【作者】 李道成；

【导师】 罗颂平；

【作者基本信息】 广州中医药大学 ， 中医妇科学， 2009， 博士

【摘要】 目的1.以宫颈癌裸鼠移植瘤为实验对象,探讨精制雄黄和白砒对宫颈癌的抑瘤效应和对裸鼠的毒性副反应。2.探讨精制雄黄和白砒对宫颈癌的细胞凋亡及细胞周期的影响,以分析精制雄黄和白砒的抑瘤机制。3.探讨精制雄黄和白砒对细胞凋亡相关蛋白Fas、Caspase-3和survivin的表达,分析精制雄黄和白砒诱导细胞凋亡的机制。方法1.将宫颈癌Hela细胞培养后,注射到裸小鼠的皮下,形成宫颈癌裸鼠移植瘤。将荷瘤小鼠随机分为模型组、卡铂组、白砒组、低剂量雄黄组、中剂量雄黄组、高剂量雄黄组、白砒+雄黄组,共7组,每组10只裸鼠。15天后处死全部裸鼠,完整取出肿瘤组织进行实验研究,取出肝、脾、肾、胃、大肠及小肠分析药物毒副作用。2.肿瘤组织匀浆后制成细胞悬液,PBS洗两遍,400目网过滤,调整细胞浓度为1×10⁶个/ml。加Annexin V-FITC和PI双染色,上流式细胞仪测定检测细胞凋亡和细胞周期分布。3.将肿瘤组织切片后,用免疫组化SP法,检测肿瘤组织中的Fas、Caspase-3和survivin的蛋白表达水平。结果1.卡铂组、白砒组、精制雄黄中剂量组、高剂量组、白砒+雄黄组的肿瘤重量与模型组比较,均明显减轻（P＜0.05）,其抑瘤率分别为34.3%、35.8%、37.0%、33.1%、37.8%。提示精制雄黄和白砒有明显的抑瘤效应,与卡铂作用相当。肝、脾、肾、胃、大肠及小肠未见明显的毒性反应。2.精制雄黄低、中、高剂量组、卡铂组、白砒组、白砒+雄黄组肿瘤细胞的凋亡率与模型组比较,细胞凋亡率上升,具有显著性差异（P＜0.05）。3.精制雄黄低、中、高剂量组、卡铂组、白砒组、白砒+雄黄组肿瘤细胞与模型组比较,在G₀/G₁周期的细胞比例明显增加,差异有显著性（P＜0.05）。提示精制雄黄和白砒对肿瘤细胞的细胞分裂起到G₀/G₁期阻滞作用。4.各药物治疗组与模型组比较,Fas蛋白表达水平升高,有统计学差异（P＜0.05）;各药物治疗组与模型组比较,Caspase-3蛋白表达水平升高,有统计学差异（P＜0.05）;各药物治疗组与模型组比较,survivin蛋白表达水平降低,有统计学差异（P＜0.05）:结论1.精制雄黄和白砒对宫颈癌裸鼠移植瘤有明显的抑瘤效果,效果与卡铂相当。雄黄灌胃+局部注射联用法抑瘤效果较好,但抑瘤效果并未显示出剂量效应,低、中、高剂量的抑瘤效应相当,这可能与其难溶于水的物理特性有关。2.精制雄黄和白砒都有诱导宫颈癌细胞凋亡的功效。诱导癌细胞凋亡可能是其发挥抑瘤效应的机制之一。3.精制雄黄和白砒都能将宫颈癌细胞阻滞在G₀/G₁周期,延缓了癌细胞增殖的过程,有利于诱导细胞凋亡。4.精制雄黄和白砒能上调Fas和Caspase-3蛋白的表达,下调survivin蛋白的表达。上调Fas和Caspase-3蛋白的表达,下调survivin蛋白的表达是精制雄黄和白砒诱导宫颈癌细胞凋亡的机制之一。更多还原

【Abstract】 Objective1.By transplanting the Hela cell to the nude mice,we study the tumor suppressive effect of purified realgar and white arsenic to cervical cancer and explore the side effects and toxicities of them to the mice.2.To explore the mechanism of inhibiting tumor growth in purified realgar and white arsenic by analyzing the apoptosis and cell cycle.3.To investigate the relation between the apoptosis and the protein expression of Fas,Caspase-3 and Survivin.Methods1.Hela cells were cultured at 37℃in a 5 mL/L CO₂ incubator.Seventy mice received subcutaneous injection with cell suspension containing 2×10⁷ cells.When tumors of about 0.5cm diameter were detected,drug administration was started.The animals were randomly divided into seven groups including model group,carboplatin group,white realgar group,low dose purified realgar group,middle dose purified realgar group,high dose purified realgar group and purified realgar combined with white arsenic group.There are 10 animals in each group.After 15d of treatment,the mice were sacrificed and the tumor masses were removed.After the weight of tumor masses was measured,some were fixed in 10%paraformaldehyde,and some were frozen.The tissues of liver,kindy,spleen,stomach,large intestine and small intestine were stained with HE and analyzed for the side effects and toxicities of the drugs.2.Tumor tissues were homogenated,washed with PBS and then filtered.The tumor cells were mixed with PBS to the concentration of 1×10⁶ cells/ml.After adding annexin V-FITC and PI,the tumor cells were analyzed by flow cytometer for the apoptosis and cell cycle.3.Fas protein,Caspase-3 protein and surviving protein were detected with Immunohistochemical SP method.Results1.The weights of the tumors in carboplatin group,white realgar group,middle dose purified realgar group,high dose purified realgar group and purified realgar combined with white arsenic group are more than in the model group（P<0.05）.In the all treatment groups,there was no differences in weights of the tumors（P>0.05）.The suppressive rates to tumor in carboplatin group,white realgar group,middle dose purified realgar group, high dose purified realgar group and purified realgar combined with white arsenic group was 34.3%、35.8%、37.0%、33.1%、37.8%respectively.These suggest that purified realgar and white arsenic can inhibit the proliferation of cervical cancer,and purified realgar and white arsenic have no toxicity to liver,lung,kidney,spleen,stomach,large intestine or small intestine.2.The apoptotic rate of cervical cancer cells treated with purified realgar and white arsenic were significantly higher than that in the model group （P<0.05）.3.The cervical cancer cells in G₀/G₁ phase in the treatment groups were much more than in the model group（P<0.05）.This suggests purified realgar and white arsenic arrest the cervical cancer cells in G₀/G₁ phase.4.The expressions of Fas protein in the treatment groups were much higher than in the model group（P<0.05）.The expressions of Caspase-3 protein in the treatment groups were much higher than in the model group（P<0.05）.The expressions of Survivin protein in the treatment groups were much lower than in the model group（P<0.05）.Conclusion1.As the carboplatin,purified realgar and white arsenic can inhibit the proliferation of cervical cancer.If realgar is given to the nude mice by filling stomach combining with local injecting method,the tumor supprcssive effect is the best.The suppressive effect of purified realgar is not in a dose-dependent manner,which may be related with its characteristic of no dissolving to water. 2.Purified realgarand white arsenic induced apoptosis of cervical cancer. Inducing apoptosis may be one of the mechanisms of the tumor suppressive effect.3.Purified realgar and white arsenic arrest the cervical cancer cells in G₀/G₁ phase.They delay the proliferation of cervical cancer,and make the apoptosis easy.4.Purified realgar and white arsenic increase protein expression of Fas and Caspase-3,and decease Survivin.UP-regulating of Fas and Caspase-3 and down-regulating of Survivin may play an important role in the apoptosis in cervical cancer cells.更多还原