| BackgroundSince the early20th century, some scholars have begun to try to do kidney transplantation in animal, Ullmann and Decastello have successfully done it in1902. In1954, a kidney transplanted from one identical twin to another was successed that had opened a new era. In the first decade of the21st century, people who waiting for kidney transplantation on the United Network of Organ Sharing (UNOS) have rose from about50,000to nearly100,000, and increasing year by year. In our country, academician Wu Jieping first completed the Renal Transplantation, in1960.By late1970, renal transplantation, as a kind of effective means for the treatment of uremia gradually promotion in big cities. After more than a century of development, kidney transplantation has become the most effective measure for the treatment of ESRD..Because of considerable progress in HLA matching and immunosuppressive agents, the is big success in renal transplantion. kidney transplant recipients still face the risk of acute rejection (AR). AR greatly reduces graft and recipient survival, thus it is crucial for the early diagnosis and treatment of AR.At present, diagnosis of AR mostly based on the change of clinical manifestations and biochemical indexes, such as the rise in serum creatinine level, decreased urine volume, weight gain, fever, proteinuria and hematuria. But at this time, there have been pathological change in renal allograft. It is obvious that the diagnosis is too late.In Europe and the United States, Procedural biopsy can detect AR timely, but it has a certain risk for it belongs to invasive examination, and has not been widely accepted in our country. The domestic and foreign researchers have utilized blood and urine markers, such as Perfori fractalkine, GB, IP21, FOXP3, PI, to diagnosis of AR, but the Sensitivity and specificity is not strong. Color Doppler evaluate the renal allograft perfusion to determine the acute rejection, but the resistance index (RI) of blood flow parameters also changed in the chronic rejection, immunosuppressant toxicity, graft artery stenosis and postoperative complications. Due to MR diffusion weighted imaging (DWI) can evaluate early graft blood perfusion, It can be used as a noninvasive instrument to monitor the acute rejection, but the apparent diffusion coefficient (ADC) values is easy Fluctuations. Therefore it needed deeply study.miRNAs are endogenous, short (18-25nucleotides in length), highly conservative, scheduling and tissue specificity, which is the key factor of the regulation of gene expression, non-coding RN As that control the expression of many genes in eukaryotes. It widely participates in the regulation of the body of all kinds of ways, including process development, virus defense, hematopoiesis, organ formation, cell proliferation and apoptosis, and lipid metabolism, The effector molecules of miRNAs involve in each stage of immune response, which are closely related with growth and differentiation of T and B cells and class switch of B cell. MiRNAs also have a close relationship with AR. A research from the Department of Organ Transplantation Zhujiang Hospital of Southern Medical University demonstrated that mir-142-5p, mir-155and mir-223are highly expressed in skin transplantation. Anglicheau et al., demonstrated that the miRNA-let-7c, miRNA-10a, miRNA-10b, miRNA-125a, miRNA-200a, miRNA-30a-3p, miRNA-30b, miRNA-30c, miRNA-30e-3p and miRNA-32are down expressed, while the miRNA-142-5p, miRNA-142-3p, miRNA-155,miRNA-223, miRNA-146b, miRNA-146a, and miRNa-342are highly expressed in AR biopsies. Sui W et al [6] demonstrated that the miRNA-324-p, the miRNA-611, the miRNA-330, the miRNA-524, microRNas-17-3p, the miRNA-483, the miRNA-663, the miRNA-516-5-p, the miRNA-326, the miRNA-197and the miRNA-197are down expressed, while the miRNA-658, the miRNA-125-a, the miRNA-320, the miRNA-381, the miRNA-628, the miRNA-602, the miRNA-629and the miRNA-125are highly expressed in Patients with acute renal transplant rejection. Putta et al demonstrated that miRNA-192by reducing collagen expression of TGF-β and fibronectin and thus slow down the process of renal fibrosis in rats. Zhou demonstrated that the urine miR-27b, miR-192are high expression in Lupus patients with renal failure. Thus, MiRNAs involved in acute rejection and renal fibrosis disease evolution. So we establish different kidney transplantation models, and to detect the changes of miR-192, miR-320and miR-223in the blood predict acute rejection through noninvasive method.According to the report from home and abroad, we selected SD-Wistar to make different renal transplantation models and detected the change of miRNAs in the blood to find some noninvasive specific biological markers for AR.Objective1. To established the different rat kidney transplantation models.2. To explore the miRNAs change in peripheral blood in different rat renal transplantation models.Methods1. The establishment of rat renal transplantation model1) Animal groups All animals were