HO-1基因转染预防慢性移植肾病的实验研究
摘要
器官移植是肾脏,胰岛,或心脏等慢性终末期疾病的主要治疗方法。虽然免疫抑制剂的进步使移植物特别是移植肾急性排斥反应的发生率能够得到较好的控制,但移植物长期生存率并没有明显改善,这主要归结于移植物慢性失功。尽管具体的分子和细胞机制尚不清楚,但目前可以肯定非免疫因素和免疫因素在慢性移植物功能障碍的发病机理中都起重要作用。
血红素氧化酶(Heme Oxygenase,HO)是降解亚铁血红素为一氧化碳、胆绿素、亚铁离子的胞内限速酶。HO 有三个亚型(HO-1, -2 and –3),其中HO-3 结构和功能与HO-2 相似,为HO 的组成型,HO-1 为诱导型,能在多种细胞中诱导表达,主要分布于脾、肝脏、骨髓等单核巨噬细胞系统。许多应激相关的刺激能够诱导HO-1 的表达,而HO-1 的细胞保护作用与它的3 种代谢产物密切相关。其中有明显的抗炎活性的CO研究最多。在既往的动物模型研究中,HO-1 能分别减轻移植物缺血/再灌注损伤和急性移植物排斥反应;在抗CD4 单抗诱导同种异体移植物长期存活的动物模型中,移植后早期诱导HO-1 表达能够改善抗体相关移植物动脉硬化。
慢性移植肾病(Chronic Allograft Nephropathy,CAN),是移植肾功能慢性失功的统称,现公认非免疫因素的缺血再灌注损伤和免疫因素的排斥反应是影响移植肾长期存活的最重要两大因素。我们在HO-1 分别具有抗缺血再灌注损伤和抗排斥反应类似保护基因作用的文献基础上,推测其在局部高表达可以同时减轻缺血损伤和排斥反应,应对CAN 的发生发展过程起到延缓的作用。因而本研究中我们使用HO-1 重组腺病毒将HO-1 基因导入供肾中,观察HO-1 能否预防慢性移植肾病的发生发展。
目的
1. 构建Ad-HO-1 重组腺病毒载体,并鉴定其安全性及生物活性;
2. 将Ad-HO-1 重组腺病毒载体感染肾小管上皮细胞(HK-2),探讨Ad-HO-1 感染靶细胞的效率、目的基因表达产物水平及在体外对刺激人单个核细(PBMC)增殖能力的影响。
3. 通过建立大鼠慢性移植肾病模型,观察Ad-HO-1 在体灌注移植肾后的转染效率及其蛋白表达情况,通过Ad-HO-1 在体灌注将HO-1 基因导入大鼠肾脏移植物,探
Background:
Organ transplantation remains the main therapeutic option for patients with renal, pancreas, or cardiac chronic end-stage diseases. However, progressive impairment of graft function that begins months or years after organ transplantation significantly influences the long-term outcomes of organ transplantation. Despite the introduction of new immunosuppressive regimens, the incidence of the chronic allograft deterioration is not significantly altered. Although the precise nature of molecular and cellular events of chronic allograft deterioration is still unclear, it is generally believed that both immune and nonimmune factors are equally important in the pathogenesis of the disease.
Heme oxygenase (HO) is the rate-limiting intracellular enzyme that degrades heme to biliverdin, free iron and carbon monoxide (CO). Three HO isoforms (HO-1, -2 and -3), products of three distinct genes, have been identified so far. The HO-2 and -3 isoforms are constitutively expressed. HO-1 isoform is inducible and can express in various types of cells. A wide range of stress-related stimuli can induce the expression of HO-1, which has potential cytoprotective effects that are likely to be mediated by three degradation products of heme: CO, biliverdin/bilirubin and free iron. Among them, CO, which has significant anti-inflammatory activities, is most extensively studied. In previous animal transplantation models, induction of HO-1 could protect grafts from ischemia reperfusion injury and acute graft rejection. In an animal model where anti-CD4 monoclonal antibody was used to induce long-term allograft survival, induction of HO-1 in the early days after transplantation could ameliorate antibody-associated graft arteriosclerosis.
