| At present,organ transplantation is the main way to save patients with end-stage disease.However,in the two years after transplantation,about 35%of patients will develop acute rejection.In the field of kidney transplant(KTx),the decline of transplant function can be detected until a significant graft injury occurs,with hysteresis.Serum creatinine can assess glomerular filtration rate.However,it is not specific for distinguishing between kidney injury and acute and chronic loss of function.Biopsy is the gold standard for the detection of transplant rejection,it is a traumatic examination often associated with complications.Currently,one of the major clinical problems faced by transplant patients is the lack of highly sensitive,highly specific and non-invasive tests for early diagnosis and continuous monitoring of graft rejection risk.Cell-free DNA(cfDNA)refers to a partially degraded endogenous DNA that is free of extracellular cells,mainly from apoptosis or necrosis of cells.Studies have shown that cfDNA can be used as the latest biomarker to indicate the health status of transplanted organs,with non-invasive,sensitive and real-time detection advantages.In this study,the Single Nucleotide Polymorphisms(SNP)hybridization capture technology was combined with high-throughput second-generation sequencing technology to quantify the cfDNA(ddcfDNA)derived from donors in patients'peripheral blood.Combined with clinical biopsy results,the correlation between ddcfDNA concentration and immune rejection in renal transplant patients was studied,and the ddcfDNA concentration threshold of rejection in renal transplant patients was determined.The dynamic changes of ddcfDNA in the early stage after kidney transplantation were also studie.The results are as follows:(1)Human plasma cfDNA samples without relatives and human plasma cfDNA samples with direct relatives were mixed and diluted proportionally into 9concentration gradients,namely 0.25%,0.5%,1%,2%,4%,8%,14%,24%,and 40%,and on the machine sequencing.The average sequencing depth of the“non-relative”standard samples was above 120 X,and the number of effective SNPs was more than1790.The unrelated standard used did not rely on donor genomic information and dependent donor gene information for heterologous cfDNA quantification.The test results were consistent and significantly correlated with the theoretical values(R~2=0.998,R~2=0.998).The average sequencing depth of the“relative”standard samples is above 140 X,and the number of effective SNPs is more than 1000.The use of unrelated criteria does not depend on donor genomic information and dependent donor gene information for heterologous cfDNA detection.The results were consistent and significantly correlated with the theoretical values(R~2=0.988,R~2=0.998).The results showed that the method of relative and non-relative transplantation without donor genotype was successfully established.(2)The content of ddcfDNA in 54 patients after renal transplantation was detected by the established method.The results showed that the concentration of ddcfDNA in the clinical rejection group(1.45%,n=37)was significantly higher than that in the non-rejection group(p<0.01)(0.53%,n=17),the threshold of ddcfDNA is 1.25%.At the same time,the degree of patient rejection was graded according to the banff standard.The results showed that ddcfDNA concentration of T-cell Mediated Rejection(TMR)(1.19%)and Antibody Mediated Rejection(AMR)(2.48%)was significantly higher than the non-rejection group(0.53%)(p=0.037,p=0.007),and there was no significant difference between the TMR and AMR groups(p=0.71).In addition,the content of ddcfDNA in the IA rejection group(0.92%)was not significantly different from the ddcfDNA content in the IB rejection group(5.3%)(p=0.99).(3)The dynamic changes of ddcfDNA in early postoperative renal transplantation were studied.The dynamic changes of ddcfDNA in 21 kidney transplant patients from day 1 to 7 were observed.The results showed that ddcfDNA concentration in the early postoperative period decreased by L type.At 3 hours after recovery of kidney blood flow,the median value of the concentration of ddcfDNA was 20.69%,then it decreased to 5.22%on the first day,then decreased to a stable level on the second day.The ddcfDNA concentration(44.99%)in the Donation of Cardiac Death(DCD)group was significantly higher than that in the Living Donor Recipient Transplant(LDRT)group(10.24%),(p<0.01).The concentration of ddcfDNA in the Delayed Graft Function(DGF)group was not significantly different from that in the non-DGF group.In addition,the results also indicate that the increase in ddcfDNA concentration first(more than 1%)is associated with rejection.If the recipient's ddcfDNA level is higher(more than 1%)but is still gradually decreasing,indicating that regardless of rejection,the treatment can still be maintained,waiting for renal function recovery.In conclusion,cfDNA has great potential as a marker indicating the health of transplanted organs.This method can better assist clinicians to monitor the health status of patients and is beneficial to the long-term survival of patients with kidney transplantation. |
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