【作者】 臧晋；

【导师】 严春寅；

【作者基本信息】 苏州大学 ， 泌尿外科， 2009， 博士

【摘要】 第一部分HIF-2α、Hsp90和VEGF在肾细胞癌中的表达及临床意义目的研究肾细胞癌中缺氧诱导因子2(HIF-2α)、热休克蛋白90(Hsp90)和血管内皮生长因子(VEGF)的表达及与临床病理特征之间的关系,并探讨三者之间及与肾细胞癌患者预后的关系。方法应用免疫组织化学(IHC)S-P法检测60例肾细胞癌及25例正常肾组织中HIF-2α、Hsp90和VEGF表达与分布,分析其间的相关性及与肿瘤临床病理特征之间的联系,根据随访资料探讨上述三者与肾细胞癌患者预后的关系。应用逆转录聚合酶链反应(RT-PCR)和免疫印迹(western blot)法检测HIF-2α及VEGF mRNA的表达。结果免疫组化S-P法检测中肾细胞癌标本HIF-2α、Hsp90和VEGF的表达明显高于正常肾组织,差别具有统计学意义。三个指标之间,两两呈显著正相关。HIF-2α、Hsp90和VEGF的表达与肾癌的分期、淋巴转移、肿瘤大小及局部侵润有关。上述三者与肾细胞癌患者生存期之间关系的分析提示HIF-2α阴性组肾细胞癌患者的生存率高于阳性组,差异有统计学意义,Hsp90阴性组生存率有高于阳性组的趋势,接近于统计学水平,VEGF阳性组病例的预后显著差于阴性组,有统计学意义。RT-PCR和western blot法检测中HIF-2α只在蛋白水平增高,VEGF在mRNA和蛋白水平均增高。结论在人肾细胞癌中HIF-2α、Hsp90和VEGF基因高表达,且HIF-2α、Hsp90和VEGF三者表达之间有显著正相关。HIF-2α和VEGF是影响肾细胞癌预后的独立因素。HIF-2α可能通过上调VEGF表达而在肾细胞癌的发生发展中起重要作用。Hsp90与HIF-2α密切相关,可能二者在肾细胞癌的发生发展中共同作用。第二部分Hsp90抑制剂17-AAG对人肾癌细胞生物学行为影响的体外研究目的观察Hsp90抑制剂17-AAG对体外培养的人肾癌细胞株Caki-1细胞的作用。方法将Hsp90抑制剂17-AAG加入人肾癌细胞株Caki-1中(实验组),分别在常氧和缺氧状态下进行培养。同时设立阴性对照组和空白对照组。MTT法检测三组细胞增殖能力。Hoechst 33342和PI双染法观察细胞凋亡,AnnexinV-FITC/PI双染法和DNA ladder检测细胞凋亡。western blot法检测HIF-2α和VEGF蛋白表达。结果对数生长周期的三组细胞在常氧状态下的增殖活性无明显区别。而实验组细胞在缺氧48小时后生长能力逐渐下降,细胞增殖明显滞后于前两组细胞,差异有统计学意义。缺氧状态下实验组的凋亡率明显增高,而阴性对照组和空白对照组细胞的凋亡率尽管也增多,但增高幅度较小,与实验组比较,差异有统计学意义。常氧状态下,实验组细胞HIF-2α和VEGF蛋白表达受到抑制,明显低于阴性对照组和空白对照组,差异有统计学意义。在模拟缺氧24小时后,阴性对照组和空白对照组的HIF-2α和VEGF蛋白表达明显增强,与常氧时比较差异有统计学意义。而实验组细胞HIF-2α和VEGF蛋白表达虽然有所增加,但幅度较小,与常氧时比较差异无统计学意义。结论在体外,Hsp90抑制剂17-AAG能抑制Caki-1细胞增殖,促进凋亡,并能抑制人肾癌细胞株Caki-1的HIF-2α和VEGF基因表达。第三部分Hsp90抑制剂17-AAG对人肾细胞癌抑制作用的体内研究目的在整体水平(裸鼠皮下移植瘤)研究Hsp90抑制剂17-AAG对人肾细胞癌生长的作用。方法建立人肾细胞癌裸鼠皮下移植瘤模型,在模型中按125mg/kg体重标准,在瘤体内分别注射17-AAG、DMSO和生理盐水,观察抑瘤效果,IHC S-P法检测HIF-2α和VEGF蛋白表达,TUNEL和透视电镜检测凋亡。结果实验组裸鼠皮下移植瘤的生长明显受到抑制。体积增长明显慢于空白对照组和阴性对照组,差别有统计学意义。同时重量明显轻于空白对照组和阴性对照组,差别有统计学意义。实验组裸鼠皮下移植瘤HIF-2α和VEGF蛋白表达弱于对照组,差别有统计学意义。实验组裸鼠皮下移植瘤凋亡指数(AI)明显小于对照组。结论在体内实验中,Hsp90抑制剂17-AAG能明显抑制人肾细胞癌的生长,促进凋亡,可能有下调HIF-2α和VEGF的表达的作用。更多还原

