摘要
急性排斥反应(acute rejection,AR)和缺血再灌注损伤(Ischemia/reperfusion injury,IRI)是影响移植肾功能的两个最重要的因素。在肾脏移植的器官过程中,不可避免地存在不同程度的缺血再灌注损伤,缺血再灌注损伤不仅能引起移植肾功能延迟恢复,而且能通过对免疫系统的间接影响促进急性排斥的发生,此外缺血再灌注损伤作为一种主要的非抗原依赖性因素,还可以直接导致慢性排斥反应。由于传统的全身免疫抑制剂在控制急性排斥反应方面的能力已近极致,所以影响移植物生存的非抗原特异性因素日益受到重视。近年来,通过转基因技术将具有细胞保护和/或免疫抑制作用的基因通过体外转染移植物的方法,逐渐成为减少移植物损伤极具希望的策略。
RNA干扰(RNA interfcrence,RNAi)技术是近年发展起来的一种基因治疗新技术,RNA干扰是一种在动植物中广泛存在的通过双链RNA分子在mRNA水平上诱导特异性序列基因沉默的过程,它通过一段双链RNA(dsRNA)导致同源mRNA特异性降解从而使目的基因失去表达。在功能基因组学研究中,它是一种可与“基因敲除”相媲美的技术。目前制备siRNA(small interfering RNAs)的方法多采用病毒表达载体,慢病毒载体(Lentivectors)是一种新型病毒表达载体,它是以HIV-1(人类免疫缺陷I型病毒)为基础发展起来的基因治疗载体,区别一般的逆转录病毒载体,它对分裂细胞和非分裂细胞均具有感染能力,慢病毒极易整合到宿主基因组,从而稳定、长期表达目的基因。
随着缺血再灌注损伤领域中基础研究的不断深入,人们发现缺血再灌注组织中存在着自由基生成增多和细胞内钙超载的现象,它们是导致缺血再灌注损伤的主要机制,并参与多器官功能衰竭的发生。自由基主要包括活性氧(reactive oxygen species,ROS)和活性氮(reactive nitrogen species,RNS),一氧化氮(NO)是活性氮的主要成分,是由一氧化氮合酶(NOS)催化下产生的一种气体自由基;一氧化氮合酶(NOS)是参与人体多种生理和病理变化的极为重要的调节酶,研究表明,在肾脏缺血再灌注损伤中,诱发性一氧化氮合酶(iNOS)表达明显升高,抑制iNOS的表达可以减轻缺血再灌注损伤。
我们利用慢病毒构建携带iNOS siRNA的转染体,在缺血再灌注损伤动物模型中转染肾脏细胞,通过干扰肾脏细胞中iNOS mRNA的表达,以期观察是否能够降低肾脏缺血再灌注损伤。
第一部分携带大鼠iNOS siRNA片断慢病毒载体的构建本研究首先通过在Genbank中找到所需靶基因的碱基序列,利用设计软件设计RNA干扰靶点siRNA,用人工合成的方法合成靶点DNA序列;对于选择的靶点siRNA,由于以前没有相关数据报道,所以要对选择的靶点siRNA逐个筛选,选择其中干扰效果最佳的靶点。
我们设计了四对靶点siRNA,首先进行体外试验筛靶。对于四对靶点分别构建vshRNA载体,通过氯化钙制备新鲜的大肠杆菌感受态细胞,然后将靶点DNA与质粒载体克隆连接、转化。通过PCR扩增,测序验证克隆载体连接序列正确。然后进行慢病毒小量包装实验:Lentivirus病毒包装,病毒转染病毒包装细胞293T,病毒大量繁殖,收获及浓缩病毒,测定病毒滴度。然后对合成的靶点进行RNA干扰有效性筛选:(1)通过Western Blot检测NRK细胞中iNos蛋白的表达情况,RNA干扰效果好的蛋白不表达,否者蛋白表达。(2)实时定量PCR检测iNos mRNA表达情况。最终选择一个干扰效果最好的靶点。选择好靶点后,对该靶点进行大量病毒包装,测定滴度,浓缩达到实验需要的浓度,为下一步进行体内实验做好准备。
第二部分siRNA干扰iNOS mRNA表达对大鼠肾脏缺血再灌注损伤的保护作用
为了观察携带iNOS siRNA片断慢病毒对大鼠肾脏缺血再灌注损伤的保护作用,我们构建了SD大鼠的肾脏缺血再灌注模型:切除右侧肾脏,在阻断左侧肾脏血流后,经肾动脉缓慢匀速灌注10ml 0～4℃的HC-A器官保存液至肾脏颜色变白,流出液澄清;1小时后恢复左肾血流。分别于肾脏复灌后2d、3d、5d、7d、14d和21d取血检测肾功能,切除肾脏作进一步检查。在治疗组,左侧肾脏灌洗后经肾动脉用带靶点的慢病毒载体灌注保存1小时用以转染肾细胞;对照组则分别用生理盐水或慢病毒空载体代替。干化学方法检测肾功能BUN和Cr;用石蜡切片HE染色和电镜观察缺血-再灌注损伤后肾脏的组织形态变化;用免疫组化方法观察肾脏内iNOS蛋白的表达;real-timePCR检测iNOS mRNA表达情况;Wester-Blot蛋白印迹检验iNOS蛋白的表达。
结果显示:(1)需要较高浓度的病毒浓度,才能有效转染肾脏细胞,使用感染增强剂可以提高转染效能。(2)经肾动脉灌注慢病毒可以成功转染大鼠肾脏。(3)被转染的大鼠肾脏能够显著降低肾组织中iNOS mRNA表达,显示干扰效果较佳。(4)治疗组大鼠较对照组大鼠肾脏肾功能生化检查明显改善;在肾脏组织形态学方面,发生缺血再灌注损伤的程度明显减轻;real-time PCR检测iNOS mRNA明显减少;蛋白印迹检验iNOS蛋白的表达明显下降。说明携带iNOS siRNA慢病毒具有保护肾脏缺血再灌注损伤的作用。结论
缺血再灌注损伤是影响移植肾功能恢复的重要因素。通过设计RNA干扰靶点siRNA,验证并筛选干扰效果,可以得到有效的干扰靶点siRNA;慢病毒是一种新型病毒载体,由慢病毒介导的携带iNOS siRNA能够有效转染大鼠肾脏,通过干扰肾脏中iNOS mRNA的表达,可以明显减弱缺血再灌注对移植物的损伤,为转基因治疗器官移植中缺血再灌注损伤提供有益的尝试。
Ischemia/reperfusion injury(IRI) and acute rejection(AR) remain two important factors affecting kidney allograft after transplantation. IRI is inherent in transplantation because every donor vascularized organ by definition suffers varied degree ischemia and reperfusion. IRI may lead to increased immunogenicity of the graft, resulting in acceleration of Ag-specific processes such as acute rejection episodes and decreased long-term graft survival due to the progression of chronic-type rejection. There is a growing recognition now that long-term graft failure is at least in part influenced by the consequence of transplantation-related IRI. With current immunosuppressive protocols resulting in significant reduction in the rate of acute rejection, increasing attention has been directed toward Ag-independent factors influencing allograft survival. Hence, it is important to develop novel strategies to limit or prevent the graft injury, which in turn should ultimately result in the improvement of the long-term allograft outcome. Recently years, with the application of gene therapy techniques to transplantation, local cytoprotection and/or immunosupression in graft may be obtained, and gene therapy has been a promising way to prevent renal from IRI.
Nitric oxide synthase (NOS) is an important mediator in both normal and pathologic renal environments. Inducible NOS (iNOS) has been found in segments of the renal tubule, glomerulus, and interlobular and arcuate arteries of normal rat kidneys . Recently, it has been suggested that NO produced from iNOS is detrimental in I/R, and that excessive amounts of NO produced I /R association with increased activity of iNOS can be cytotoxic.
RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism, which is triggered by double-stranded RNA and causes degradation of mRNAs homologous in sequence to the dsRNA. Recently, RNA silencing has attracted much attention as a tool in biology. A unifying feature of all RNA silencing phenomena is the production of small RNAs that act as specificity determinants for downregulating gene expression.
Lentiviruses such as HIV are members of the family of retroviruses. They are able to integrate into the genome of nondividing cells, which makes them an attractive tool for gene transfer in the central nervous system. An important aspect of lentivectors is that they can be used for gene transfer in ES cells and preimplantation embryos, which is the basis of lentiviral transgenesis. In this study, we combined lentiviral transgenesis with RNAi to evaluate the therapeutic potential of anti-iNOS siRNAs in rat.
Part 1 Construction of a recombinant Lentiviruse carrying iNOS siRNAs gene Find the rat gene sequence from the Genbank and design the sequence of siRNA by the manufacturer. select effctive sequence of siRNA and transfer the sequences into cells effectively. Contrivances four target sequence, first we do the experiment in in vitro:uses the plasmid Construct Clon carrying agent, shRNAs were synthesized using the AmpliScribe, then link the target sequence and Clon agent. The approach described here enabled us to select highly effective rat siRNA. Amplificat the Clon sequence by polymerase chain reaction and ensure correct of the target sequence.
Minimal packaging plasmids of Lentiviruse: Lentiviruse packag, Lentiviruse transfect 293T cell, viral replication, concentrate the viral, lentivector transfer plasmids were cloned with a variety of RNA export/transport elements to determine their ability to generate lentivectors titers, through the Western Blot and real-time PCR detect the efficacy of RNA interference. At last, select the best one that its efficacy is biggest and to pack it. Concentrate lentivectors titers make it can be use the sequentia experiment.
Part 2 The protection of recombinant Lentiviruse carrying iNOS siRNAs gene against renal ischemia-reperfusion in rats
In order to study the protection against renal ischemia/reperfusion with the recombinant adenovirus, the model of kidney ischemia-reperfusion was established with Sprague-Dawley rats. In brief, the right kidney was cut off firstly, and the left kidney was flushed via abdominal aorta with 0～4℃HC-A organ storage solution. 1 hour late, the left kidney was reperfused。The blood samples were collected at2d、3d、5d、7d、14d and 21d after the kidney reperfusion for the BUN、Cr assaying, meanwhile, the left kidney was cut off at the same time after the kidney reperfusion and prepared for further analyses. In the therapy team, every left kidney was perfused and preserved with Lentiviruse carrying iNOS siRNAs after flushed with 0～4℃HC-A organ storage solution via renal artery. The rats in control groups were perfused with 0.9% normal saline solution slide chemical methods. The kidney samples of rats were harvested for assaying of histology, immunohistochemistry. The results showed that perfusion and preservation with Lentiviruse via renal aorta could transfect renal cells of rats effectively. By immunohistochemical and enzymatic activity of iNOS assay, we found that the rats treated with Lentiviruse carrying iNOS siRNAs degraded iNOS mRNA in kidneys at a high level. According to this, the level of BUN、Cr in serum were decreased, and the change in histology (HE staining and transmission electron microscoping) was ameliorated.
Conclusion:
We draw a conclusion that recombinant Lentiviruse carrying iNOS siRNAs gene can be introduced into kidney during cold preservation effectively in IRI model. The target organ can degrade iNOS mRNA in kidneys, which can attenuate the kidney ischemia/reperfusion injury in rats. These findings provide some experimental evidences that gene delivery of sequences encoding graft cytoprotection can be full considered for clinical application in renal transplantation.
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