【作者】 郑建明；

【导师】 徐勇； 宋文利； 莫春柏；

【作者基本信息】 天津医科大学 ， 外科学， 2008， 博士

【摘要】 目的:构建与免疫基因相关的基因芯片,并利用其筛选出与急性排异反应相关的差异表达基因。对筛选出表达量增高的基因,通过聚类分析,确定高表达基因,并用定量PCR对该高表达基因进行定量分析,从而验证构建基因芯片的稳定性及准确性。对具有代表性的基因进行临床应用的初步研究。方法:通过自行设计的包含有CD分子、细胞因子、趋化因子、粘附因子、FasL、穿孔素、颗粒酶B等共475个免疫相关基因的基因芯片,对临床上肾移植术后发生急性排斥反应和非急性排异反应患者进行了研究。以手术后第四天和移植肾肾穿刺活检当日/随访日收集外周血作为对照血样和实验血样,用Ficoll技术提取外周血淋巴细胞,并采用一步法提取总RNA,逆转录合成cDNA探针,荧光染料标记后,与芯片进行杂交,扫描后筛选出差异表达基因。对筛选出表达量增高的基因,通过聚类分析,确定高表达基因,并用定量PCR对该高表达基因进行定量分析。根据发现与急性排斥反应相关的基因,初步探讨其在移植肾急性排斥反应发生中的机理;对具有代表性的穿孔素、颗粒酶B、IL-4进行PCR定量,并对穿孔素、颗粒酶B进行临床应用研究。结果:研究发现外周血淋巴细胞中与急性排斥反应可能相关的免疫基因,筛选出急性排斥反应时表达量出现差异的基因,其中急性排斥发生时外周血淋巴细胞免疫相关基因差异表达上调20条,包括:IL-4、IL-6、IL-10、IL-2、IL-15、INF-γ、TNF-α、CD126、CTLA-4、IL-5、IL-13、IL-18、CD40L、Perforin、GranzymeB、FasL、MCP1、CD30、HLA-DR、MIP-1。下调11个基因,包括:TGFB1、RAB5C、RAB6C、RAB10、IL-17、IL-12、CD4、CD47、CD24、CD79B、CD81。而在非急性排异组患者中并没有发现上述基因的显著改变。对其中的穿孔素、颗粒酶B、IL-4进行实时免疫荧光PCR定量验证,证实他们在急性排异反应时较肾功能稳定时出现量上出现显著性升高(P＜0.05),治疗后显著下降(P＜0.05)。应用实时荧光定量PCR对临床上38例移植肾急性排异反应和20例移植肾功能稳定患者进行研究,发现应用外周血淋巴细胞的穿孔素mRNA水平来诊断移植肾急性排异反应的敏感度为86.8%,特异度为80%。临床应用外周血淋巴细胞的颗粒酶mRNA水平来诊断移植肾急性排异反应的敏感度为86.8%,特异度为90%。临床应用外周血淋巴细胞的IL-4的mRNA水平来诊断移植肾急性排异反应的敏感度为86.8%,特异度为75%。结论:本课题通过自行设计的具有475个免疫相关基因的基因芯片,对临床上肾移植术后发生急性排斥反应患者进行了研究。并应用PCR定量验证,结果显示此基因芯片具有良好的稳定性、准确性。发现了外周血淋巴细胞中与急性排斥反应可能相关的免疫基因,筛选出急性排斥反应时表达量出现差异的基因,其中急性排斥发生时外周血淋巴细胞免疫相关基因差异表达上调20个、下调11个。对具有代表性的穿孔素、颗粒酶B、IL-4进行PCR定量,发现三者在移植肾急性排异反应时明显升高。临床应用外周血淋巴细胞的穿孔素、颗粒酶、IL-4mRNA水平来诊断移植肾急性排异反应的具有较好的敏感度和特异度。更多还原

【Abstract】 Object:To construct the gene array related with immunology gene and bolting the deference related with acute rejection by gene array to express gene.for bolting express-dose increasing gene,through cluster analysis,to confirm high-express gene and make quantitative analysis for the high-express gene by quantitation PCR,in order to validate stability and accuracy of the constructed gene array.Method:Through self-designed gene array related with 475 immunology gene including CD molecule,cell factor,chemotactic factor,adherence factor,Fasl, perforin,granzyme B,etc.,to make clinical study for renal post-operated patients that with acute rejection or non-acute rejection.Collecting periph blood at the day of 4-day after operation and renal graft puncture/follow-up day as the compare sample and lab sample,extracting periph blood lymph cell by Ficoll technology,and extracting total RNA by one step method,reverse transcription composition cDNA probe,fluor colorant marked,make array hybridization,bolting the deference after scan,express gene.For bolting express-dose increasing gene,through cluster analysis, to confirm high-express gene and make quantitative analysis for the high-express gene by quantitation PCR.According to the discovered gene related with acute rejection,initially approach the mechanism when renal graft acute rejection happens, make PCR quantitation to representative perforin,granzyme B,IL-4,make clinical application study to perforin,granzyme B.Result:From the study,discover immunology gene in periph blood lymph cell which is possibly related with acute rejection and bolting the gene whose express-dose appears deference at the acute rejection.Periph blood lymph cell immunology related gene’s deference expresses up-regulation 20,including IL-4、IL-6、IL-10、IL-2、IL-15、INF-γ、TNF-α、CD126、CTLA-4、IL-5、IL-13、IL-18、CD40L、Perforin、Granzyme B、FasL、MCP1、CD30、HLA-DR、MIP-1;down-regulation 11,including TGFB1、RAB5C、RAB6C、RAB10、IL-17、IL-12、CD4、CD47、CD24、CD79B、CD81.There is no markedly change for the above gene in the non-acute rejection patients group.To make real-time immunology fluor PCR quantitation validation for perforin,granzyme B,IL-4,it proves that their express-dose at acute rejection increases more markedly than at renal function stability(P＜0.05),deceases less markedly after treated(P＜0.05).Using real-time fluor PCR quantitation,make clinical study of 38 renal graft acute rejection patients and 20 renal graft function stable patients,we find that sensitiveness is 86.8%and specificity is 80%by perforin mRNA in periph blood lymph cell to diagnose renal graft acute rejection;sensitiveness is 86.8%and specificity is 90%by granzyme mRNA in periph blood lymph cell to diagnose renal graft acute rejection;sensitiveness is 86.8%and specificity is 75%by IL-4 mRNA in periph blood lymph cell to diagnose renal graft acute rejection.Conclusion:Through self-designed gene array with 475 immunology related gene, this article studies clinical patients who have acute rejection after renal transplantation operation.Using PCR quantitation validation,the result is that the gene array has good stability and accuracy.Discover immunology gene in periph blood lymph cell which is possibly related with acute rejection and bolting the gene whose express-dose appears deference at the acute rejection.Periph blood lymph cell immunology related gene’s deference expresses up-regulation 20 and down-regulation 11.Make PCR quantitation to representative perforin,granzyme B, IL-4,then discover them increase markedly at renal graft acute rejection It will have good sensitiveness and specificity on clinical application to diagnose renal graft acute rejection through perforin,granzyme B,IL-4mRNA level in periph blood lymph cell.更多还原