Chronic allograft nephropathy(CAN) is the predominant cause of allograft function loss during the first decade after transplantation. The pathogenesis remains uncertain, but both immune-mediated and nonimmune-mediated factors are thought to play a role. In this study, we transferred HO-1 gene into rat donor kidney using a recombinant adenovirous
vector, an approach that might be potential for clinical transplantation. This allowed us to study whether HO-1 by itself could prevent chronic allograft nephropathy and lead to long-term survival of organ grafts. Objective: 1. To construct and identify HO-1 gene recombinant replication-deficient adenovirus (Ad-HO-1). 2. To observe the cytotoxicity of Ad-HO-1 and its capacity of mediating the expression of HO-1 in cultured cells. 3. To investigate the effects of HO-1 on renal allografts after HO-1 gene transfection in rat chronic allograft nephropathy model. Methods and Results: 1. The construction and identification of HO-1 gene recombinant replication deficient adenovirus. In this study, we used AdEasy system to construct HO-1 recombinant replication-deficient adenovirus. Firstly, the whole HO-1 gene was obtained from the PRHO-1 plasmid via XhoⅠ+HindⅢdigestion, and inserted into Puc18 plasmid. Then HO-1 gene was digested from Puc 18-HO-1 by KpnⅠ+HindⅢand subcloned into shuttle vector of pAdTrack-CMV. The resulting plasmid pAdTrack-CMV-HO-1 was linearized with PmeⅠand cotransformed into BJ5183 cells together with adenovirus genomic plasmid of pAdEasy-1. The pAdEasy-1 was E1 and E3 deleted and its E1 function could be complemented in 293 cells. Recombinants were selected with kanamycin and screened by restriction enzyme analysis. The recombinant adenoviral construct was then cleaved with PacI to expose its ITR (Inverted Terminal Repeats) and transfected 293 cells to produce viral particles. Recombinant adenoviruse was purified by Double Cesium Chloride Gradient and titered (infectious particles, IP) as TCID50 (tissue culture infectious dose 50). Adenovirus stocks were tested for the absence of replication-competent adenoviruses by PCR amplification of the E1 adenoviral region. The resulting recombinant adenovirus was identified by PCR of HO-1 gene and expression of HO-1 protein by western blot. 2. Cytotoxicity of Ad-HO-1 and its capacity mediating the expression of HO-1 in cultured cells. The cell lines HK-2 transfected by Ad-HO-1.Significant difference of the survival
rates of the cells were not observed between the transfected cells and the controls 2 and 4 days after transfection. The results suggest Ad-HO-1 has little cytotoxic affect on non-packaging cells. The expression of HO-1 and GFP in the Ad-HO-1 transfected cell lines was determined by Laser Scan Confocal Microscope.The results showed that the expression rates of HO-1 in HK-2 cell line transfected at 37℃were over 90%. Both GFP and HO-1 genes should be coexpressed in pAdTrack-pAdEasy system, so the experssion extent of GFP could represent the experssion extent of HO-1, were directly identified by fluorescence microscope. The results showed that there was strong expression of GFP in tubular epithelial cells, which suggested that Ad-HO-1 has the ability mediating HK-2 to express HO-1. 3. Ad-HO-1 mediated HO-1 gene transfection in rat donor kidney. The transfection efficiency of Adv HO-1 in kidney of F344 rats was determined with fluorescence microscope by observing the expression of GFP in kidney. Evident green fluorescence was observed in sections of kidneys when the vessel of liver, the aorta and vena cava below the renal vessel were ligated and Ad-HO-1 was injected systemically in vivo via the aorta for 3h. The transfection occurred in tubular cells and glomerular structure. The results suggested that the rat kidney could be transfected effeciently by high titer Ad-HO-1 in circulation at body temperature. The results showed that the expression had occurred on the third day after transplantation, and lasted for 3~4 weeks, which suggested that Ad-HO-1 gene could be expressed for a short time in HO-1 gene-transfected kidney after transplantation. 4. The effect of renal allografts transfected with HO-1 gene in chronic allograft nephropathy model. The effect of HO-1 gene transfection in kidney on the renal allografts was observed in the standardized Fisher/Lewis transplant model of CAN. The results showed that control recipients without HO-1 transfection developed histologic changes characteristic of CAN and that at the same time the inflammation in gene-transfected grafts was less sever than that in controls.The results presented here suggest that HO-1 gene transfection in kidney can reduce immune injury to renal allografts,It is a promosing candidate for the prevention of CAN,but the mechanism of HO-1 in preventiong the CAN warrants further studies.
Conclusion: 1. HO-1 gene recombinant replication-deficient adenovirus was successfully constructed. 2. HO-1 gene recombinant replication-deficient adenovirus had the qualities of high efficiency, stable expression of HO-1 and safety in cultured tubular epithelial cells. 3. Ad-HO-1 could efficiently transfer HO-1 gene into rat donor kidney. 4. After transfected by HO-1 gene, the renal grafts showed lower expression of α-SMA, TGF-βand PDGFB, decreased level of glomerulosclerosis and tubulointerstitial fibrosis and better renal fanction. All above suggests that HO-1 can prevent CAN efficiently.
