联合抑制人肺腺癌细胞的MCT1基因及NHE1基因表达对其增殖生长的影响

Effect of Co-inhibition of MCT1Gene and NHE1Gene on Proliferation and Growth of Human Lung Adenocarcin oma Cells

背景与目的:以往的研究中我们发现,用反义基因分别抑制肿瘤细胞膜上的单羧酸转运蛋白第1亚型(monocarboxylatetransporter-1,MCT1)基因和Na+/H+交换蛋白第1亚型(Na+/H+exchanger-1,NHE1)基因的表达,可使肿瘤细胞内pH(intracellularpH,pHi)降低,细胞酸化死亡。而同时抑制这两种基因的表达,对肿瘤细胞pHi及其增殖生长的影响尚不清楚。方法:用电穿孔法将MCT1基因及NHE1基因相应的反义基因表达载体pLXSN-MCT1和pLXSN-NHE1同时转染于人肺腺癌细胞系A549中,经G418筛选阳性克隆,用PCR及RT-PCR方法鉴定MCT1和NHE1的反义基因与A549基因组的整合。pHi及乳酸含量用分光光度法测定;细胞生长周期用流式细胞仪测定;并将联合转染反义基因的细胞、转染MCT1反义基因的细胞、A549细胞分别接种于裸鼠皮下,观察移植瘤的生长。结果:与A549细胞比较,联合转染反义基因的细胞pHi降低、乳酸显著升高(P<0.001),细胞周期阻滞在G0-G1期。接种联合转染反义基因细胞和转染MCT1反义基因细胞的裸鼠移植瘤明显比接种A549细胞的轻且小(P<0.001)。结论:MCT1基因和NHE1基因可能通过影响pHi对肿瘤细胞的增殖生长起重要的调节作用。

Background &Objective:The authors previously discovered t hat the intracellular pH values(pHi )of tumor cells could be decreased,whic h led tumor cells to acidify and die,w hen the expressions of the first subt ype of monocarboxylate transporter(MCT1)gene and the first subtype of Na + /H + exchanger(NHE1)gene in tumor cell membranes had been inhibited by each corresponding antisense gene respectively.However it was not clear whether there were co-effects on the pHi,proliferation ,and growth of tumor cells,aft er two genes were inhibited at the sam e time.Methods:pLXSN-MCT1and pLXSN-NHE1,the corresponding antisense gene expressio n vectors of MCT1and NHE1genes,were introduced into human lu ng adenocarcinoma A549cells at the s ame time with electroporation.The p ositive colonies were selected by G418.Usin g PCR and RT-PCR technique,it was con firmed that antisense genes of MCT1a nd NHE1genes integrated with A549genomic DNA.The pHi and lactate content were detected with spectrophotometr y.Cell growth cycle was measured with flow c ytometer.The cells co-transfected antisense genes,cells transfected MCT1antisense gene and A549cells were respectively inoculated in nude mouse subcutaneous to observe the growth o f transplantation tumors.Results:Compared with A549cells,the pHi of c ells co-transfected antisense genes was remarkably decreased(P<0.05),while lactate content was remarkab ly increased(P<0.001).The cell cycle was blocked at G 0 -G 1 period.And the transplantation tumors of nude mice inoculated co-transfected antisense gene-cells and inoculate d MCT1-antisense-gene-transfecte d cells were significantly lighter a nd smaller than ones inoculated A549cells(P<0.001).Conclusion:MCT1and NHE1genes play important re gulation roles in proliferation and growth of tumor cells,probably b y affecting pHi.