The Experimental Study On Gene Therapy Of The Acute Rejection Of Renal Allografts With Lentiviral Ector-mediated Down-regulation Of Intercellular Adhesion Molecule-1 In The Rats

Part one The establishment of a renal allogrft model in ratsObjective: To establish an acute renal allograft rejection model in rats.Methods: Rat renal transplantations were performed with hypothermia infusion conducting at dystopy, donor kidney grafted at the left side, end-side anastomosis of the donor left renal artery valve to recipient abdominal aorta, end-side anastomosis of the donor left vena cava to the recipient vena cava. To suture the donor ureter with ureterocystic flap to the recipient's bladder. The rats were randomly divided into 2 groups, Wistar rats were served as donors and SD rats as recipients. Group A without any treatment , whereas Group B were injected CsA 6mg/kg/d×10d. . The pathological changes of the grafts were observed 7 days after transplantation. Meanwhile, recorded the survival time.Results: After three months training, the rate of succeeding reached 90%. Pathological results showed that severity of acute reject reaction after 7 days was gradeⅡB-Ⅲin Group A. At the same time, survival time of rats exceeded 15 days in Group B, and was 7.3 days in Group A. Conclusion: A stable and reliable model of renal allograft in rats was established with high acute rejection rate.Part two Construction and identification of the lentiviral RNAi vector of rat ICAM-1 gene in vitro and in vivoObjective: To construct the recombinated lentivirus which carrying siRNA of rat ICAM-1 gene. To identificat the most effective lentivirus.Study the efficacy of lentivirus transfecting animal organs in vivo. Methods: Four specific target sequences were selected according to rat ICAM-1 mRNA sequence, and non-specific served as control group. The DNA oligonucleotide were cut with Hpa I and Xho I, then subcloned it into the plasmid pGCL-GFP which was also cut with Hpa I and Xho I ,ligated by DNA ligase,and the acquired recombinants were subsequently transfected into E coli strain DH53 for preparing recombinant.The polymerase chain reaction(PCR) was used to determine whether there were integration of needed DNA oligonucleotide and examined the sequence of the plasmid.This recombinanted plasmid was cotransfected along with Helper 1.0 and Helper 2.0 into HEK293T to package lentivirus particles. According to the GFP expression, the functional titer of recombinated lentivirus is determined by FCM after transduction into HEK293T cells.Then Compare with the RNAi effect of four specific target sequences inhibited the expressing of ICAM-1 in NRK cells by Real-time RT-PCR and Western Blot.The best lentivirus was amplified and concentrated,then determined the functional titer again. SD rats were injected lentivirus by vena .Examine the expression of GFP 96 hours and 25 days later.Results: It is confirmed by digestion and sequencing that ICAM-1 shRNA expression structure is correctly cloned to pGCL-GFP. After cotransfection, lentiviral vector can be packaged in HEK293T cells.The No.3 lentivirus was the most effective one.When MOI=50,the rate of No.3 lentivirus inhibiting the gene expression was 88.4% in vitro.In vivo,injecting 20×108 TU lentivirus by vena could effective transfect to the most organs of rats.The GFP gene could keep expression over 25 days. Conclusion:Successfully constructed the recombinated lentivirus carrying siRNA of rat ICAM-1 gene .Part three Gene therapy of the acute rejection of renal allografts with Lentiviral ector-mediated down-regulation of ICAM-1 in ratsObjective:To study the efficiency of rICAM-lentivirus in prevent renal allografts acute rejection in rats.Methods: The rats were randomly divided into 4 groups.The Group a(syngraft), SD to SD rats; Group b to d, Wistar rats to SD rats. Group a and Group b without any treatment , whereas donors were injected 20×108 TU lentivirus 4 days before transplant operation at Group c and d. While Group c injected with non-silence cortrol lentivirus differed from Group d injected with rICAM lentivirus. After kidney transplantation, the peripheral blood were obtained to exam the kidney function .At the same time, the level of IL-2 and sICAM were examed using enzyme-linked immunosorbent assay(ELISA). The pathological character and ICAM-1 mRNA of the grafts were observed after transplantation 7 days. Meanwhile, the survival time was recorded.Results: The levels of creatinine of serum were significantly higher in Group b and c than in Group a and d in 7 days post transplantation. Pathological results showed that severity of acute reject reaction after 7 days was gradeⅡB-Ⅲin Group b and Group c, grade 0-ⅠA in Group a and Group d. The copy of ICAM-1 mRNA in allografts was remarkably high in Group b and c. The results of ELISA showed that there were higher level of IL-2 and sICAM-1 in Group b and Group c,meanwhile survival time of rats exceeded 100 days in Group a, and was 7.3 days in Group b, 7.0 days in Group c, 23.8 days in Group d.Conclusion: Lentiviral vector-mediated down-regulation of intercellular adhesion molecule-1 is a effective method to therapy the acute rejection of renal allografts with promise well.