The distribution of neuronal and inducible nitric oxide synthase in urethral stricture formation

PURPOSE: The distribution of neuronal (n) and inducible (i) nitric oxide synthase (NOS) may have a role in the maintenance of normal urethral spongiosum and during the development of spongiofibrosis in urethral stricture disease. MATERIALS AND METHODS: Eight normal and 33 strictured human bulbar urethras were studied by histological and immunohistochemical techniques for the neuronal markers S-100, nNOS and iNOS. The smooth muscle-to-collagen ratio was calculated by morphometric analysis of Masson's trichrome sections. Immunohistochemical staining patterns of the neuronal markers in normal urethral tissue was compared to that in urethral stricture tissue with spongiofibrosis. RESULTS: The smooth muscle-to-collagen ratio was significantly lower in the strictured urethra compared to that in the control group (p = 0.001). In the strictured bulbar urethra nNOS immunoreactivity was decreased compared to that in normal urethral tissue. The severity of spongiofibrosis corresponded to the loss of nNOS immunoreactivity. iNOS immunoreactivity was found in strictured urethral epithelium and spongiosal tissue, whereas the control group was nonimmunoreactive to iNOS. CONCLUSIONS: Urethral stricture formation is a fibrotic process associated with significant changes in NOS metabolism. Abnormal collagen synthesis following urethral trauma may be stimulated by inappropriate iNOS activity. A functional nerve supply to the urethral spongiosum seems to be crucial in the maintenance of the unique ultrastructure of the urethral spongiosum.     

Gene expression of inducible nitric oxide synthase in injured spinal cord tissue

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Role of inducible nitric oxide synthase in pig liver transplantation

BACKGROUND: Previously, we clarified the role of inducible nitric oxide synthase (iNOS) and the protective effect of an iNOS inhibitor in warm ischemia and reperfusion model. In this study, we investigated whether the same effects would be obtained by iNOS inhibitor in liver transplantation model. MATERIAL AND METHODS: Orthotopic liver transplantation was performed in pigs in the usual manner after about 6 h of cold preservation in University of Wisconsin solution. Aminoguanidine hemisulfate (AG) was used as the iNOS inhibitor and AG was administered intraportally at the dose of 10 mg/kg just after reperfusion. Two experimental groups were subjected, control group (n = 10), and AG group (n = 10). We investigated changes of serum nitrite/nitrate (NOx) and aspartate aminotransferase (AST). Expression of iNOS was examined by immunohistochemistry, including a double immunofluorescnce technique in combination with cofocal laser scanning microscopy. RESULTS: Serum NOx and AST were significantly lower in the AG group. Histological hepatic damage and thrombocyte thrombi were attenuated in the AG group. Expression of iNOS was recognized strongly at Kupffer cells and neutrophils in the centrilobular region of liver after reperfusion by cofocal laser scanning microscopy. Moreover, iNOS staining was attenuated in AG group compared with control group. CONCLUSIONS: These results indicate that hepatic ischemia and reperfusion injury in liver transplantation might be triggered by iNOS expression of Kupffer cells and neutrophils, and attenuated by administration of an iNOS inhibitor. Moreover, AG showed down regulation of iNOS expression after reperfusion.     

