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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A5 49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A5 49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A5 49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 0 0 1) ;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
Objective To use antisense technology to inhibit the expression of the first subtype of the monocarboxylate transporter gene (MCT1) in tumor cells, and to observe the effect on the regulation of pHi (pHi), lactate transport and growth properties of tumor cells. Methods (1) Reverse transcription polymerase chain reaction (RT PCR) was used to amplify the MCT1 target gene fragment from the human lung cancer cell line A5 49, and the cloned gene fragment was inserted reversely into the retroviral vector pLXSN to construct antisense recombinant expression. The vector pLXSN MCT1 was verified by DNA sequencing. (2) The pLXSN and recombinant vector pLXSN MCT1 were transfected into the lung adenocarcinoma cell line A5 49 by electroporation, and the positive cell clones were transfected with G418. PCR method was used to identify the transfection integration and expression of pLXSN MCT1 recombinant vector in the genome. Spectrophotometric method was used to determine the change of intracellular pH and lactic acid content, and cell growth curve was used to study cell growth. Results (1) Double enzyme electrophoresis analysis and DNA sequence analysis of the constructed recombinant vector proved that the antisense vector was successfully constructed. (2) Compared with non-transfected A5 49 cells, the pHi of cells transfected with MCT1 antisense recombinant expression was significantly lower and lactic acid was significantly increased (P < 0.01); and cell growth was significantly inhibited. Conclusion The monocarboxylate transporter MCT1 plays an important role in the regulation of pHi, lactate transport and cell growth in tumor cells.