【作者】 潘浩；

【导师】 温端改；

【作者基本信息】 苏州大学 ， 泌尿外科， 2008， 博士

【摘要】 第一部分:大鼠同种异体肾移植模型的建立目的:建立一种稳定、可靠、实用的大鼠同种异体肾移植急性排斥反应模型。方法:1.采用移植肾置于左侧,供肾原位低温灌洗;移植肾带主动脉瓣与受体腹主动脉端-侧吻合;供体左肾静脉连其上方下腔静脉断端与受体下腔静脉端-侧吻合;供体输尿管带膀胱瓣与受体膀胱吻合。2.技术熟练后,行Wistar→SD大鼠肾移植,随机分为2组(每组12只):A组(排斥组),不作任何治疗;B组(CsA治疗组),术后使用CsA 10d。两组均行病理检查和观察生存期。结果:1.经过3个月的练习,建立了比较稳定的大鼠同种异体肾移植模型。手术成功率可达到90%。2. A组平均存活7.33d;B组平均存活21.2d。术后7天病理检查示A组出现明显的急性排斥反应。结论:此模型较容易掌握,除常规显微外科器械外不需要特殊的器械或设备。第二部分:携带大鼠ICAM-1基因RNA干扰慢病毒载体的构建、体外鉴定与初步体内实验目的:1.构建携带大鼠ICAM-1 RNA干扰的重组慢病毒。2.筛选出基因抑制效率最高的重组慢病毒。3.研究慢病毒载体体内转染主要脏器的有效性。方法:1.根据基因序列,设计并合成针对4个靶点的寡核苷酸,制备双链DNA oligo;Hpa I和Xho I酶切pGCL-GFP载体以使其线性化,将其与双链DNA oligo在DNA连接酶作用下进行连接反应,将连接产物转化DH5α大肠杆菌感受态细胞,PCR筛选获得阳性克隆并进行基因测序。2.将重组载体与病毒包装质粒共转染人胚肾293 T细胞,获得的病毒颗粒经浓缩后测定滴度。3.通过重组慢病毒体外转染NRK细胞,以RT-PCR和Western Blot法筛选出基因抑制效率最高的重组慢病毒,并将其再次扩增、浓缩后测定滴度。4. SD大鼠静脉注射不同浓度重组慢病毒,分别于96小时、25天后检测报告基因表达情况。结果:1.经过酶切、连接、转化后获得阳性克隆;基因测序结果完全正确。2.获得的针对4个靶点的重组慢病毒,经RT-PCR和Western Blot法证实,3#病毒在MOI=50时,基因抑制率即达到88.4%。3.大鼠体内实验表明,20×108 TU重组慢病毒可有效转染各主要脏器,转染25d后大鼠肾组织仍在不断表达报告基因。结论:1.成功构建了含有GFP报告基因的大鼠ICAM-1 RNA干扰慢病毒载体。2.体外实验证实了该慢病毒载体能高效转导NRK细胞,并能有效阻断目的基因的表达。3.体内实验初步确定了该慢病毒载体的有效性。第三部分:慢病毒介导的ICAM-1基因沉默治疗大鼠肾移植模型急性排斥反应目的:1.研究携带大鼠ICAM-1 RNA干扰的重组慢病毒抑制移植肾ICAM-1基因表达的作用。2.探讨慢病毒介导的ICAM-1基因沉默防治大鼠肾移植模型急性排斥反应的作用效果和机理。3.初步探讨慢病毒载体结合RNA干扰应用于同种异体移植排斥的效果和前景。方法:1.供体为Wistar大鼠、受体为SD大鼠肾移植,随机分为4组(每组12只):a组(同系对照组);b组(排斥组),不作任何治疗;c组(空白对照病毒组):供体术前96小时尾静脉输注空白对照病毒20×108 TU;d组(治疗组):供体术前96小时尾静脉输注rICAM重组慢病毒20×108 TU。2.肾移植术后观察生存期,并抽取外周血,检测肾功能,ELISA法检测血清IL-2、ICAM-1的浓度。3.肾移植术后7天取移植肾,RT-PCR和Western Blot法检测移植肾ICAM-1的表达;HE染色观察病理变化。结果:1.肾移植术后治疗组生存期达23.8天,与b、c两组(仅为7.3、7.0天)相比有统计学差异。2.肾移植术后治疗组血清Cr、IL-2、ICAM-1的浓度与同系对照组相比无显著差别,而与b、c两组差别巨大。3.肾移植术后7天,治疗组移植肾ICAM-1表达明显被抑制。HE染色提示移植肾Banff 0~ⅠA级,而b、c组移植肾BanffⅡB-ⅢA级。结论:1.携带ICAM-1 RNA干扰的重组慢病毒可以有效地、特异性地抑制移植肾ICAM基因的表达。2.被植入经rICAM重组慢病毒转染的供肾后,受体的生存期明显延长。3.慢病毒介导的ICAM-1基因沉默治疗大鼠肾移植模型急性排斥反应效果良好,具有潜在的应用价值。更多还原

【Abstract】 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.更多还原