血管内皮生长因子对关节软骨细胞诱导型一氧化氮合酶的影响

目的 观察血管内皮生长因子(VEGF)对体外培养的关节软骨细胞诱导型一氧化氮合酶(iNOS)表达的影响。方法 体外培养SD乳鼠关节软骨细胞,用白细胞介素(IL)-1β诱导的方法建立骨关节炎(OA)体外模型,实验分为4组,每组加入不同处理因素进行干预,A组:(正常对照组)不加任何处理因素;B组:10 μg/L VEGF;C组:10 μg/L IL-1β;D组:10 μg/L VEGF+ 10 μg/LIL-1β。采用实时荧光定量PCR( Real Time PCR)检测iNOS mRNA的表达,采用蛋白免疫印迹法( Western blot)检测iNOS蛋白的表达。结果 iNOS mRNA的表达:A组iNOS mRNA无表达,B组(9.64±1.64)、C组(17.27±2.01)及D组(28.93±6.63),3组的iNOS mRNA表达量显著升高,进一步组间比较,D组软骨细胞iNOS的mRNA表达水平明显高于B组(P＜0.01)及C组(P＜0.05),C组软骨细胞iNOS的mRNA表达水平高于B组(P＜0.05)。iNOS蛋白的表达:A组iNOS蛋白无表达,B组(0.44±0.12)、C组(0.74±0.07)及D组(1.38±0.38),3组的iNOS蛋白表达量显著升高,进一步组间比较,D组软骨细胞iNOS的蛋白表达水平明显高于B组(P＜0.01)及C组(P＜0.05),C组软骨细胞iNOS的mRNA表达水平高于B组(P＜0.01)。结论 在OA的发病过程中,VEGF可能通过上调软骨细胞iNOS的表达发挥重要作用。     

Expression of nitric oxide and inducible nitric oxide synthase in acute renal allograft rejection in the rat

BACKGROUND: Recent studies have shown that nitric oxide (NO) synthases, particularly inducible nitric oxide synthase (i-NOS), are induced in acute rejection episodes following heart, liver, pancreas and kidney allotransplantation. Furthermore, tissue and cellular injury has been demonstrated to be mediated by peroxynitrite (ONOO-), a metabolite of NO as well as a potent oxidant. However, a detailed relationship between NO, i-NOS and graft injury in transplantation remains elusive. METHODS: The present study used the following models of renal transplantation in rats: allografts (n = 5, Brown-Norway to Lewis [LEW] rats), isografts (n = 5, LEW to LEW) and allografts treated with aminoguanidine (AG), an i-NOS inhibitor (n = 5). Blood urea nitrogen (BUN), serum creatinine (SCr) and urinary and serum nitrosocompounds (NOx) were measured on days 2, 4 and 7 post-transplant. Western blot analysis of i-NOS protein expression and measurement of i-NOS activity were carried out in grafts harvested on Day 7, along with immunohistochemical and histopathological examinations. RESULTS: In the allograft group, both BUN and SCr levels increased markedly on Day 7, in parallel with a sharp increase in NOx. A band stained by anti-i-NOS antibody was detected at approximately 130 kDa, along with high levels of i-NOS activity and diffusely distributed i-NOS-positive cells (macrophages). Histologically, an acute rejection episode was confirmed (Grade 3 according to Banff classifications). In the AG group, reduced renal function and graft injury were significantly less severe than in the allograft group. CONCLUSIONS: In rat renal allograft acute rejection, markedly increased levels of serum NOx were observed, along with enhanced tissue i-NOS activity, together resulting in graft injury. AG administration suppressed the increase of serum NOx levels, with concomitant mitigation of tissue injury and renal function impairment.     

Caspase-3-dependent apoptosis in Escherichia coli-infected urothelium: involvement of inducible nitric oxide synthase

OBJECTIVE: To investigate if nitric oxide (NO) derived from inducible NO synthase (iNOS) regulates apoptosis and/or cell shedding in uroepithelial cells in vivo and in vitro, as bladder epithelial cells undergo shedding during urinary tract infection, which is considered a part of the host's defence and is thought to take place via an apoptotic pathway. MATERIALS AND METHODS: Bladders and kidneys of mice infected with Escherichia coli were used to study caspase-3 immunoreactivity at different times after infection. Wild-type (E. coli 1177) and type-1 recombinant (E. coli PKL4) bacteria were used. iNOS-deficient mice were used to study the association of caspase-3 with iNOS. Isolated human uroepithelial cells were used to examine the effect of the NO donor DETA/NO and the peroxynitrite generator SIN-1 on caspase-3 activity and cell shedding in vitro. RESULTS: Many caspase-3 immunoreactive neutrophils were found soon after infection and some superficial bladder epithelial cells were also immunoreactive for caspase-3. CONCLUSIONS: Caspase-3 immunoreactivity was also detected in neutrophils and bladder epithelial cells of infected iNOS-deficient mice. There was no co-expression between iNOS- and caspase-3 in bladder epithelial cells. DETA/NO and SIN-1 did not stimulate caspase-3 activity or cell shedding in isolated human uroepithelial cells. Caspase-3 and iNOS are not co-expressed in uroepithelial cells and apoptosis is evident in the absence of iNOS. Exogenous NO did not induce apoptosis or cell shedding in isolated human uroepithelial cells.     

