RNA干扰hTERT基因治疗喉鳞状细胞癌的实验研究

Experimental Studies on the Effect of RNA Interference on hTERT Gene in the Treatment of Laryngeal Squamous Carcinoma

目的:探讨RNA干扰hTERT(人端粒酶逆转录酶)基因对人喉鳞状细胞癌的治疗作用。方法:根据hTERTcDNA序列构建表达hTERTmRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1、pshRNA2。将shRNA质粒分别转染人喉癌Hep-2细胞株及荷瘤裸鼠瘤体内。以激光共聚焦显微镜观察质粒在Hep-2细胞及瘤体内的表达;以MTT法观察质粒对Hep-2细胞增殖的抑制作用;以蛋白印迹法(Westernblot法)检测Hep-2细胞中hTERT蛋白的表达;以免疫组化SP法检测裸鼠瘤体内hTERT蛋白的表达。结果:pshRNA1、pshRNA2转染Hep-2细胞及pshRNA1转染瘤体后,共聚焦显微镜下见大量的癌细胞表达绿色荧光,hTERT蛋白表达明显下降。细胞受pshRNA1、pshRNA2转染后,其生长活性受到明显抑制。体内抑瘤实验表明,与空质粒载体组(pshRNA4)和生理盐水组相比,pshRNA1组移植瘤生长明显受到抑制。结论:表达shRNA的、hTERT基因特异的真核表达质粒能有效转染体内、外喉鳞癌细胞,并可有效抑制人喉鳞癌细胞的生长,为今后应用RNA干扰基因治疗喉癌提供重要的实验参考。

Objective： To assess the effect of RNA interference on the human telomerase reverse transcriptase （hTERT） mRNA in treatment of laryngeal squamous carcinoma. Methods：Two types of plasmids, pshRNA1 and pshRNA2, as well as the involved fluorescein gene, were synthesized according to the structures of hTERT cDNA. The tumor model of nude mice was established by subcutaneous implantation of Hep-2 cells, shRNA plasmids were transfected into Hep-2 cells and the transplantation tumors. Fluorescence expression in Hep-2 cells and the tumor was detected by confocal microscopy. The proliferation of Hep-2 cells was detected by MTT assay. Confirmation of hTERT protein expression in vitro and in vivo was performed by Western Blotting and immunohistochemistry, respectively. Resuits： After the Hep-2 cells and the tumors were transfected by pshRNA1 and pshRNA2, many green fluorescent cells were observed by confocal microscopy. The viability of Hep-2 cells was inhibited by pshRNA1 and pshRNA2, pshRNA1 and pshRNA2 could detect a low level of TERT protein expression in Hep-2 cells and the transplantation tumors. Studies of in vivo experiment demonstrated that pshRNA1 significantly inhibited the growth of the established tumors in nude mice. Conclusion： These results demonstrate shRNA plasmids, which express specific sequences of hTERT gene, are effectively transfected into Hep-2 cells in vivo and in vitro. This data can also support the potential application of RNA interference to treat the laryngeal squamous carcinoma.