| BackgroundAfter decades of development, kidney transplantation has become the most effective measure for the treatment of ESRD. In spite of the big success in renal transplantion and considerable progress in HLA matching and immunosuppressive agents, 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. 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. New biomarkers in blood and urine such as ICAM 21, IL-24, soluble IL-22R and soluble CD30 can detect AR but the sensitivity and specificity are not very high for clinical application. New imaging techniques such as ultrasonic contrast and PW IMRI can reflect the shape and blood perfusion of kidney to monitor the acute rejection. Nonetheless, it still has hysteretic nature.The cellular immune response is the main immulogical mechanisms for renal allograft rejection. Regulatory T cells (Treg) and T helper 17 (Thl7) are both T lymphocyte subsets, which play an important role in transplantation, autoimmune diseases, inflammatory diseases and tumor. Treg can maintain immune balance and induce immune tolerance, while Th17 are able to defense extracelluar pathogen infection, mediate inflammatory response and transplant rejection. The balance of Treg/Th17 is conducive to maintain immune homeostasis, but the imbalance of Treg/Th17 could cause diseases, which may be rejection in transplantation. So, we expect to find a simple, effective, noninvasive and specific way to predict acute rejection through detection of Treg/Th17 balance in peripheral blood.MicroRNAs (miRNAs) are a recently discovered class of endogenous single stranded noncoding RNAs, which are believed to play a critical role in growth and development, oncogenesis, cell proliferation, differentiation and apoptosis by regulating gene expression at the posttranscriptional level. 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 Weill Cornell Medical Centre in New York demonstrated that mir-142-5p, mir-155 and mir-223 are highly expressed in AR biopsies, which tell us miRNA expression profiles of renal biopsies can reflect acute rejection. Thus, we hypothesize that miRNA expression profiles in blood can predict acute rejection through noninvasive methodSkin transplantation is the strongest transplant rejection model in the present time, which is widely used in transplant model establishment for its short observation period, easy operation, high success rate and good controllability. According to the report from home and abroad, we selected a relatively simple method with higher graft survival to make different mouse skin transplantation models and detected the change of Treg/Th17 balance and miRNAs in the blood to find some noninvasive specific biological markers for AR.Objective1. To establish different mouse skin transplantation models.2. To explore the Treg/Th17 change in peripheral blood in different mouse skin transplantation models.3. To investigate the miRNA expression in the blood in different mouse skin transplantation models.Methods1. The establishment of mouse back to back skin transplantation modela) Animal groups60 Balb/c mouse recipients, in accordance with the same weight, age and gender, were randomly divided into three groups:autograft group (control group), allograft group (acute rejection group) and CsA group,20 mice for each group. Balb/c mice were performed skin transplantation with their own back skin in control group and with skin from C57BL/6 mice in acute’rejection group and CsA group. CsA (20mg/Kg·d) was injected peritoneally from the day of surgery in CsA group while the equal amount of 0.9% saline was used in control group and acute rejection group.b) Skin transplantationThe graft was skin from donor’s back and the transplant bed was in recipient’s back. After anesthesia, we sutured the graft on the transplant bed with 6-8 interrupted stitches and dressed it with vaseline gauze and sterile common gauze.c) Postoperative observationThe bandage was removed 7 days after transplantation. The survival of skin graft was observed and evaluated with mean survival time (MST). The graft was considered with no rejection if it was healed on the recipient’s back without inflammation and congestion and had the same color and good hair growth in hair skin. But if the skin graft tended to be black, dry, crusted and wrinkled, we regarded it as rejected. The graft would be considered totally rejected in case that it had the rejection behavior and the rejection area accounted for over 80% of the total graft.2. Explore the Treg/Th17 change in peripheral blood in different mouse skin transplantation modelsa) Animal groups 30 Balb/c mouse recipients, in accordance with the same weight, age and gender, were randomly divided into three groups:autograft group (control group), allograft group (acute rejection group) and CsA group,10 mice for each group. The treatment for each group was same as above.b) Skin transplantationThe detailed procedures were as before.c) The detection of Treg/Th 17 in peripheral bloodThe peripheral blood was collected from mouse epicanthic vein before operation and in day 1, day 3, day 7 after operation with a volume about 100 μ1. Anticoagulated by heparin, the blood was added complete medium, PMA, Ionomycin and Golgistop and then cultured in the incubator under 37℃ and 5% CO2 condition. 