诱导型一氧化氮合成酶在迟发性血管痉挛中的作用

目的　以大鼠迟发性脑血管痉挛模型为基础研究诱导型一氧化氮合成酶 (iNOS)在迟发性血管痉挛发展中的作用。方法　 3 2只雄性SD大鼠随机分为实验组和对照组 ,实验组枕大池二次注血诱导迟发性脑血管痉挛 ,对照组枕大池注射生理盐水。第 8天行脑血管造影 ,枕大池抽取脑脊液测一氧化氮 (NO)浓度。逆转录 聚合酶链反应 (RT PCR )法和免疫组织化学法测定并评价iNOSmRNA和蛋白质在基底动脉、大脑中动脉和皮质中的表达。结果　颅内动脉血管减影提示对照组颈内动脉颅内段、大脑中动脉 (MCA)明显变细 ,大脑中动脉中段直径 (MD)与镫骨动脉中段直径 (SD)之比衡量大脑中动脉的管径显示实验组MCA管径较对照组MCA管径减少 3 0 %。对照组脑脊液中NO浓度为 (11.70± 2 .62 ) μmol/L ,实验组脑脊液中NO的浓度为(5 5 .67± 12 .84)μmol/L。iNOSmRNA和蛋白质表达于基底动脉、大脑中动脉和皮质 ,其中基底动脉表达最强。 结论　iNOS作为迟发性脑血管痉挛发展中的关键因素参与血管壁的迟发性损伤。     

镁硅玉人工髋关节假体周围骨溶解与诱导型一氧化氮合酶、过氧亚硝基阴离子的关系

[目的]观察诱导型一氧化氮合酶（iNOS）和过氧亚硝基阴离子（ONOO^-）在假体周围各区的表达和分布变化，各区iNOS和ONOO^-表达与骨溶解程度之间的关系。[方法]临床选取6例镁硅玉人工全髋关节翻修术，手术中按Delee-Charnley髋臼分区法和Gruen股骨分区法，取出松动假体周围各区的假体-骨间界膜，免疫组化法检测iNOS和ONOO^-体内生成标志物硝基酪氨酸（NT）的表达，同时以术前X线片，区分假体周围非骨溶解区和骨溶解区。分析并比较iNOS和NT在各个分区中的阳性表达，与骨溶解程度的关系。[结果]髋臼侧Ⅲ区的iNOS阳性细胞率高于Ⅰ、Ⅱ区，股骨侧1、2、6、7区染色阳性细胞率高于3、4、5区（P〈0．05）；髋臼侧Ⅲ区的NT阳性细胞率明显高于Ⅰ、Ⅱ区，股骨侧阳性细胞率由高到低依次为1、7区，2区，6、4、3、5区（P〈0．05）；骨溶解区界膜组的iNOS和NT阳性细胞率均明显高于非溶骨区界膜组和OA滑膜组（P〈0．01）。[结论]假体周围iNOS和ONOO^-的表达具有一定规律性，并与骨溶解程度密切相关。iNOS和ONOO^-的异常表达可能是磨损颗粒造成界面骨重建受阻和骨溶解的关键环节之一。     

