【作者】 潘翠翠；

【导师】 罗春丽；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 第一部分hepaCAM在TCCB中表达及临床意义目的探讨肝细胞粘附分子(hepatocyte cell adhesion molecule, hepaCAM)在膀胱移行细胞癌(transitional carcinomal of the bladder, TCCB)中表达及与临床病理参数关系。方法采用半定量RT-PCR法检测hepaCAM基因在膀胱癌细胞株(T24、BIU-87)和55例TCCB组织及癌旁组织中hepaCAM mRNA表达、并分析其与临床病理参数间的关系。结果hepaCAM mRNA在膀胱癌细胞株(T24、BIU-87)中表达缺失,hepaCAM mRNA在TCCB组织中的表达水平明显低于癌旁组织(P<0.001), hepaCAM mRNA表达与TCCB临床分期有关(P<0.05),而与病理分级、性别、年龄无关。结论hepaCAM mRNA在膀胱癌细胞株(T24、BIU-87)及TCCB组织中表达明显下降或缺失,且与临床分期有关,推测hepaCAM可能是TCCB新的候选抑癌基因。第二部分TCCB中hepaCAM基因外显子2甲基化状态分析目的检测膀胱癌细胞株(T24、BIU-87)和TCCB组织中hepaCAM外显子2的甲基化情况,探讨hepaCAM基因在TCCB中表达下调是否与hepaCAM外显子2的甲基化有关,从表观遗传学角度探讨膀胱癌发病的机制。方法采用甲基化敏感性限制性内切酶PCR法检测膀胱癌细胞株(T24、BIU-87)和TCCB组织中hepaCAM外显子2甲基化情况,分析其与临床病理参数之间的关系,并探讨TCCB组织中hepaCAM外显子2甲基化与hepaCAM mRNA表达之间的关系。结果甲基化敏感性限制性内切酶PCR检测发现膀胱癌细胞株(T24、BIU-87)和52.7%(29/55)TCCB组织中存在hepaCAM外显子2甲基化,而相应的癌旁组织甲基化率为5.5%(3/55),hepaCAM外显子2甲基化率在癌及癌旁组织中的差异具有显著统计学意义(P<0.001)。hepaCAM外显子2甲基化与hepaCAM mRNA表达呈负相关(r=﹣0.403,P<0.05)。结论hepaCAM外显子2甲基化是导致TCCB组织中hepaCAM mRNA表达下调的主要原因之一,为探讨揭示TCCB发生发展机制提供新的理论依据。第三部分DNA甲基化调控膀胱癌细胞株hepaCAM表达的机制目的探讨5-氮2-脱氧胞苷(5-Aza-CdR)(DNA甲基化转移酶抑制剂)对膀胱癌细胞株(T24、BIU-87)中hepaCAM表达的调控作用及对细胞生长增殖的影响。进一步探讨hepaCAM外显子2甲基化导致hepaCAM下调TCCB发生的机制。方法分别用不同药物浓度的5-Aza-CdR处理T24和BIU-87细胞株,MTT法检测5-Aza-CdR对细胞增殖的影响,采用甲基化敏感性限制性内切酶PCR法hepaCAM基因外显子2区域甲基化状态,RT-PCR法检测5-Aza-CdR处理前后hepaCAM mRNA的表达情况。结果5-Aza-CdR具有抑制T24和BIU-87细胞增殖作用。未经5-Aza-CdR处理的T24和BIU-87细胞hepaCAM mRNA不表达,且hepaCAM基因外显子2区高甲基化;经5-Aza-CdR处理后T24和BIU-87细胞hepaCAM基因表达,细胞中hepaCAM基因外显子2区甲基化状态得到逆转。结论5-Aza-CdR能有效逆转膀胱癌T24和BIU-87细胞hepaCAM基因外显子2的异常甲基化,从而诱导hepaCAM基因的重新表达,抑制肿瘤细胞生长,结果表明:hepaCAM外显子2甲基化是促进hepaCAM表达下调从而导致膀胱癌发生的新的分子机制,为TCCB治疗提供新的思路。更多还原

【Abstract】 PART ONE: THE EXPRESSION OF HEPACAM IN TCCBObjective: To explore the expression of hepaCAM (hepatocyte cell adhesion molecule) mRNA in TCCB(transitional carcinomal of the bladder)and the relationship between hepaCAM mRNA and the clinical pathology parameters.Methods: To detect expression of hepaCAM mRNA by RT-PCR in bladder cancer cell lines (T24,BIU-87), 55 TCCB tissues and corresponding adjacent tissues and analyze the relationship between hepaCAM mRNA and the clinical pathology parameters.Results: The expression of hepaCAM mRNA was deficient in bladder cancer cell lines(T24,BIU-87). the expression level of hepaCAM in TCCB tissues was lower than that in adjacent tissues (P<0.001). No significant difference of hepaCAM mRNA was detected in the pathological grade, gender and age, but there was statistically significant differences in clinical stage (P<0.05).Conclusion: The expression of hepaCAM mRNA is obviously decrease or deficient in TCCB tissues and bladder cancer cell lines (T24,BIU-87). HepaCAM may be a putative tumor suppressor gene in TCCB.PART TWO THE ANALYSIS OF THE MEHYLATION STATUS OF HEPACAM EXON2Objective: To detect the methylation status of hepaCAM