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华中科技大学 博士学位论文 LRIG2基因和蛋白在膀胱肿瘤中的表达及重组质粒载体pCMV- LRIG2转染BIU-87细胞株的效应研究 姓名:李国灏 申请学位级别:博士 专业:外科学(泌尿外科) 指导教师:叶章群 20070501 华中科技大学同济医学院 博士学位论文 5 第一部分第一部分 LRIG2 基因在膀胱肿瘤中的表达基因在膀胱肿瘤中的表达 Part I The Expression of LRIG2 Gene in Bladder Cancer 摘摘 要要 目的目的:了解 LRIG2 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 方法方法: 利用半定量 RT-PCR 技术检测 31 例膀胱肿瘤组织和 16 例正常膀胱粘膜组 织内 LRIG2 基因 mRNA 表达情况,同时结合病例的分级、分期等对结果进行统 计学分析。 结果结果:38 例肿瘤标本中,34 例检测到 LRIG2mRNA 的表达,表达水平(灰度比值) 平均为 0.390.22( s);16 例正常粘膜标本中均检测到 LRIG2mRNA 的表达, 表达水平平均为 0.540.16。两者相比较 P=0.012,有统计学差异,肿瘤组织内 LRIG2mRNA 的表达水平显著低于正常膀胱组织。肿瘤组织中,浅表型肿瘤表达水 平为 0.440.16,浸润型为 0.210.12,两者相比较 P=0.012,有统计学差异; 低级别肿瘤表达水平为 0.490.18,中高级别为 0.330.16,两者相比较 P=0.033,有统计学差异。 结论结论:LRIG2 基因在膀胱肿瘤组织中表达水平明显下调,且与肿瘤的分级分期相 关,提示其有可能为一种抑癌基因,与肿瘤的发生、发展有密切关系。 关键词关键词:LRIG2, 膀胱肿瘤, RT-PCR 华中科技大学同济医学院 博士学位论文 16 第二部分第二部分 LRIG2蛋白在膀胱肿瘤中的表达蛋白在膀胱肿瘤中的表达 Part II The Expression of LRIG2 Protein in Bladder Cancer 摘摘 要要 目的目的:了解 LRIG2 蛋白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 方法方法:利用 Western blot 技术检测 38 例膀胱肿瘤组织和 16 例正常膀胱粘膜组织 内 LRIG2 蛋白 A 表达情况,并对结果进行统计学分析。 结果结果:38 例肿瘤标本中,33 例检测到 LRIG2 蛋白的表达,表达水平(灰度比值) 平均为 0.410.15 ( s);16 例正常粘膜标本中均检测到 LRIG2 蛋白的表达, 表达水平平均为 0.500.08。两者相比较 P=0.038,有统计学差异,膀胱肿瘤组 织内 LRIG2 蛋白的表达水平显著低于正常膀胱组织。肿瘤组织中,浅表型肿瘤表 达水平为 0.450.13,浸润型为 0.270.13,两者相比较 P=0.027,有统计学 差异;低级别肿瘤表达水平为 0.490.15,中高级别为 0.350.12,两者相比 较 P=0.040,有统计学差异。 结论结论:LRIG2 蛋白在膀胱肿瘤组织中表达水平明显下调,且与肿瘤的分级分期相 关, 提示其有可能是一种具有抑癌功能的蛋白, 与肿瘤的发生、 发展有密切关系。 关键词关键词:LRIG2, 膀胱肿瘤,Western blot 华中科技大学同济医学院 博士学位论文 29 第三部分第三部分 重组质粒载体重组质粒载体pCMV-LRIG2转染转染BIU-87细胞株的效应研究细胞株的效应研究 Part III The Research on The Effect of BIU-87 Cell Transfected by Recombinated Plasmid pCMV-LRIG2 摘摘 要要 目的目的:研究携带有 LRIG2 基因的重组质粒载体 pCMV-LRIG2 对膀胱癌细胞株 BIU-87 生物学行为的影响。 方法方法:用脂质体介导的基因转染技术,将携带有 LRIG2 基因的重组质粒载体 pCMV-LRIG2 转染至膀胱癌细胞株 BIU-87, 用 G418 筛选出抗性克隆后, Western 检测转染前后 LRIG2 和 EGFR 蛋白表达水平变化,MTT 法绘制转染前后细胞生 长曲线,Transwell 法检测转染前后细胞侵袭力,同质粘附实验观察转染前后细 胞粘附能力。 结果结果:转染 pCMV-LRIG2 组 LRIG2 蛋白表达水平显著高于未转染组和空载体转 染组; 转染 pCMV-LRIG2 组细胞生长速度较未转染组和空载体转染组明显减慢, 侵袭力显著低于未转染组和空载体转染组, 而粘附能力显著高于未转染组和空载 体转染组。 结论结论:LRIG2 有类似于抑癌基因的作用,通过对 EFG-EGFR 信号转导途径发挥负 反馈抑制作用,从而抑制 BIU87 细胞的生长、侵袭和转移。 关键词:关键词:LRIG2, 膀胱肿瘤, 效应 华中科技大学同济医学院 博士学位论文 6 Abstract Purpose: To investigate the expression of LRIG2 gene between bladder cancer and normal bladder tissue, and to identify the different expression in dissimilar grading and staging. Methods: RT-PCR technique was used to detect the expression of LRIG2 mRNA in 31 cases of bladder cancer tissue and 16 cases of normal bladder tissue. Statistics analysis was combined with the staging and grading of the tumor. Results: In the 31 cases of bladder cancer tissue, 34 cases were detected the expression of LRIG2 mRNA, and the average level was 0.390.22. All the 16 cases of normal bladder tissue were detected the expression of LRIG2 mRNA, and the average level was 0.540.16. There was significant difference between the bladder cancer group and the normal bladder tissue group (P=0.012).The expression of LRIG2 mRNA in bladder cancer was significantly lower than in the normal bladder tissue. In the bladder cancer group, the average expression level of superficial group and invasive group were 0.440.16 and 0.210.12, and there was significant difference between the two group (P=0.012). The average expression level of lower grading and higher grading were 0.490.18 and 0.330.16, and there was significant difference between the two group (P=0.033). Conclusion: The expression of LRIG2 gene was significantly down-regulated in bladder cancer, and the expression level was correlated with the grading and staging of the tumor, suggesting that the gene maybe a kind of cancer inhibiting gene and participating the arising and developing of the cancer. Key words: LRIG2, bladder cancer, RT-PCR 华中科技大学同济医学院 博士学位论文 17 Abstract Purpose: To investigate the expression of LRIG2 protein between bladder cancer and normal bladder tissue, and to identify the different expression in dissimilar grading and staging. Methods: Western blot technique was used to detect the expression of LRIG2 protein in 31 cases of bladder cancer tissue and 16 cases of normal bladder tissue. Statistics analysis was combined with the staging and grading of the tumor. Results: In the 31 cases of bladder cancer tissue, 33 cases were detected the expression of LRIG2 protein, and the average level was 0.410.15. All the 16 cases of normal bladder tissue were detected the expression of LRIG2 protein, and the average level was 0.500.08. There was significant difference between the bladder cancer group and the normal bladder tissue group (P=0.038). The expression of LRIG2 protein in bladder cancer was significantly lower than in the normal bladder tissue In the bladder cancer group, the average expression level of superficial group and invasiv
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