level of NHE 1 mRNA in A549 cells and transfected A549 cells Intracellular pH(pHi) values were measured with fluorescence spectrophotometer The growth characteristics of the cells was observed Tumor cell apoptosis was observed with in situ apoptosis assessment Results PCR verified the integration of antisense vector with the genomic DNA of transfected cells in positive clones Semi quantification RT PCR showed that the NHE 1 gene expression level was significantly lower in transfected A549 cells(0 42±0 06) than in those untransfected (0 71±0 08, P <0 01) and those transfected with pLXSN(0 69±0 16, P <0 01) Compared with A549 cells, pHi values decreased significantly in transfected A549 cells at 12 h, 24 h and 48 h(6 990±0 005,6 840±0 005 and 6 750±0 005 respectively vs 7 150±0 004,7 140±0 007 and 7 120±0 008 respectively, P <0 001) The growth of transfected A549 cells was significantly slower ( P <0 01), the double time was longer (3 20 vs 4 97) and the apoptosis rate was markedly higher [(24 50±3 62)% vs (1 50±0 55)%, P <0 01] ConclusionThe antisense vector was successfully transfected to A549 cells with liposome and integrated with transfected cell genome, and the expression of NHE 1 mRNA of A549 cells was inhibited, thereby resulted in a decrease of the pHi value by means of reduction of Na +/H + exchanging activity, with the inhibition of proliferation and growth of A549 cells and increased of A549 cell apoptosis
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2004年第3期191-194,共4页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
重庆市应用基础研究课题 ( 64 2 3 )
