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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: In the previous study, we found that antisense gene could inhibit the expression of monocarboxylatetransporter-1 (MCT1) gene and Na + / H + exchanger type 1 (Na + / H + exchanger-1, NHE1) gene expression, the intracellular pH (intracellular pH, pHi) decreased tumor cell death. At the same time, the inhibition of the expression of these two genes on the growth of tumor cells pHi and its impact is not clear. Methods: The antisense gene expression vectors pLXSN-MCT1 and pLXSN-NHE1 corresponding to MCT1 gene and NHE1 gene were transfected into human lung adenocarcinoma cell line A549 by electroporation. The positive clones were screened by G418, PCR method to identify the integration of the antisense gene of MCT1 and NHE1 with the A549 genome. The pHi and lactic acid content were determined by spectrophotometry. The cell growth cycle was determined by flow cytometry. The cells transfected with antisense gene, the transfected MCT1 antisense gene and the A549 cells were inoculated subcutaneously in nude mice respectively Transplanted tumor growth. RESULTS: Compared with A549 cells, the cells transfected with antisense gene had lower pHi, higher lactate (P <0.001) and cell cycle arrest at G0-G1 phase. Transplanted nude mice transplanted with antisense gene transfected cells and antisense MCT1 cells were significantly lighter and smaller than that of A549 cells (P <0.001). Conclusion: MCT1 gene and NHE1 gene may play an important regulatory role by affecting pHi on the proliferation and proliferation of tumor cells.