【作者】 袁晓奕；

【导师】 叶章群； 杨为民；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 近年来研究发现，LRIGs基因家族在多种肿瘤中具有抑癌作用，其可能的机制是下调EGFR的表达，从而抑制EGF对人体肿瘤所带来的促细胞增殖、侵袭等作用。但其在膀胱癌中的作用报道甚少，本研究检测了LRIG3基因及蛋白在膀胱癌中的表达，并将其重组腺病毒质粒感染人膀胱癌T24细胞系，观察了LRIG3对T24细胞生长、凋亡及迁移的影响等，分三个部分简述如下。第一部分LRIG3蛋白及基因在膀胱肿瘤中的表达及意义目的：了解LRIG3蛋白及基因在膀胱肿瘤和正常膀胱组织中的表达及其与膀胱肿瘤不同分级、分期之间的关系。方法：采用免疫组化SP法和逆转录．聚合酶链反应(RT-PCR)法检测64例膀胱尿路上皮癌组织和12例正常膀胱粘膜中LRIG3蛋白及mRNA的表达情况，并结合临床资料进行分析。结果：膀胱癌组织中LRIG3蛋白和mRNA的表达水平显著低于正常膀胱黏膜组织；且分级和分期越高，LRIG3蛋白和mRNA的表达水平越低。结论：LRIG3基因在膀胱癌中存在表达缺失和低表达，提示LRIG3基因在膀胱癌的发生发展中可能具有抑癌基因的作用，有可能成为肿瘤基因治疗的又一靶点。第二部分LRIG3基因特异性腺病毒表达载体的构建与鉴定目的：构建并鉴定含有LRIG3基因的腺病毒表达载体rAd-LRIG3，并感染人膀胱癌细胞T24，为后期检测LRIG3对膀胱癌生物学行为的影响提供靶细胞。方法：通过质粒提取、琼脂糖凝胶电泳、酶切、连接、转化等多种基因工程技术，从感受态大肠杆菌DH5α菌株中提取并大量制备pLRIG3-EGFP真核表达质粒，然后对其行限制性内切酶酶切、凝胶电泳法进行鉴定。测序正确后，利用腺病毒载体系统Adeno-X构建重组腺病毒质粒pAd-LRIG3，酶切、PCR鉴定重组腺病毒质粒，鉴定正确后，将pAd-LRIG3转染至HEK293细胞，包装成复制缺陷型腺病毒质粒rAd-LRIG3。大量扩增rAd-LRIG3，测病毒滴度。实验分为三组：实验组(T24-LRIG3组，感染rAd-LRIG3)、对照组(T24组，未感染腺病毒质粒)和感染空载体组(T24-HK组，感染rAd-HK)。感染48小时后，应用RT-PCR及Western-Blotting鉴定该质粒在细胞中是否得到表达。结果：酶切分析、测序鉴定表明rAd-LRIG3构建成功；RT-PCR及W-B分析表明，T24-LRIG3组中LRIG3mRNA及蛋白的表达水平较T24-HK组和T24组明显升高，差异有显著性(P＜0．05)。结论：携带有LRIG3基因的重组腺病毒构建成功；建立了rAd-LRIG3感染人膀胱癌细胞T24的长期稳定细胞系。可能为寻找膀胱癌的有效基因治疗途径奠定基础。第三部分LRIG3基因对膀胱癌细胞T24生物学行为的影响目的：研究LRIG3基因对膀胱癌细胞T24生物学行为的影响。以判断LRIG3基因在膀胱癌的发生和进展方面是否具有作用；进而说明LRIG3基因是否是膀胱癌病因学中的一个抑癌基因。方法：利用重组腺病毒质粒rAd-LRIG3感染膀胱癌细胞T24，得到稳定细胞株T24-LRIG3，细胞计数并绘制细胞生长曲线；Hoechst33258染色、透射电镜、流式细胞仪检测细胞凋亡情况；细胞迁移实验观察感染后细胞侵袭能力的变化。实验分为三组：实验组(T24-LRIG3组，感染rAd-LRIG3)、对照组(T24组，未感染腺病毒质粒)和感染空载体组(T24-HK组，感染rAd-HK)。结果：MTT法观察显示，感染rAd-LRIG3后细胞生长增殖速度较未感染组和空载体感染组明显减慢；Hoechst33258染色及透射电镜显示部分细胞呈现凋亡形态学改变；流式细胞仪定量检测表明感染LRIG3基因后，T24细胞出现较为明显的凋亡峰；细胞迁移力显著低于未转染组和空载体转染组。结论：LRIG3基因能有效抑制膀胱癌细胞的生长增殖和迁移，并诱导细胞的凋亡，是膀胱癌病因学中的一个抑癌基因。将来可望为膀胱癌的基因治疗提供新方法。更多还原

【Abstract】 PartⅠ. The expression of the LRIG3 gene in Urothelial carcinoma of thebladder and its clinical significanceObjective: To investigate the relationship between the expression of leucine-richrepeats and immunoglobulin-like domain protein 3 (LRIG3) gene and the biologicalbehavior of bladder urothelial carcinoma (BUC).Methods: Expressions of LRIG3 in 64 specimens of bladder urothelial carcinoma and12 specimens of normal bladder tissues were detected by SP of immunohistochemistry andRT-PCR. The results were analyzed in relation to the clinical data.Results :The expression of LRIG3 protein and mRNA in bladder cancer weresignificantly lower than that of normal bladder epithelium. The expression of LRIG3protein and mRNA were significantly decreased in higher staging and grading tumorscomparing to lower ones.Conclusions: The expression of LRIG3 gene was lost or reduced in bladder urothelialcarcinoma, which indicates that the LRIG3 gene may play an important role in suppressingthe tumorigenesis and development in bladder urothelial carcinoma.It may be a new targetfor tumor gene therapy. PartⅡ. The construction and identification of Adenovirusexpression vector targeting LRIG3 geneObjective : To construct and identify adenovirus vector that expresses the LRIG3 gene(rAd-LRIG3) and to establish a monoclone T24 cell line transfected with plasmidrAd-LRIG3, which will enable development of a gene therapy protocol for the treatment ofhuman bladder