纳米雄黄混悬液诱导Siha细胞凋亡及对HPV16E6/E7表达的影响

Realgar nanometer suspension inducing apoptosis of Siha cell and its effect on expression of HPVE6/E7 oncogene

目的：探讨纳米雄黄混悬液对人宫颈癌Siha细胞的抑制增殖、诱导凋亡作用及其对HPV16E6和E7致瘤基因的影响。方法：采用微射流法制备纳米雄黄混悬液,应用不同质量浓度（6.25,12.5,25,50 mg.L^-1）的纳米雄黄混悬液作用于Siha细胞不同时间（12,24,48,72 h）,光镜和透射电镜观察实验组（不同质量浓度的纳米雄黄混悬液处理组）和对照组（不加药物的细胞）细胞的形态学变化;琼脂糖凝胶电泳观察凋亡细胞DNA Ladder形成;MTT比色法测定细胞生长抑制情况;流式细胞仪检测细胞凋亡率及细胞周期改变;RT-PCR方法检测HPV16E6和E7的表达。结果：纳米雄黄混悬液25~50 mg.L-1处理Siha细胞48,72 h后,细胞存活率降低,凋亡增加,作用呈时间-剂量依赖关系。电镜显示细胞呈典型的凋亡形态学改变,琼脂糖凝胶电泳显示凋亡细胞DNA Ladder形成;流式细胞术检测证实细胞凋亡率与纳米雄黄混悬液作用浓度呈明显相关,与对照组相比,差异有极显著意义（P〈0.01）,且细胞呈G0-G1期阻滞。RT-PCR检测显示随着纳米雄黄混悬液作用浓度的增加,HPV16E6和E7的表达不同程度地受到抑制。结论：纳米雄黄混悬液对于Siha细胞具有诱导凋亡、抑制增殖的作用,其机制可能与其抑制HPV16E6和E7致瘤基因的表达有关。

Objective： To study the growth-inhibitory and apoptosis-inducing effects of realgar nanometer suspension in human carcinoma cervical cell Siha line,and the effect on HPV16E6/E7 oncogene expression.Method： A ＂micro-jet efflux＂ strategy was used for the preparation of realgar nanometer suspension.Siha cells were treated with various concentrations（6.25,12.5,25,50 mg·L-1） of realgar nanometer suspension for different hours（12,24,48,72 h）.The effect of realgar nanometer suspension on Siha cell growth suppression was detected by MTT method.Special morphological changes of apoptosis were observed by light and transmission electron microscopy（TEM） and DNA fragments electrophoresis.The apoptotic rates were quantified by flow cytometry（FCM）.The expression of HPV16E6/E7 mRNA was assayed by RT-PCR.Result： After being treated with 25-50 mg·L-1 realgar nanometer suspension for 48,72 h,the survival of Siha cells decreased,and the rate of apoptosis markedly increased.With TEM and DNA electrophoresis,the special morphological changes were found.The apoptotic rates of Siha cells treated with realgar nanometer suspension were significantly higher than those in the control group（P〈0.01）.G0-G1 phase arrest appeared following the treatment with realgar nanometer suspension in 25 and 50 mg·L-1 48 h.RT-PCR assay revealed that realgar nanometer suspension reduced HPV16E6/E7 gene expression.Conclusion： Realgar nanometer suspension can inhibit the proliferation of human carcinoma cervical cell Siha line and induce the cell apoptosis.The mechanism may be related to the downregulation of HPV16E6/E7 oncogene expression.