血红素氧化酶(Heme Oxygenase,HO)是降解亚铁血红素为一氧化碳、胆绿素、亚铁离子的胞内限速酶。HO 有三个亚型(HO-1, -2 and –3),其中HO-3 结构和功能与HO-2 相似,为HO 的组成型,HO-1 为诱导型,能在多种细胞中诱导表达,主要分布于脾、肝脏、骨髓等单核巨噬细胞系统。许多应激相关的刺激能够诱导HO-1 的表达,而HO-1 的细胞保护作用与它的3 种代谢产物密切相关。其中有明显的抗炎活性的CO研究最多。在既往的动物模型研究中,HO-1 能分别减轻移植物缺血/再灌注损伤和急性移植物排斥反应;在抗CD4 单抗诱导同种异体移植物长期存活的动物模型中,移植后早期诱导HO-1 表达能够改善抗体相关移植物动脉硬化。
慢性移植肾病(Chronic Allograft Nephropathy,CAN),是移植肾功能慢性失功的统称,现公认非免疫因素的缺血再灌注损伤和免疫因素的排斥反应是影响移植肾长期存活的最重要两大因素。我们在HO-1 分别具有抗缺血再灌注损伤和抗排斥反应类似保护基因作用的文献基础上,推测其在局部高表达可以同时减轻缺血损伤和排斥反应,应对CAN 的发生发展过程起到延缓的作用。因而本研究中我们使用HO-1 重组腺病毒将HO-1 基因导入供肾中,观察HO-1 能否预防慢性移植肾病的发生发展。
目的
1. 构建Ad-HO-1 重组腺病毒载体,并鉴定其安全性及生物活性;
2. 将Ad-HO-1 重组腺病毒载体感染肾小管上皮细胞(HK-2),探讨Ad-HO-1 感染靶细胞的效率、目的基因表达产物水平及在体外对刺激人单个核细(PBMC)增殖能力的影响。
3. 通过建立大鼠慢性移植肾病模型,观察Ad-HO-1 在体灌注移植肾后的转染效率及其蛋白表达情况,通过Ad-HO-1 在体灌注将HO-1 基因导入大鼠肾脏移植物,探
Background:
Organ transplantation remains the main therapeutic option for patients with renal, pancreas, or cardiac chronic end-stage diseases. However, progressive impairment of graft function that begins months or years after organ transplantation significantly influences the long-term outcomes of organ transplantation. Despite the introduction of new immunosuppressive regimens, the incidence of the chronic allograft deterioration is not significantly altered. Although the precise nature of molecular and cellular events of chronic allograft deterioration is still unclear, it is generally believed that both immune and nonimmune factors are equally important in the pathogenesis of the disease.
Heme oxygenase (HO) is the rate-limiting intracellular enzyme that degrades heme to biliverdin, free iron and carbon monoxide (CO). Three HO isoforms (HO-1, -2 and -3), products of three distinct genes, have been identified so far. The HO-2 and -3 isoforms are constitutively expressed. HO-1 isoform is inducible and can express in various types of cells. A wide range of stress-related stimuli can induce the expression of HO-1, which has potential cytoprotective effects that are likely to be mediated by three degradation products of heme: CO, biliverdin/bilirubin and free iron. Among them, CO, which has significant anti-inflammatory activities, is most extensively studied. In previous animal transplantation models, induction of HO-1 could protect grafts from ischemia reperfusion injury and acute graft rejection. In an animal model where anti-CD4 monoclonal antibody was used to induce long-term allograft survival, induction of HO-1 in the early days after transplantation could ameliorate antibody-associated graft arteriosclerosis.
Chronic allograft nephropathy(CAN) is the predominant cause of allograft function loss during the first decade after transplantation. The pathogenesis remains uncertain, but both immune-mediated and nonimmune-mediated factors are thought to play a role. In this study, we transferred HO-1 gene into rat donor kidney using a recombinant adenovirous
vector, an approach that might be potential for clinical transplantation. This allowed us to study whether HO-1 by itself could prevent chronic allograft nephropathy and lead to long-term survival of organ grafts. Objective: 1. To construct and identify HO-1 gene recombinant replication-deficient adenovirus (Ad-HO-1). 2. To observe the cytotoxicity of Ad-HO-1 and its capacity of mediating the expression of HO-1 in cultured cells. 3. To investigate the effects of HO-1 on renal allografts after HO-1 gene transfection in rat chronic allograft nephropathy model. Methods and Results: 1. The construction and identification of HO-1 gene recombinant replication deficient adenovirus. In this study, we used AdEasy system to construct HO-1 recombinant replication-deficient adenovirus. Firstly, the whole HO-1 gene was obtained from the PRHO-1 plasmid via XhoⅠ+HindⅢdigestion, and inserted into Puc18 plasmid. Then HO-1 gene was digested from Puc 18-HO-1 by KpnⅠ+HindⅢand subcloned into shuttle vector of pAdTrack-CMV. The resulting plasmid pAdTrack-CMV-HO-1 was linearized with PmeⅠand cotransformed into BJ5183 cells together with adenovirus genomic plasmid of pAdEasy-1. The pAdEasy-1 was E1 and E3 deleted and its E1 function could be complemented in 293 cells. Recombinants were selected with kanamycin and screened by restriction enzyme analysis. The recombinant adenoviral construct was then cleaved with PacI to expose its ITR (Inverted Terminal Repeats) and transfected 293 cells to produce viral particles. Recombinant adenoviruse was purified by Double Cesium Chloride Gradient and titered (infectious particles, IP) as TCID50 (tissue culture infectious dose 50). Adenovirus stocks were tested for the absence of replication-competent adenoviruses by PCR amplification of the E1 adenoviral region. The resulting recombinant adenovirus was identified by PCR of HO-1 gene and expression of HO-1 protein by western blot. 2. Cytotoxicity of Ad-HO-1 and its capacity mediating the expression of HO-1 in cultured cells. The cell lines HK-2 transfected by Ad-HO-1.Significant difference of the survival