诱导型一氧化氮合成酶对同系大鼠移植肠运动功能的作用

目的 观察诱导型一氧化氮合成酶(iNOS)对同系原位全小肠移植术后早期移植肠运动功能的影响.方法 分为对照组、移植组、L-NIL治疗组,每组12只大鼠.对照组行十二指肠造瘘术,另两组均行同系原位全小肠移植及十二指肠造瘘术,术后分别给予生理盐水、L-N6-(1-亚氨乙基)-赖氨酸(L-NIL).于术后2 d,各组取6只获取肠段行病理组织学检查,观察炎性损伤程度,并采用RT-PCR和免疫组织化学方法检测iNOS mRNA及蛋白表达水平.各组另6只行小肠传输实验,观测小肠传输功能.结果 移植组呈明显炎性损伤改变,iNOS mRNA及蛋白表达水平(1.278±0.142)%,(56.33±5.16)%较对照组(0.066±0.016)%,(9.17±3.17%)上调(P＜0.01),较对照组小肠传输延迟(P＜0.01).L-NIL治疗组炎性损伤程度较移植组减轻,iNOS mRNA及蛋白表达水平(0.588±0.096)%,(26.17±4.14)%较移植组下调(P＜0.01),且小肠传输延迟有改善(P＜0.01).结论 iNOS在术后早期移植肠炎症损伤及其引发的肠运动功能障碍中可能起重要作用.     

Effects of intravesical instillation of cyclooxygenase-2 inhibitor on the expression of inducible nitric oxide synthase and nerve growth factor in cyclophosphamide-induced overactive bladder

OBJECTIVE: To examine the effects of intravesical cyclooxygenase-2 (COX-2) inhibitors on the expression of inducible nitric oxide synthase (iNOS) and nerve growth factor (NGF) in cyclophosphamide (CYP)-induced overactive bladder (OAB). MATERIALS AND METHODS: In all, 40 Sprague-Dawley rats were divided into control, OAB, and COX-2 inhibitor-treated groups. OAB was induced by an intraperitoneal injection with CYP. Cystometry was performed in all rats and, in half of the OAB rats, a COX-2 inhibitor was administered intravesically. The bladders of all rats were stained immunohistochemically for iNOS and NGF. RESULTS: In the OAB rats, the contraction interval and intercontraction interval were significantly shorter than in control rats, and the contraction time and pressure were significantly greater. In the COX-2 inhibitor-treated rats, the contraction interval and intercontraction interval were significantly longer than in the OAB rats, and the contraction time was significantly shorter. On immunohistochemical staining, there was no iNOS activity and NGF activity was minimally localized in the mucosa and submucosa in the control group. In the OAB rats, NGF activity in the mucosa and submucosa were increased, and there was greater expression of iNOS in all layers and of NGF in detrusor; in the COX-2 inhibitor-treated rats, their expression was less in all layers. CONCLUSIONS: Intravesical instillation with COX-2 inhibitors can reduce CYP-induced bladder hyperactivity and expression of iNOS and NGF. Intravesical instillation with COX-2 inhibitors can be considered as a possible treatment for OAB.     

诱生型一氧化氮合酶在胆道感染大鼠肝细胞中的表达

目的　了解诱生型一氧化氮合酶 (induciblenitricoxidesynthase ,iNOS)在胆道感染大鼠肝细胞中表达的情况及其规律。方法　制作大鼠胆道感染模型 ,采用还原型辅酶Ⅱ黄递酶组织化学法检测大鼠肝细胞中iNOS的表达。结果　大鼠胆道感染 2h后肝细胞即有iNOS的表达切片积分光度(13 5 8± 0 6 4) ,与对照组切片积分光度 (3 5 9± 0 2 8)相比 ,P <0 0 1。 8h达到峰值切片积分光度(2 9 2 7± 0 90 ) ,2 4h至 48h仍有较高表达切片积分光度分别为 (19 47± 0 6 5 )和 (19 96± 0 78)。结论　胆道感染时肝细胞可持续高效地表达iNOS ,提示胆道感染时肝脏是合成NO的重要器官 ,并可能对胆道感染的转归具有重要影响     

