hTERT-siRNA抑制胆囊癌细胞端粒酶活性及增殖运动的研究

Inhibition of hTERT Activity for Suppression of Gallbladder Carcinoma Cells Proliferation and Migration Using hTERT-siRNA Technique

目的:了解hTERT-siRNA对GBC-SD细胞代谢、侵袭和端粒酶活性、hTERT mRNA、hTERT蛋白、β-catenin mRNA、-βcatenin蛋白的作用,并探讨其相关机制。方法:在质粒pGCsi-H1/GFP-hTERT干扰后,采用端粒酶TRAP、半定量RT-PCR和Western blot方法检测胆囊癌细胞GBC-SD端粒酶活性、hTERT基因和-βcatenin基因mRNA、蛋白质表达水平;四甲基偶氮唑蓝(MTT)光吸收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 evaluate the inhibitory effect on telomerase activity,SDH activity,and invasion efficiency and to investigate the effect on hTERT mRNA and hTERT protein by transfecting pGCsi-H1/GFP-hTERT vector into gallbladder carcinoma GBC-SD cells to reveal the molecular mechanism about the gallbladder carcinoma effect of hTERT-siRNA. Methods. Telomerase activity,mRNA and protein of hTERT and β-catenin in GBC-SD cells were determined using telomerase TRAP, RT-PCR and Western blot techniques respectively. SDH activity was assayed by MTT and invasion assay was determined by Transwell chamber after GBC-SD cells were interfered in pGCsi-H1/GFP-hTERT vector. Results： pGCsi-H1/GFP-hTERT could inhibit telomerase activity,SDH activity,invasion efficiency,and expression of hTERT mRNA and protein. The inhibition increased according with the increase of pGCsi-H1/GFP-hTERT vector concentrations. The results indicated that pGCsi-H1/NEGative vector failed to inhibit telomerase activity, SDH activity,invasion efficiency,and expression of mRNA and protein of hTERT. A statistically significant difference in the inhibition was found between pGCsi-H1/GFP-hTERT vector and pGCsi- HI/NEGative vector （P〈0.01）. The dose of 0.25-2 mg/L pGCsi-H1/NEGative vector did not have the ability to inhibit expression of mRNA and protein of β-catenin. Conclusion： pGCsi-H1/ GFP-hTERT has the ability to inhibit telomerase activity, SDH activity and invasion efficiency and reduces the expression of hTERT, but does not alter the content of mRNA and protein of β-catenin.