【作者】 李立杰；

【导师】 薛敏；

【作者基本信息】 中南大学 ， 妇产科学， 2012， 博士

【摘要】 目的：探讨纳米雄黄对不同宫颈癌细胞株Hela(HPV18阳性,腺癌),Caski (HPV16阳性,鳞癌),C33A (HPV阴性,腺癌)增殖、凋亡及其对HPV16/18E6、E7mRNA及E6、E7蛋白的影响。方法：体外培养三种不同宫颈癌细胞,采用不同质量浓度(5,10,20,40ug/ml)纳米雄黄作用于不同宫颈癌细胞不同时间(24487296h)/(48h),相差显微镜下观察不同质量浓度组与对照组细胞形态学变化；MTT法测定药物对细胞生长的抑制作用；流式细胞仪检测细胞凋亡及细胞周期；实时荧光定量PCR法检测HPV16/18E6/E7mRNA的表达；免疫组化法检测HPV16/18E6/E7蛋白的表达。结果：不同浓度纳米雄黄处理不同细胞株,均不同程度抑制细胞生长,促进细胞凋亡,具有时间浓度依赖性,其中对Caski细胞作用最强,对C33A细胞作用最弱；流式细胞仪检测结果表明,不同质量浓度的纳米雄黄可促进细胞凋亡,且呈浓度依赖性；细胞周期结果显示高质量浓度组(20,40ug/ml)对HPV阳性宫颈癌细胞有G2/M期阻滞；实时荧光定量PCR结果显示不同浓度纳米雄黄可不同程度抑制E6E7mRNA的表达,且随浓度增大,抑制作用增大,对E6基因的抑制作用强于E7基因,对于HPV16型E6E7mRNA的抑制作用强于HPV18型；免疫组化结果显示,E6E7蛋白定位于胞浆内,随着药物质量浓度的增加,蛋白表达量减少。结论：纳米雄黄混悬液可抑制不同宫颈癌细胞株的增殖,促进细胞凋亡,其抑制细胞增殖、促进细胞凋亡的作用对Caski细胞最强,对Hela细胞次之,对C33A细胞相对最弱。对HPV阳性的细胞可产生G2/M期阻滞。纳米雄黄对宫颈癌细胞的增殖抑制及凋亡诱导作用可能与抑制E6E7基因及蛋白的表达有关,其中E6基因可能起主导作用。更多还原

【Abstract】 Purposes:To investigate the proliferation inhibitory and apoptosis induction effects of nano-realgar on cervical cancer cell Caski (HPV16+, adeno carcinoma)、Hela (HPV18+, squmous carcinoma) and C33A (HPV-, adeno carcinoma).Methods:Treat the cells with different concentration (5,10,20,40ug/ml) and time (24,48,72,96h)/(48h), observe the morphology under phase contrast microscope; MTT to test the growth inhibitory; flow cytometry to measure the percentage of apoptosis and different stages of cell cycles; real-time PCR for the measurement of the level of E6E7mRNA; immunohistochmistry for the detection of the level of E6and E7oncoprotein.Results:The cells’ growth rates was decreased and they got more apoptosis on an concentration-time dependened behavior, what is more, Caski cells seems more sensitive to the drug where as the C33A cells were the last sensitive one; The flow cytometry test shows that the nano-realgar could induce more apoptosis as the concentration increased (P<0.05); high concerntration (20,40ug/ml) of nano-realgar can induce G2/M stage block; Real time PCR shows the level of E6and E7mRNA was decreased in an concentration depended behavior and the E6gene was inhibited more that E7gene and so as both the E6and E7gene of HPV16to HPV18; IHC shows the E6and E7oncoprotein was decreased as the concentration of nano-realgar increased.Conclusion:Nano-realgar can inhibit the growth and induce apoptosis on different kinds of cervical carcinoma cell line, the effects goes as follows Caski> Hela>C33A, it can also induce G2/M stage block on HPV positive cervical cancer cells when the concentration of drug is high enough (20,40ug/ml). The E6E7mRNA and oncoprotein may contribute to these effects, and E6may take the leading role.更多还原