论文部分内容阅读
目的了解端粒酶逆转录酶(hTERT)-siRNA对GBG-SD细胞代谢、侵袭和端粒酶活性、hTERT mRNA、hTERT蛋白、β-catenin mRNA、β-catenin蛋白的作用,并探讨其相关机制。方法在质粒pGCssi-H1-GFP-hTERT干扰后,采用端粒酶TRAP、半定量逆转录-聚合酶链反应(RT—PCR)和Westem blot方法检测GBC-SD端粒酶活性、hTERT基因和β-catenin基因mRNA、蛋白质表达水平。噻唑蓝(MTT)比色法检测琥珀酸脱氢酶(SDH)活性、运用Transwell小室了解癌细胞侵袭情况。结果pGCsi-H1/GFP-hTERT对GBC-SD端粒酶活性、SDH活性、侵袭、hTERT mRNA、hTERT蛋白均有抑制作用并随浓度升高而增强。pGCsi-H1/NEGative对上述指标无明显抑制作用,与pGCsi-H1/GFP-hTERT抑制作用比较差异有统计学意义(P＜0．01)。结论pGCsi-H1/GFP—hTERT可以抑制端粒酶、SDH活性及其侵袭力,其机制与hTERT mRNA及蛋白表达降低有关。
Objective To investigate the effect of telomerase reverse transcriptase (hTERT) -siRNA on the metabolism, invasion and telomerase activity, hTERT mRNA, hTERT protein, β-catenin mRNA and β-catenin protein in GBG-SD cells and to explore its mechanism . Methods After the interference of plasmid pGCssi-H1-GFP-hTERT, telomerase TRAP, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the telomerase activity of GBC- -catenin gene mRNA, protein expression level. The activity of succinate dehydrogenase (SDH) was detected by MTT colorimetric assay. Transwell chamber was used to detect the invasion of cancer cells. Results pGCsi-H1 / GFP-hTERT could inhibit the telomerase activity, SDH activity, invasion, hTERT mRNA and hTERT protein of GBC-SD and increase with increasing concentration of pGCsi-H1 / GFP-hTERT. The inhibition of pGCsi-H1 / NEGative on pGCsi-H1 / NEGative had no significant difference with that of pGCsi-H1 / GFP-hTERT (P <0.01). Conclusion pGCsi-H1 / GFP-hTERT can inhibit the activity of telomerase and SDH and its invasiveness, and its mechanism is related to the decrease of hTERT mRNA and protein expression.