Construction Of Renal Graft Acute Rejection Related Immunology Gene Array, Test And Application On Clinical Study

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.