单羧酸转运蛋白基因对肿瘤细胞内pH值及生长特性的调节作用

目的 以反义技术抑制肿瘤细胞的单羧酸转运蛋白基因第一亚型(MCT1)表达,观察其对肿瘤细胞内pH值(pHi)调节、乳酸转运及生长性质的影响.方法 (1)应用逆转录-聚合酶链式反应(RT-PCR)从人肺癌细胞系A549中扩增MCT1目的 基因片段,将克隆的基因片段反向插入逆转录病毒载体pLXSN,构建反义表达重组载体pLXSN-MCT1,并对其进行DNA测序验证.(2)通过电穿孔法将pLXSN、重组载体pLXSN-MCT1转染于肺腺癌细胞系A549中,经G418筛选转染细胞阳性克隆.用PCR方法 鉴定pLXSN-MCT1重组载体在基因组的转染整合及表达;以分光光度法测定细胞内pH及乳酸含量变化,并以细胞生长曲线研究细胞的生长情况.结果 (1)对所构建的重组载体进行双酶切电泳分析及DNA序列分析证明反义载体构建成功.(2)与未转染的A549细胞比较,转染MCT1反义表达重组体的细胞pHi降低、乳酸显著升高(P＜0.001);且细胞的生长受到明显抑制.结论 单羧酸转运蛋白MCT1基因在肿瘤细胞pHi调节、乳酸转运及细胞的生长中起着重要的调节作用.

The effect of monocarboxylate transporter gene on the regulation of pHi and growth character in cancer

Objective To study the influence of the first subtype of monocarboxylate transporter ( MCT1) gene on pHi regulation,lactate transport and cell growth in tumor cells Methods (1)With RT PCR technique,MCT1 cDNA fragment was cloned from human lung cancer cells A549,and the cloned fragment MCT1 was reversely inserted into the vector pLXSN to acquire antisense expression recombinant vector pLXSN MCT1. (2) pLXSN,pLXSN? MCT1 were respectively introduced into the A549 cells by electroporation. The transfected A549 cell resistant to G418 drug was selected as positive clones and proved by PCR . The changes of intracellular pH and lactate in the transfected A549 cells were detected by spectrophotometric method. Cell growth was studied by cell growth curve. Results (1) The cloned fragment was in the length of 640 bp and successfully bound to pLXSN. It was also proved to be the objective one by DNA sequencing. (2) Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN MCT1 ,comparing to A549 cells without transfection( P <0.001). The growth of A549 cells transfected pLXSN MCT1 was also inhibited remarkably. Conclusion MCT1 gene could play an important role in pHi regulation,lactate transport and cell growth in tumor cells.