divided into two groups; Wistar rats as donors, SD rats as recipients, to established the allogeneic transplantation group (acute rejection group). Given cyclosporine A (CsA) immunosuppressive to establish allogeneic transplantation group (2mg/kg-day i.p. from the Postoperative day)(normal group).10mice for each group.2)Renal transplantationWith1%sodium pentobarbital anesthesia, then with heparin sodium perfusion the left kidney at low temperature in situ, end-to-end coincide the artery, renal vein(the epidural catheter auxiliary completed) and ureter from donor and recipient.3) postoperative observationThe later stages of the pre-experiment, two groups of rats remove the transplanted kidney and immersed in10%neutral formalin after renal transplantation days1,3,5,7, then slice and HE staining for pathological examination. During the formal experiment, Observed the general living conditions every day, such as the diet, urine, gums, fur, activities. Urine output>5mL/d, the operation is successful, urine output <1mL/d, which needed to biopsy.2. To investigate the miRNA expression in the blood in different rat renal transplantation models.1) Animal groups:The detailed procedures were as before.2) Renal transplantation:The detailed procedures were as before.3) The detection of miRNAs in the blood.The postoperative rats selected in each group before operation and in day1, day3, day5, day7after operation. The blood was collected by Cut off the tail with a volume about2ml. Anticoagulated by EDTA, the blood was preserved at-80℃. mir-192、mir-320、mir-223were extracted and purified with miRNA purification kit, underwent RT PCR and finally detected by real time PCR. Ct value was calculated by measuring the cycle number required for the fluorescence to reach the set threshold according to ampliation curve and melting curve. The data was analyzed by means of2-ΔΔCt.3. Statistical methodsSPSS13.0was used to for the statistical analysis of the data. The measurement data was showed as mean±SD (X±s). The mouse skin graft survival was analyzed with Kaplan-Meier method and Log-Rank test. Single factor data were compared with one way ANOVA between groups. Repeated measurement data were compared with ANOVA for the repeated measures. Factorial designed data were compared with factorial analysis. Bonferroni method was used for multiple comparisons if equal variances assumed and Dunnett’s T3method if equal variances not assumed. The difference was consider statistically significant when P<0.05.ResultsSurvival status and complications of two groups ratsPre-experimental stage had used100rats, SD and Wistar were50rats respectively. Pre-experimental stage focuses on familiar with the microscopy operation and mastery and stable model of rat renal transplantation in the pre-experimental stage. Entered the experimental stage after more than three months microscopy skills and modeling training. The total of20pairs in formal experiments, in which the SD, Wistar the mice were20,recorded survival status as well as a variety of postoperative complications (including mortality in rats represent the rats were not sacrificed according to experimental plan)(Table2). In a formal experimental stage, donor surgery taked to1to1.5h, the receptors in kidney transplant time were1.5to2h, The Vascular anastomosis (30±3) min, The ureteral anastomosis Time (12±2)min.2. The miRNA change in the blood in different time of two groups The relative mir-192expression level was significantly different between two groups (F=23.153, P=0.000) and among different time (F=24.019, P=0.000). The interaction effect between group and time was also significant (F=13.639, P=0.000).In the day5, the relative mir-192expression level was significantly different between two groups (F=19.490, P=0.002). The AR group was significantly higher than normal group (P=0.002).The relative mir-320expression level was significantly different between two groups (F=16.925, P=0.001) and among different time (F=13.334, P=0.000). But the interaction effect between group and time was not significant (F=2.978,P=0.128).In the day5, the relative mir-320expression level was significantly different between two groups (F=10.172, P=0.020).The relative mir-223expression level was not significantly different between two groups (F=1.279, P=0.360), but was significant in different time (F=13.987, P=0.000). The interaction effect between group and time was not significant (F=0.479, P=0.870).Conclusion:1. Wistar rats as donors, SD rats as recipients, end-to-end coincide the artery, renal vein(the epidural catheter auxiliary completed) and ureter from donor and recipient could make a stable acute rejection model by this approach in our experiment.2. Mir-192and mir-320expression in rat blood increased in early AR and could be used for the early diagnosis of AR.3. Mir-223expression in rat blood does not change significantly in early AR and cannot be applied in the early prediction of AR. |
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