【作者】 谢宇；

【导师】 杨罗艳；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 膀胱癌是泌尿系统最常见的恶性肿瘤。Ezrin被研究发现同许多恶性肿瘤的发生、发展、浸润、转移有关。但是ezrin同膀胱移行细胞的作用不清楚,并且未见有用RNAi技术研究ezrin同膀胱移行细胞癌关系的研究。本实验我们首次使用siRNA抑制膀胱移行细胞癌细胞株T24中ezrin基因的表达,研究ezrin在膀胱癌发生、发展、侵袭中的作用.本实验同时探讨了RhoA与ezrin在膀胱癌中的关系。为探讨膀胱癌基因治疗新途径提供理论基础。第一部分膀胱移行细胞癌组织中ezrin、CD44的表达目的探讨ezrin、CD44在膀胱移行细胞癌中的表达与意义方法1、采用免疫组织化学检测60例膀胱移行细胞癌与14例正常膀胱组织中ezrin、CD44的蛋白表达。2、采用RT-PCR方法检测40例膀胱移行细胞癌与10例正常膀胱组织中ezrin、CD44的mRNA表达.结果1、ezrin、CD44在膀胱移行细胞癌组织中的蛋白表达明显高于正常膀胱组织(P<0.05), ezrin、CD44的蛋白表达与膀胱移行细胞癌组织的病理分级、临床分期密切相关,ezrin、CD44蛋白表达的阳性表达随着膀胱肿瘤病理分级、临床分期的增高而增高(P<0.05)。2、ezrin、CD44在膀胱移行细胞癌组织中的mRNA表达明显高于正常膀胱组织(P<0.05), ezrin、CD44的mRNA表达与膀胱移行细胞癌组织的病理分级、临床分期密切相关,ezrin、CD44mRNA的阳性表达随着膀胱肿瘤病理分级、临床分期的增高而增高(P<0.05)。3、ezrin、CD44的蛋白表达在膀胱移行细胞癌中有正相关性。结论ezrin、CD44蛋白均在BTCC中表达异常增强,在BTCC发生、发展过程中起重要作用。ezrin可作为诊断、判断预后、指导治疗、随访检测的指标。ezrin基因可能成为BTCC新的治疗靶点。第二部分应用RNAi沉默ezrin基因对膀胱移行细胞癌株生物学行为的影响目的探讨ezrin基因表达与膀胱移行细胞癌株生物学行为的关系。方法1、设计siRNA ezrin,用于干扰并下调T24细胞中目的基因的表达。2、采用RT-PCR以及western-blot方法检测实施siRNA干扰后,T24细胞中ezrin的mRNA以及蛋白量。3、采用绘制细胞生长曲线、MTT实验、流式细胞检测、细胞划痕实验以及细胞迁徙实验、细胞粘附实验,观察siRNA干扰后,细胞生物学行为变化。结果1、设计的siRNA能够很好的对目的基因产生沉默效应。2、转染siRNA后的实验组T24细胞,ezrin的mRNA表达量较对照组明显下调,差异有统计学意义(P<0.05)。ezrin蛋白表达量显著下调,差异有统计学意义。3、转染siRNA后的实验组T24细胞,细胞生长速度较对照组明显减慢,差异有统计学意义(P<0.05)；实验组T24细胞凋亡率为(30.37±8.37)%,实验组细胞凋亡率同空白组、阴性对照组比较,凋亡率明显增加,(p<0.05)。24小时后,实验组T24细胞迁移到划痕区的细胞明显低于空白组和阴性对照组。实验组发生迁移细胞数目明显降低,迁移能力明显下降,与对照组比较差异有显著性(P<0.05)。siRNA干扰可以降低细胞的粘附能力,空白组、阴性对照组、实验组细胞粘附率分别为72.3±5.1%,70.5±5.7%以及39.2±4.6%,差异有统计学意义(p<0.05)结论siRNA可以明显下调T24细胞中ezrin的mRNA以及蛋白表达量,且导致细胞生物学行为的改变,表现为细胞的生长减慢、凋亡增加,细胞的迁移能力及粘附能力降低。第三部分ezrin同RhoA信号传导通路关系的研究目的探讨RhoA及ezrin在膀胱癌中的关系。方法1)EGF作用于膀胱移行细胞癌T24细胞,采用Western-blot检测ezrin、RhoA、磷酸化RhoA的蛋白含量变化。2)将EGF及RhoA激酶抑制剂Y-27632作用于膀胱移行细胞癌T24细胞,检测ezrin、磷酸化RhoA的蛋白含量变化。结果Western-blot结果显示,在给予EGF刺激后,p-RhoA水平逐渐升高,于10、30、60、90min时分别升高了70±11%,250±23%,420±47%以及490±43%。在EGF刺激下RhoA蛋白表达无明显变化。Western-blot结果显示,在给予EGF刺激后,ezrin水平逐渐升高,于8、16、24小时分别升高了170±60%,310±73%,750±47%。用Y-27632预处理T24细胞,EGF诱导的RhoA磷酸化水平明显受到抑制,同时ezrin蛋白表达水平也受到抑制。结论RhoA可能作为ezrin的上游调控元件。更多还原

【Abstract】 Bladder cancer was the most common cancer in urology.Ezrin has been thought that it may play an important role in genesis、development、invasion metastasis of variety tumor. What kind of role ezrin playing was not yet clear in development of BTCC..Experimental study on siRNA of ezrin transfecting BTCC cell lines was not yet reported at home and abroad. The first time we used siRNA to inhibit BTCC cell line T24 to reduced the ezrin gene expression,to study fuction of ezrin in BTCC genesis、development、invasion.The relationship of RhoA and ezrin in BTCC also was studied in this study. Providing foundation of theory which can explore a new way of gene therapy in BTCC.PartⅠThe expression of ezrin and CD44 in bladder transitional cell carcinomaObjectiveTo explore the expression of ezrin and CD44 in bladder transitional cell carcinoma.MethodsImmunohistochemical method was used to assess the expression of ezrin and CD44 in 60 cases of bladder transitional cell carcinoma and 14 cases of control. RT-PCR was used to determine the mRNA of ezrin and CD44 in 40 cases of bladder transitional cell carcinoma of and 10 cases of control.ResultsThe protein expression of ezrin and CD44 were significantly higher in BTCC than that of the controls (P<0.05). The protein expression of ezrin and CD44 were positively