| OBJECTIVEThe aim of this study was, by establishing a murine kidney transplantation model, to investigate the effects of exogenous overexpression of Nrpl of dendritic cells on inducing immune tolerance to kidney allografts in mice. The present study will help enrich the theoretical basis of transplant immune tolerance and provide a new target for prevention and treatment of renal allograft rejection in clinic.METHODS1. To investigate and evaluate all the Chinese and English literatures on surgical techniques of mouse kidney transplantation model, three English databases including EMbase, PubMed,Cochrane Library and four Chinese databases including Sinomed, CNKI, Wanfang, VIP were retrieved. Two investigators independently screened and included the relevant literatures and extracted information on author, country, anesthesia, vascular anastomosis,urinary system anastomosis, technical contribution, ect. Finally, we summarized the techniques and notes of the surgery and qualitatively evaluate the literatures. This part is aimed at providing theoretical basis for murine transplantation surgery.2. This part is aimed to explore suitable training method and techniques of mouse kidney transplantation for beginners. Firstly, soft hoses and rat-tail artery suture training under the surgical microscope should be performed proficiently. Murine renal transplantations were then operated after the microsurgery training. C57BL/6 male mice, 8-10 weeks of age, were randomly adopted as the donor or recipient. The methods of transplant operation included two surgical procedures. As for surgical approach 1, we used the inferior vena cava (inferior vena cava group) and the aortic valve (oblique incision) to perform the anastomosis. Both venous and arterial anastomosis were performed by the Rong’s microvascular suturing method, and we set the arterial anastomosis method as the consecutive surturing group. By surgical approach 1, a total of 70 cases were estabilshed to observe the postoperative thrombosis, hemorrhage, hydronephrosis and other complications. As for surgical approach 2, we used exactly the renal vein (renal vein group) of the graft to perform the venous anastomosis and the Rong’s microvascular surturing method as the surturing approach; the treatment of renal artery was the same as surgical approach 1, but the surturing approach was changed to intermittent suturing method (intermittent suturing group). By surgical approach 2, a total of 20 cases were acomplished to compare the venous and arterial anastomosis complications and suturing time with the last 20 cases of the surgical approach 1. All surgical urinary system reconstruction was performed using ureteral implantation method. The successful establishment of murine transplantation model will provide technical support for the transplant immune tolerance research.3. The mice bone marrow-derived DC were randomly divided into DC group, GFP-12-DC group and Nrp1-GFP-12-DC group via different intervention. Flow cytometry analysis of DC phenotype and MTT analysis of effects of DC on stimulating the T lymphocytes proliferation in different groups were observed. C57BL/6J (H-2Kb) were randomly divided into four groups by intravenous injection of 100 μl PBS, DC (2×106/ml), GFP-DC (2×106/ml)and Nrp1-GFP-DC (2×106/ml) 3 days pre-transplantation. BALB/c (H-2Kd) mice were used as donors to establish the life-supporting transplantation model with the pre-treated C57BL/6J (H-2Kb) mice as recipients. Excluding transplant mice with surgical complications post-transplantation, 9 cases in each group were established. In each group, 6 cases were randomly selected to oberserve the survival time. Other 3 cases in each group were removed of transplanted kidneys 14 days after transplantation to observe the renal pathological changes and to detect Nrp1,IL-10 and IFN-γ mRNA and protein expression.RESULTS1. A total of 24 articles were included in the present study, including 8 Chinese and 16 English literatures. From 2011 to 2015, 12 relevant articles were published, accounting for 50% of the total number of literatures, indicating that murine kidney transplantation technique has been concerned again in recent years. China, the United States and Australia are the top three literature publishing countries. The contents of the literatures basically describe the types of mouse kidney transplantation model, vascular anastomosis, urinary system reconstruction, fluid replacement, postoperative care or learning curve. The vascular anastomosis and urinary system reconstruction techniques are emphasized mostly,accounting for 34% and 38% respectively. There were 2 literatures providing a learning curve for mouse kidney transplantation. There are 3 articles providing surgical videos, which can be more intuitive to show technical points to peer. In addition, some of the literatures fail to specify the mouse strain, are not clear with the dose, route and frequency of the medication, and does not mention the suture specifications and suture methods;2. After soft hoses surture training for 1 week, the final suture speed reached 2.2±0.31 min/needle, this phase helped the surgeon to be familiar with the basic micro surgical techniques. The rat tail artery suture training lasted for 2 weeks and suture skills became stable at day 11 and 13 at a speed of 1.3±0.36min/needle and 1.1±0.23min/needle respectively. This phase helped further improve the microsurgical techniques. Researchers can move to the murine kidney transplantation once the microsurgical training is finished. Among the 70 cases operated by the surgical approach 1,the first successful transplanted mouse was achieved at the 20th case. There were 28 mice being successfully transplanted in total number. There were 7 cases of hemorrhage (10%), 2 cases of stenosis (2.9%), 31 cases of thrombosis (44.3%) and 2 cases of hydronephrosis(2.9%). Among the last 10 cases, the successful rate was 80% (survival time>7 days), the rate of achieveing long time survival reached 70% (survival time>30 days), the warm ischemia time was 28.6±3.1 min,the cold ischemia time was 29.7±2.3min,the donor operation time was 30.1±3.9min, and the recipient operation time was 53.2±5.7min.Compared with the surgical approach 1, the incidence of arterial complications of the surgical approach 2 had no significant difference when different venous patches were used.As for the arterial anastomsis, the rate of thrombosis of the interrupted suturing group was not statistically significant compared with the consecutive suturing group, but had higher incidence of hemorrhage and longer suturing time.3. According to the flow cytometry analysis, the proportion of CD83+,CD86+and MHC-Ⅱ+DC was 19.96±3.77%, 18.54±1.74% and 10.55±0.94% respectively in Nrpl-GFP-12-DC group,which were significantly lower than those in GFP-12-DC group (45.11±1.44%,36.09±2.17% and 31.34±1.34%) and 12-DC group (33.72±1.10%,42.47±5.27% and 29.40±0.80%), which indicated that Nrpl transfection could inhibit the maturation of DC.The subsequent MTT assay showed that the ability of Nrpl-GFP-DC to stimulate lymphocyte proliferation was inhibited compared with other groups. The median survival time of PBS group, DC group, DC-GFP group and Nrpl-GFP-DC group was 25 days, 30 days, 32 days and 48 days respectively. Adoptive transfer of Nrp1-GFP-DC to transplanted mice was proved to prolong the kidney graft survival associated with lower degree of renal injury, fewer inflammatory cells infiltration, lower IFN-y and higher IL-10, Nrpl expression in the grafts (P<0.05).CONCLUSION1. Recently, murine kidney transplantion surgical techniques have regained the attention, but the relevant literatures still lack and most technical improvements has not been largely used or compared across different studies. In addition, the medication dosage, route of administration, suture methods need to be more clearly described. It can be concluded that,despite the continuous improvement and modification of surgical techniques, murine kidney transplantation is still a challenging operation. This part summarized the surgical techniques commonly used in the relevant articles and provided a theoretical basis for further murine kidney transplantation surgery;2. Althoug there are literatures reporting that nearly 40 cases need to be done to successfully establish a mouse kidney transplantation model, the training process and surgery method mentioned in this part can quickly improve the microsurgical basic skills and shorten the learning curve for beginners to establish the model;3. Overexpression of Nrpl on dendritic cells can remain its immaturation status and inhibit its potential on stimulating T lymphocyte proliferation,reduce the inflammatory response in renal allograft and extend the survival time after kidney transplantation in mice. |
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