carcinoma.Methods: Genetic engineering techniques, including plasmid extraction, agarose gelelectrophoresis, restriction enzymolysis, connection, transformation of competent cells,were used to construct the recombinant plasmid of pLRIG3-EGFP from competentescherichia coli DH5α, which is confirmed by restriction enzyme digestion, agarose gelelectrophoresis and sequencing identification. The adenovirus plasmid rAd-LRIG3 wasconstructed by Adeno-x expression system, and the control adenovirus plasmid rAd-HKwas constructed by the same system. After verification by enzymolysis and PCR, thepAd-LRIG3 vector was cotransfected into 293 cells where they were packed as thereplication-deficient adenovirus rAd-LRIG3, rAd-LRIG3 was abundantly amplified andthen virus titer was evaluated. The whole experiment was divided into 3 groups. TherAd-LRIG3 was used to infect the human bladder cancer T24 cells. For controlexperiments, the vector rAd-HK was also transfected into T24 cells and nontransfected T24cells. RT-PCR and Western Blotting were used to detect the LRIG3mRNA and proteinexpression in T24 cell lines.Results:rAd-LRIG3 was successfully constructed and verified by enzymolysis andsequencing. RT-PCR and W-B analysis showed that LRIG3mRNA and protein level inrAd-LRIG3 transfected T24 cells was significantly higher than that in rAd-HK transfectedand nontransfected T24 cells; the difference was significant(P＜0.05).Conclusions: Successful construction of the recombinant adenovirus vector containingthe LRIG3 gene was achieved. A T24 cell line transfected with rAd-LRIG3 was obtained, which could stably express the LRIG3 protein in cells, and it will establish a foundation forthe future research about bladder cancer gene therapy.PartⅢ. The Effects of LRIG3 Gene on the Biological Behaviors ofHuman Bladder Cancer Cell (T24)Objective: To investigate whether the expression of gene LRIG3 can effect thegrowth, proliferation and apoptosis of bladder cancer T24 cells.Methods: The whole experiment was divided into 3 groups. The rAd-LRIG3 was usedto infect the human bladder cancer T24 cells. For control experiments, the vector rAd-HKwas also transfected into T24 cells and nontransfected T24 cells. The effect of LRIG3 onthe cellular proliferation capacity of T24 cells was assayed by the growth curve. The cellapoptosis was detected by Hoechst33258 staining, electron microscope, and flow cytometryanalysis. The cell migration assay was used to detect the difference of invasion andmetastases between transfected and non-transfected cells.Results: MTT showed the cell proliferation was markedly inhibited compared with thecontrol cells. Partial cancer cells presented morphological changes of apoptosis byHoechst33258 staining , electron microscope and flow cytometry analysis. Contrast tocontrol cells and rAd-HK transfected T24, the invasion ability of rAd-LRIG3 transfectedcells decreased obviously.Conclusion: LRIG3 gene can suppress T24 cell growth and proliferation, in additionto acceleration of its apoptosis. LRIG3 may act as a tumours suppressor gene in bladdercancer progression. This implied the possibility of LRIG3 as the potential method for genetherapy of bladder cancer.更多还原