The potential role of inducible nitric oxide synthase (iNOS) activity in the testicular dysfunction associated with varicocele: An experimental study

Nitric oxide (NO) has been reported to be increased in the spermatic veins of men affected by varicocele. The aim of the present study was to determine whether iNOS (inducible nitric oxide synthase) has a role in testicular dysfunction associated with varicocele, immunohistochemistry analyze was used to study iNOS activity in testis of adolescent rats with experimental left varicoceles. Rats were randomly divided into three groups. The first group consisted of rats undergoing partial ligation of left renal vein (n:12). The second group consisted of rats undergoing a sham operation (n:6) and, the third group referred to as control rats (n:7). Immunohistochemistry slides were evaluated by counting the number of positive cells and expressed as percents (% iNOS activity). We found that iNOS was predominantly expressed in the cytoplasm of Leydig cells in each group and only a small amount of iNOS was expressed in Sertoli cells. There were significant differences in % iNOS activity between both testes of varicocele group and both of testes control group(p < 0.01), but no significant differences were noted between other groups (p > 0.05). Because of iNOS activity was markedly increased in the Leydig cells of varicocele bearing rats, we suggest that iNOS activity may play a role in the testicular dysfunction associated with varicocele during adolescence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protective effect of a novel and selective inhibitor of inducible nitric oxide synthase on experimental crescentic glomerulonephritis in WKY rats.

BACKGROUND: Nitric oxide (NO) plays important roles in a variety of pathophysiological processes. It has been reported that inducible NO synthase (iNOS) is upregulated in the glomeruli of patients with glomerulonephritis, although there has been no direct evidence that NO generated by iNOS contributes to the progression of glomerulonephritis. ONO-1714, a novel cyclic amidine analog, is a selective inhibitor of iNOS. To elucidate the role of iNOS in the pathogenesis of experimental crescentic glomerulonephritis, we examined the effect of ONO-1714 given to rats with nephrotoxic serum (NTS) nephritis. METHODS: We induced NTS nephritis in Wistar-Kyoto (WKY) rats. These rats were given ONO-1714 or physiological saline intraperitoneally for 14 days using an osmotic pump after intraperitoneal injection with NTS. RESULTS: Glomerular expression of iNOS and urinary excretion of NO metabolites (nitrite/nitrate) were increased in rats after injection of NTS. As compared with the control group, ONO-1714 significantly reduced proteinuria, crescent formation, glomerular infiltration of macrophages and urinary excretion of nitrite/nitrate. CONCLUSION: The present results suggest that NO radicals generated by iNOS contribute to the progression of experimental crescentic glomerulonephritis in WKY rats. The selective iNOS inhibitor ONO-1714 may be beneficial for the treatment of crescentic glomerulonephritis.     

抗氧化剂抑制内毒素性休克大鼠诱导型一氧化氮合酶基因表达

感染性休克时循环机能障碍的显著特点表现为进行性、顽固性低血压,伴对血管活性药物敏感性降低,以至组织器官灌注不足、重要器官机能代谢障碍,病死率仍高达30％～70％。休克时循环机能障碍所涉及的病理生理机制极其复杂,脂质过氧化物的堆积和NO的爆发生成是其中的关键病理因素之一。我们以往的研究中发现抗氧化剂能部分逆转内毒素休克大鼠的血管低反应性,为了进一步探讨其治疗学机制,本实验对抗氧化剂在内毒素性休克大鼠iNOS mRNA表达中的作用进行了观察。     