6h later, it was added into FITC Rat Anti-Mouse CD4 and incubated for 30mins in the dark. After fixation and permeablization, it was added Alexa Fluro 647 Rat Anti-Mouse Foxp3 and PE Rat Anti-Mouse IL-17A and then incubated for 30mins in the dark. Finally, it was resuspended in PBS and detected by FACS.3. Investigate the miRNA expression in the blood in different mouse skin transplantation models.a) Animal groups36 Balb/c mouse recipients, in accordance with the same weight, age and gender, were randomly divided into three groups:autograft group (control group), allograft group (acute rejection group) and CsA group,12 mice for each group. The treatment for each group was same as above.b) Skin transplantationThe detailed procedures were as before.c) The detection of miRNAs in the blood3 mice were randomly selected in each group before operation and in day 1, day 3, day 7 after operation. The blood was collected from the heart with a volume about lml and the mice were sacrificed. Anticoagulated by EDTA, the blood was preserved at -20℃. MiRNAs were 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 amplication curve and melting curve. The data was analyzed by means of2-△△Ct.4. Statistical methodsSPSS 13.0 was 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 T3 method if equal variances not assumed. The difference was consider statistically significant when P<0.05.Results1. The mouse skin graft survival in three groupsThere was no skin displacement or falling off in three groups when the bandages were removed 7 days after transplantation. The MST in control group, AR group and CsA group is more than 30 days,8 days and 20 days respectively.18 of 20 skin grafts in control group survived more than 30 days with a high survival rate of 90%.The MST were significantly different in three groups (P=0.000). The control group was longer than AR group and CsA group (χ2=39.112, P=0.000; χ2=15.898, P=0.000). The CsA group was longer than AR group (χ2=35.786, P=0.000).2. The Treg/Thl7 change in peripheral blood in different time of three groupsThere was no significant difference of Treg/CD4+T cells in different groups (F=1.987, P=0.157) and in different time (F=1.109, P=0.350). The interaction effect between group and time was also not significant (F=0.221, P=0.969).There was no significant difference of Thl7/CD4+T cells in different groups (F=0.513, P=0.605) and in different time (F=1.367, P=0.259). The interaction effect between group and time was also not significant (F=0.188, P=0.979).There was no significant difference of Treg/Thl7 in different groups (F=0.890, P=0.422) and in different time (F=0.374, P=0.772). The interaction effect between group and time was also not significant (F=0.087, P=0.997).3. The miRNA change in the blood in different time of three groupsThe relative mir-142-5p expression level was significantly different among three groups (F=15.185, P=0.000) and among different time (F=44.018, P=0.000). The interaction effect between group and time was also significant (F=7.709, P=0.000).In the day3, the relative mir-142-5p expression level was significantly different among three groups (F=26.490, P=0.001). The AR group was significantly higher than control group and CsA group (P=0.002, P=0.003) while there was no significant difference between CsA group and control group (P= 1.000).The relative mir-155 expression level was significantly different among three groups (F=9.876, P=0.001) and among different time (F=11.228, P=0.000). But the interaction effect between group and time was not significant (F=l.924, P=0.118).In the day7, the relative mir-155 expression level was significantly different among three groups (F=8.002,.P=0.020). The AR group was significantly higher than control group (P=0.022), but had no statistical difference with CsA group (P=0.387). And no significant difference was found between CsA group and control group (P=0.021).The relative mir-223 expression level was not significantly different among three groups (F=1.196, P=0.320), but was significant in different time (F=13.792, P=0.000). The interaction effect between group and time was not significant (F=0.408,P=0.867).Conclusion1. Treg, Thl7 and Treg/Thl7 in mouse peripheral blood have no obvious change in the early stage of AR in skin allograft, so Treg/Th17 balance is not proper to be used for the early prediction of AR.2. Mir-142-5p and mir-155 expression in mouse blood increased in early AR and could be used for the early diagnosis of AR.3. Mir-223 expression in mouse blood does not change significantly in early AR and cannot be applied in the early prediction of AR. |
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