exon 2 in bladder cancer cell lines(T24,BIU-87) and TCCB tissues and explore the potential mechanism of the down-regulation of hepaCAM expression in TCCB.Methods: We use the methylation-specific restriction-PCR-assay to detect the methylation status of hepaCAM exon 2 in TCCB and bladder caner cell lines(T24,BIU-87), analyze the relationship between the methylation of hepaCAM exon 2 and the clinical pathology parameters and explore the relationship between the methylation of hepaCAM exon 2 and expression of hepaCAM mRNA from the perspective of epigenetics.Results: The methylation status of hepaCAM exon 2 was observed in bladder caner cell lines(T24,BIU-87) and 29 of 55 primary cancers, whereas in the adjacent tissues by methylation-specific restriction-PCR-assay , only 3 of 55 cases exhibited hepaCAM exon 2 methylation, and the methylation rate of hepaCAM exon 2 was significant higher in TCCB than that in adjacent renal tissues(52.7% vs. 5.5%,P<0.001). the methylation of hepaCAM exon 2 was negatively correlated to the expression of hepaCAM(r=-0.403,P<0.05).Conclusion: The methylation of hepaCAM exon 2 may be one important reasion for down-regulation of hepaCAM expression in TCCB. The study of hepaCAM methylation provides a new theoretical evidence for the mechanism of the occurrence and development of TCCB.PART THREE THE MECHANISM OF DNA METHYLATION REFULATION HEPACM EXPRESSION IN BLADDER CANCER CELLSObjective: The study is to explore the transcription regulation 5-Aza-2-deoxycytidine (5-Aza-CdR) on hepaCAM tumor suppressor gene in bladder cancer cell lines(T24,BIU-87)and to investigate its effect on the growth of T24 and BIU-87 cells and to further explore the mechanism of hepaCAM exon2 resulting in down-regulation of hepaCAM expression in TCCB.Methods: T24 and BIU-87 cell lines were separately treated with different dose of 5-Aza-CdR.MTT was used to detect the effect of 5-Aza-CdR on the growth of T24 and BIU-87 cells. The status of hepaCAM exon 2 methylation was analyzed using methylation-specific restriction-PCR-assay. RT-PCR was used to examine the expression of hepaCAM gene in before and after treated T24 and BIU-87 cells.Results: The growth of the T24 and BIU-87 cells was suppressed by treatment with 5-Aza-CdR.Before 5-Aza-CdR treatment, hypermethylation of hepaCAM exon 2 exised and hepaCAM mRNA was not expressed in T24 and BIU-87 cells. HepaCAM mRNA was re-expressed and the methylation status of hepaCAM exon 2 was obviously reversed after treatment with 5-Aza-CdR.Conclusion: 5-Aza-CdR may effectively cause the demethyaltion of hepaCAM exon 2, induce the expression of hepaCAM, and inhibit the growth of tumor cells.The result suggest that the methylation of hepaCAM exon2 is a new mechanism of resulting in down-regulation of hepaCAM expression in TCCB.This provides new ideas for the treatment of TCCB.更多还原