The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

BACKGROUND: Production of nitric oxide (NO) by graft infiltrating macrophages has been suggested as an important effector mechanism of allograft rejection. Expression of the gene for the inducible NO synthase (iNOS) and the production of NO in rejected graft has been demonstrated in various models of allotransplantation. However, whether NO plays a role in rejection of skin xenografts has not been documented. METHODS: Explants of rejected skin allografts or xenografts (rat to mouse) were cultivated in vitro and the production of NO, interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) by graft infiltrating cells was determined by the Griess reaction or ELISA. Effects of supernatants from cultures of xenograft explants on the expression of gene for iNOS, accumulation of iNOS protein and NO production were determined by RT-PCR or Western blots. Molecular mass of the factor with the suppressive activity was characterized by filtration on chromatography Sephacryl S-200 Superfine column. In addition, the effects of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a selective iNOS inhibitor, on survival of skin xenografts were tested. RESULTS: While explants of rejected mouse skin allografts produced substantial amounts of NO, undetectable or only very low levels of NO were found in supernatants from cultured rat skin xenografts. Cocultivation of bacterial lipopolysaccharide (LPS)-stimulated mouse macrophages which produce high quantities of NO, with pieces of rejected xenografts, but not of syngeneic grafts, allografts or normal rat skin, completely inhibited production of NO. Production of IL-6 and IL-10 by LPS-stimulated macrophages was not inhibited under the same conditions. The inhibition of NO production was mediated by a factor which was produced by rejected rat xenograft and which was eluted from chromatography Sephacryl S-200 Superfine column in a fraction representing a molecular mass of 67 kDa. The factor did not inhibit the expression of the gene for iNOS, reduce the level of iNOS protein in stimulated macrophages, or function as a scavenger of NO. Rather, the factor inhibited the function of iNOS. The finding that NO does not play an important role during rejection of skin xenografts is supported by the observation that treatment of graft recipients with AMT, a specific iNOS inhibitor, did not enhance xenograft survival, while the same treatment resulted in prolongation of survival of skin allografts. CONCLUSION: The results thus demonstrate that a 67-kDa molecule produced by rejected rat skin xenografts selectively inhibits iNOS activity in graft infiltrating macrophages. We suggest that NO does not play a significant role in rejection of skin xenografts as it does in the case of allograft rejection.     

iNOS/NO对结直肠肿瘤发生发展的影响

一氧化氮（nitric oxide,NO）具有广泛的生物学活性。近年来发现一氧化氮（NO）、一氧化氮合成酶（nitric oxide synthase-2,NOS-2）与结直肠肿瘤的发生、发展密切相关,它与环氧化酶（cy-cloxygenase-2,COX-2）之间存在复杂的调控机制。对NO清除剂、NOS抑制剂和释放NO的非甾体抗炎药的研究为结直肠肿瘤的防治提供了一个思路。     

诱导型一氧化氮合酶在前列腺增生组织中表达的研究

目的：探讨诱导型一氧化氮合酶(iNOS)与前列腺增生(BPH)病理生理变化的关系。方法:用免疫组化法检测10例正常和30例有膀胱出口梗阻的BPH患者膀胱和前列腺组织中iNOS的表达。结果：BPH组前列腺组织中iNOS呈阳性染色，主要分布于前列腺的上皮细胞及上皮下组织中，间质平滑肌组织中均为阴性；对照组的膀胱壁、膀胱颈部和前列腺组织中均无iNOS阳性染色。结论：iNOS仅在BPH患者前列腺组织中有特异性表达，提示它参与了前列腺增生的病理生理过程。     

Selective inducible nitric oxide synthase (iNOS) inhibition attenuates remote acute lung injury in a model of ruptured abdominal aortic aneurysm