correlated with the histological grade and clinical stage of the BTCC(P<0.05). The mRNA expression of ezrin and CD44 were significantly higher in BTCC than that of controls. The mRNA level of ezrin and CD44 were positively correlated with the histological grade and clinical stage of the BTCC(P<0.05).ConclusionBoth expression of ezrin and CD44 were higher in BTCC. The ezrin and CD44 play important role in the development of BTCC. The ezrin can serve as indicators of diagnosis,prognosis and guide treatment and the new target of the BTCC therapy.PartⅡsiRNA on ezrin effect on biological behavior of bladder carcinoma cellObjectiveTo explore the relationship between the ezrin gene expression in BTCC and the biological behavior of strain.MethodsThe siRNA ezrin probe was designed to interfere and reduce the ezrin gene expression in T24 cell. The RT-PCR and western-blot were used to determined the mRNA and protein of ezrin in the cells, which were treated by siRNA ezrin. Cell growth curve,MTT assay,The flow cytometry,cell scratch experiments and cell migration experiments cell adhere experiments were used to test the biological behavior of cells, which were treated by siRNA ezrin.ResultsThe siRNA silencing effect was observed by the treatment of siRNA ezrin. After the siRNA transfection, the mRNA expression of ezrin in experimental group was significantly lower than that of control (P<0.05). The ezrin protein expression was significantly reduced by the transfection (P<0.05).The growth rate of T24 cells, which was treated by siRNA was much slower than the control group, the difference was statistically significant. The apoptosis rate of experimental group was 30.37±8.37%, which was signicantly increased than those of blank control and negative control(P<0.05). The migrating cells was significantly lower than the control group and negative control group. The number of migrating cells in the experimental group was signicantly smaller than those of control (P<0.05). The treatment of siRNA can reduce the cell adhesion capacity.The adhesion rate of the blank control and negative control and experiment group were 72.3±5.1%,70.5±5.7% and 39.2±4.6% respectively. The difference was statistically different (P<0.05).ConclusionThe siRNA can signicantly decreased the mRNA and protein expression of ezrin in T24 cells and cause the different behavioral. The decreased growth rate, adhesion and migrating capacity and increased apoptosis can be observed after the siRNA treatment.PartⅢThe study of the relationship of ezrin and RhoA in BTCCObjectiveTo explore the the relationship of ezrin and RhoA in BTCCMethodWestern-blot was used to determine the protein expression of Ezrin and RhoA and p-RhoA after the treatment of EGF. The protein expression of ezrin and p-RhoA after the treamtment of EGF and Y-27632.ResultsThe p-RhoA protein expression was increased by 70±11%, 250±23%,420±47% and 490±43%, after the treatment of EGF for 10、30、60、90mins. No difference of the RhoA protein expression was observed after the treatment of EGF. The ezrin protein expression was significantly increased by 170±60%,310±73%,750±47% after the treatment of EGF for 8、16、24 hours. The Y-27632 treatment can decreased the effect of EGF on the expression of p-RhoA and ezrin.ConclusionRhoA may regulatd ezrin as a upstream regulatory signal.更多还原