发夹状核酶阻断HPV16转化口腔上皮细胞的研究

Inhibition of HPV16 transformation of oral epithelial cells by hairpin ribozyme

目的:探讨发夹状核酶阻断人乳头状瘤病毒16(HPV16)转化口腔上皮细胞的能力及可能的机制。方法:设计合成HPV16E6特异性发夹状核酶基因;构建重组真核表达载体;脂质体介导转染HPV16诱导的永生化口腔上皮细胞(HIOEC);空白载体对照;潮霉素筛选;斑点杂交检测细胞中核酶的表达;PCR、RT-PCR和免疫细胞化学分别检测细胞中HPV16E6的DNA、RNA和蛋白表达;荧光染色、流式细胞仪检测、生长曲线绘制观察细胞生物学特性的改变。结果:成功构建含HPV16E6特异性发夹状核酶的重组真核表达载体pcDNA-RZE6;转导筛选后,获得阳性克隆HIOEC-RZE6和空白载体对照HIOEC-pcDNA;HIOEC-RZE6中核酶转录、HPV16E6RNA和蛋白表达下降,细胞增殖减缓,出现凋亡;HIOEC-pcDNA中未见同样变化。结论:HPV16E6特异性发夹状核酶可以抑制HPV16E6表达,并阻断HIOEC进一步恶变,诱导细胞凋亡。

PURPOSE:To evaluate the possibility and the potential mechanisms of inhibition of HPV16transformation of oral epithelial cells by hairpin ribozyme.METHODS:We constructed recombinant eukaryotic expression vectors pcD-NA-RZE6,which contained hairpin ribozyme gene against HPV16E6RNA.The recombinants were introduced into im-mortalized oral epithelial cells HIOEC by Lipofectamine and the positive clones HIOEC-RZE6were selected by hy-gromycin B.Ribozymes expression was confirmed by RNA dot blot.HPV16E6RNA and protein before and after transfec-tion were detected by RT-PCR and immunocytochemistry,respectively.Cell apoptosis was examined by fluorescent stain-ing and flow cytometry assay.RESULTS:Hairpin ribozymes stably transcripted in HIOEC-RZE6.HPV16E6transcrip-tion and expression were down regulated.No similar results were detected in HIOEC-pcDNA.The growth of HIOEC-RZE6cells was slower than those of HIOEC and HIOEC-pcDNA.The apoptotic rate of HIOEC-RZE6cells was higher than those of HIOEC and HIOEC-pcDNA.CONCLUSION:Hairpin Ribozyme against HPV16E6can efficiently inhibit HPV16E6expression in HIOEC,which may lead to apoptosis of immortalized oral epithelial cells induced by HPV16and block their further transformation.