OBJECTIVE: Abdominal aortic aneurysm rupture is associated with a systemic inflammatory response syndrome and acute lung injury. Using a selective inducible nitric oxide synthase (iNOS) inhibitor, N(6)-(iminoethyl)-lysine (L-NIL), we explored the role of iNOS in the early pro-inflammatory signaling and acute lung injury in experimental abdominal aortic aneurysm rupture. MATERIALS AND METHODS: Anesthetized rats were randomized to sham control or shock and clamp (s + c) groups, which underwent one hour of hemorrhagic shock, followed by 45 minutes of supramesenteric aortic clamping, and then two hours resuscitated reperfusion. Animals in s + c were randomized to receive intravenous L-NIL at 50 microg/kg/h or saline at the start of reperfusion. Pulmonary permeability to (125)I-labeled albumin, myeloperoxidase (MPO) activity, cytokine levels, and semi-quantitative RT-PCR for mRNA were indicators of microvascular permeability, leuco-sequestration, and pro-inflammatory signaling, respectively. RESULTS: Lung permeability index were significantly increased in s + c compared to sham (4.43 +/- 0.96 versus 1.30 +/- 0.17, P < 0.01), and attenuated by L-NIL treatment (2.14 +/- 0.70, P < 0.05). Lung tissue MPO activity was significantly increased in s + c compared to sham (2.80 +/- 0.32 versus 1.03 +/- 0.29, P < 0.002), and attenuated by L-NIL treatment (1.50 +/- 0.20, P < 0.007). Lung tissue iNOS activity was significantly increased in s + c compared to sham animals (P < 0.05), and attenuated by L-NIL treatment (P < 0.05). Lung tissue iNOS mRNA was upregulated 8-fold in s + c compared to sham (P < 0.05). Data represents mean +/- standard error mean, comparisons with ANOVA. CONCLUSIONS: These data suggest that in our model of ruptured abdominal aortic aneurysm iNOS plays a crucial role in reperfusion lung injury. Selective inhibition of iNOS during early reperfusion prevents neutrophil mediated acute lung injury.     

靶向大鼠诱导型一氧化氮合酶基因的shRNA重组腺病毒载体的构建及鉴定

目的 构建携带增强型绿色荧光蛋白基因(EGFP)标志针对大鼠诱导型一氧化氮合酶(iNOS)基因的shRNA重组腺病毒载体并在人胚肾-293细胞中扩增制备重组病毒.方法 利用AdMax包装系统,用先期构建的带有EGFP标记基因的穿梭质粒pDC316-iNOS-shRNA-EGFP,与骨架质粒pBHGlox_El,3Cre共转染293包装细胞,同源重组产生复制缺陷型重组腺病毒载体Ad5-inosshRNA-EGFP,反复感染293细胞扩增病毒后,离子交换法纯化病毒,并测定病毒颗粒数及滴度.结果 经聚合酶链反应(PCR)检测和EGFP表达证明已成功构建了携带iNOS-shRNA-EGFP的重组腺病毒载体;扩增纯化后,测得重组腺病毒颗粒数为3.6×1011 VP/ml,A260/A280值约为1.25,病毒活性为7.94×109IU/ml.结论 已成功构建重组腺病毒载体Ad5-inos-shRNA-EGFP,为利用基因激活技术治疗勃起功能障碍的研究奠定实验基础.

Abstract：

Objective To construct an adenovirus vector expressing short hairpin RNA (shRNA)of rat inducible oxide synthase (iNOS) carrying enhanced green fluorescent protein (EGFP) and amplify the adenovirus vector in HEK-293 cells. Methods The shuttle plasmid pDC316-inos-shRNA-EGFP that was constructed at an earlier date, was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad5-inosshRNA-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method, then the virus particles were counted and the purity and titer were determined.Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by polymerase chain reaction (PCR) and GFP expression. After amplification and purification, the virus particle count, A260/A280 and titer of recombinant adenovirus were 3.6 × 1011 VP/ml, 1.25 and 7.94 ×109 IU/ml,respectively. Conclusion Recombinant adenovirus vector Ad5-inos-shRNA-EGFP is successfully constructed, which laid a foundation for gene activation in erectile dysfunction treatment.