【Abstract】 PartⅠThe clinicopathologic significance of HSP90,HIF-2α,and VEGF expression in renal cell carcinoma.Objective To investigate the expression of heat shock protein 90(Hsp90), hypoxia-inducible factor 2α(HIF-2α),and Vascular endothelial growth factor (VEGF).Analyze the relationship among these protein and the clinicopathologic features of human renal cell carcinoma.To explore whether Hsp90,HIF-2α,and VEGF play an important role in the outcome of renal cell carcinoma.Methods Measure the expressions of Hsp90,HIF-2α,and VEGF In 60 case of resected specimens of renal cell carcinoma and 25 case their corresponding nomal renal tissues by immunohistochemical S-P method.Analyze the significance of these protein with the clinicopathologic features and outcome in human renal cell carcinoma.RT-PCR and western blot were used to detect the expression levels of HIF-2αand VEGF mRNAs and proteins.Results Increased expressions of Hsp90,HIF-2α,and VEGF were detected in renal cell carcinoma,and significant correlations were observed among them.the expressions of HSP90、HIF-2α、and VEGF were closely related with the stage,LN metastasis and the size of tumor of renal cell carcinoma.The overall survival rate is higher in renal cell carcinoma,which have low expression of HIF-2αand VEGF.The survival rate is slightly higher in renal cell carcinoma,which have low expression of Hsp90.Higher expression of HIF-2αwas detected only at protein level,and for VEGF both at mRNA and protein level via RT-PCR and western blot. Conclusions The expressions of Hsp90,HIF-2α,and VEGF are overexpressed in renal cell carcinoma.It is likely that HIF-2αis involved in the angiogenesis of renal cell carcinoma via upregulation of VEGF expression.Furthermore,Hsp90 and HIF-2αlikely plays an important role in renal cell carcinoma together.PartⅡStudy of the impact of biological behaviors on human renal cell carcinoma cells by 17-AAG in vitro.Objective To explore the effects of 17-AAG on human renal cell carcinoma cells in vitro.Methods human renal cell carcinoma cell line Caki-1 cells were cultured with or without 17-AAG under normoxic and hypoxic conditions,respectively.MTT assay was used to determine the proliferation rate of Caki-1 cells.Apoptosis was detected by the means of Hoechst 33342/PI double staining、AnnexinV-FITC/PI double staining and DNA ladder.Western blot were used to detect the expression of HIF-2αand VEGF protein.Results In experimented group cells,ability of proliferation was inhibited in hypoxic state,meanwhile,apoptotic rate increased.Expressions of HIF-2αand VEGF protein were significantly inhibited in Caki-1 cells of experimental group,compared with in that of negative control group or blank control group.Conclusions 17-AAG can inhibit cell proliferation,and promote apoptosis in human renal cell carcinoma cell of Caki-1 in vitro,and likely due to the inhibition of HIF-2αand VEGF expression.PartⅢResearch for inhibition human renal cell carcinoma by 17-AAG in vivoObjective To research growth of human renal cell carcinoma by 17-AAG in vivo via transplanted subcutaneously in nude mouse.Methods human renal cell carcinoma transplanted subcutaneously in nude mouse. 17AAG,DMSO,and NS were intratumorally injected with 125mg/kg respectively in subcutaneous xenograft model,inhibitory effect of tumor growth was observed,HIF-2αand VEGF protein expressions were detected by immunohistochemical S-P method, TUNEL stain and transmission electron microscopy was used to determine apoptosis.Results Growth rate of subcutaneous xenograft significantly decreased experimental group,the tumors size and weight of experimental group were significantly lesser than those of negative control group and blank control group.For experimental group, expressions of HIF-2αand VEGF proteins were weaker than those of negative control group and blank control group.Conclusions 17-AAG can dramatically inhibit the growth of human renal cell carcinoma,likely due to upregulation the expression of HIF-2αand VEGF.更多还原