rates of the cells were not observed between the transfected cells and the controls 2 and 4 days after transfection. The results suggest Ad-HO-1 has little cytotoxic affect on non-packaging cells. The expression of HO-1 and GFP in the Ad-HO-1 transfected cell lines was determined by Laser Scan Confocal Microscope.The results showed that the expression rates of HO-1 in HK-2 cell line transfected at 37℃were over 90%. Both GFP and HO-1 genes should be coexpressed in pAdTrack-pAdEasy system, so the experssion extent of GFP could represent the experssion extent of HO-1, were directly identified by fluorescence microscope. The results showed that there was strong expression of GFP in tubular epithelial cells, which suggested that Ad-HO-1 has the ability mediating HK-2 to express HO-1. 3. Ad-HO-1 mediated HO-1 gene transfection in rat donor kidney. The transfection efficiency of Adv HO-1 in kidney of F344 rats was determined with fluorescence microscope by observing the expression of GFP in kidney. Evident green fluorescence was observed in sections of kidneys when the vessel of liver, the aorta and vena cava below the renal vessel were ligated and Ad-HO-1 was injected systemically in vivo via the aorta for 3h. The transfection occurred in tubular cells and glomerular structure. The results suggested that the rat kidney could be transfected effeciently by high titer Ad-HO-1 in circulation at body temperature. The results showed that the expression had occurred on the third day after transplantation, and lasted for 3~4 weeks, which suggested that Ad-HO-1 gene could be expressed for a short time in HO-1 gene-transfected kidney after transplantation. 4. The effect of renal allografts transfected with HO-1 gene in chronic allograft nephropathy model. The effect of HO-1 gene transfection in kidney on the renal allografts was observed in the standardized Fisher/Lewis transplant model of CAN. The results showed that control recipients without HO-1 transfection developed histologic changes characteristic of CAN and that at the same time the inflammation in gene-transfected grafts was less sever than that in controls.The results presented here suggest that HO-1 gene transfection in kidney can reduce immune injury to renal allografts,It is a promosing candidate for the prevention of CAN,but the mechanism of HO-1 in preventiong the CAN warrants further studies.
Conclusion: 1. HO-1 gene recombinant replication-deficient adenovirus was successfully constructed. 2. HO-1 gene recombinant replication-deficient adenovirus had the qualities of high efficiency, stable expression of HO-1 and safety in cultured tubular epithelial cells. 3. Ad-HO-1 could efficiently transfer HO-1 gene into rat donor kidney. 4. After transfected by HO-1 gene, the renal grafts showed lower expression of α-SMA, TGF-βand PDGFB, decreased level of glomerulosclerosis and tubulointerstitial fibrosis and better renal fanction. All above suggests that HO-1 can prevent CAN efficiently.
引文
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55. Braudeau C, Bouchet D, Tesson L, et al. Induction of long-term cardiac allograft survival by heme oxygenase-1 gene transfer. Gene Ther. 2004, 11:701.
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57. Savikko J, Taskinen E and Von-Willebrand E. Chronic allograft nephropathy is prevented by inhibition of platelet-derived growth factor receptor: tyrosine kinase inhibitors as a potential therapy. Transplantation, 2003 , 75(8): 1147-1153.
58. Ostendorf T, Kunter U, Grone HJ, et al. Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis. J-Am-Soc-Nephrol, 2001 ,12(5): 909-927.
59. Azuma H, Binder J and Heemann U ,et al. Effects of RS61443 on functional and morphological changes in chronically rejecting rat kidney allografts. Tansplantation, 1995,59(4):460-466.
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65. 陈孝文等,肾内分泌学,人民卫生出版社,1994 年10 出版.
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79. Hewitson TD,Becker GJ. Interstitial myofibroblasts in IgA glmerulonephritis. Am J Nwphrol, 1995, 15(2):111-117.