沉默Toll样受体4基因表达的RNAi慢病毒载体的构建

背景：Toll样受体4（toll-like receptor4,TLR4）是介导内毒素/脂多糖应答的主要受体,在由内毒素诱导的炎性反应的信号通路中发挥着重要作用。目的：构建人TLR4基因的RNA干扰慢病毒载体,并观察其对人脐静脉内皮细胞TLR4在蛋白水平的沉默效应。方法：利用Invitrogen在线软件设计人TLR4基因shRNA序列,合成、退火形成双链寡核苷酸后克隆到线性载体pENTRTM/H1/TO的黏性末端,并进行DNA测序。得到的阳性重组子再与慢病毒载体进行重组反应,从而获得干扰TLR4基因真核表达的慢病毒载体。在脂质体的介导下将慢病毒包装辅助复合体和TLR4基因的真核表达慢病毒载体导入293FT细胞包装病毒,测定病毒滴度,感染人脐静脉内皮细胞,检验其干扰TLR4基因表达的有效性。结果与结论：实验成功构建TLR4基因真核表达慢病毒干扰载体并获得相应的慢病毒,病毒滴度为8.7×106U/mL。免疫印迹杂交结果表明,所获得的慢病毒感染人脐静脉内皮细胞TLR4基因在蛋白水平的表达显著降低。实验成功构建了人TLR4基因慢病毒RNA干扰表达载体,并验证了其在人脐静脉内皮细胞上的有效性。     

核转运蛋白2慢病毒RNA干扰载体的构建及鉴定

目的探索构建针对人类核转运蛋白2(KPNA2)基因的重组慢病毒干扰RNA(sh RNA)载体的方法,研究其对人骨肉瘤细胞株MG63的沉默效率。方法针对KPNA2基因设计小片段干扰序列。加入AgeⅠ酶和Eco RⅠ酶酶切位点,根据设计好的序列合成单链DNA oligo并退火合成双链DNA oligo。将合成好的双链DNA oligo与GV115载体重组,形成GV115-sh RNA慢病毒载体。聚合酶链式反应(PCR)法鉴定筛选出阳性重组子,经测序后包装慢病毒载体。荧光法测定病毒滴度,重组慢病毒感染骨肉瘤细胞株MG63,通过显微镜观察细胞中绿色荧光蛋白(GFP)的表达来计算其转染效率。RT-PCR法检测重组慢病毒RNA干扰载体对人骨肉瘤细胞株MG63中KPNA2表达的沉默效率。实验结果数据以均数±标准差(sx±)表示,应用SPSS 13.0统计软件分析,两组均数间的比较采用t检验,以P<0.05为差异有统计意义。结果经PCR分析和测序证实,成功构建KPNA2基因重组慢病毒RNA干扰载体;荧光法测定病毒滴度为4×108 TU/ml;荧光显微镜下观察GFP,显示细胞感染效率达到80%以上;RT-PCR检测对照组及实验组KPNA2 m RNA表达分别为0.991±0.087 vs.0.216±0.012,差异有统计学意义(P<0.01)。结论 KPNA2基因重组慢病毒RNA干扰载体构建成功,并有效干扰人骨肉瘤细胞株MG63中KPNA2 m RNA的表达。     

慢病毒介导TIEG1基因乳腺癌靶向载体的构建及活性测定

目的:构建慢病毒介导的TIEG1基因乳腺癌特异性靶向载体,并鉴定其活性。方法:将乳腺癌特异性survivin启动子片段和TIEG1基因先后克隆进带有绿色荧光蛋白的慢病毒载体中,构建survivin启动子驱动的TIEG1基因慢病毒表达载体,酶切及测序鉴定。包装纯化慢病毒颗粒,感染人脐静脉内皮细胞HUVEC和乳腺癌细胞SK-BR-3,观察绿色荧光蛋白的特异性表达。结果:成功构建带有肿瘤特异性survivin启动子驱动的TIEG1基因慢病毒表达载体。感染细胞后,乳腺癌SK-BR-3细胞可观察到绿色荧光表达,而人正常脐静脉内皮细胞基本无表达。半定量RT-PCR证实TIEG1基因在乳腺癌SK-BR-3细胞中过表达。结论:构建的survivin启动子驱动的慢病毒表达载体具有一定的肿瘤特异性,将为实现以慢病毒为载体的TIEG1基因肿瘤靶向治疗提供良好的实验基础。     

核糖体结合蛋白1干扰慢病毒载体的构建及其对人肝癌细胞的干扰效应

目的构建核糖体结合蛋白1(RPN1)的干扰慢病毒载体,建立下调RPN1表达的肝癌细胞系。方法运用分子克隆针对RPN1基因构建干扰慢病毒载体,通过与病毒包装辅助质粒共感染293T细胞包装病毒,高速离心浓缩病毒,免疫荧光方式测定病毒滴度,感染Huh7.5.1肝癌细胞系,实时荧光定量PCR(RTqPCR)测定病毒干扰效果。结果成功构建并包装RPN1RNA干扰慢病毒,病毒滴度为2×10~9 TU/mL,感染Huh7.5.1细胞后阳性率大于90.0%。相对于阴性对照,RPN1RNA干扰慢病毒组RPN1基因表达敲减效率达到92.4%(P0.05)。结论成功构建并包装了RPN1干扰慢病毒,建立下调RPN1表达的人肝癌细胞株。     

靶向Egr-1基因RNA干扰慢病毒载体的构建及鉴定

目的构建靶向人早期生长反应因子(early growth response-1,Egr-1)基因RNA干扰(RNA interference,RNAi)慢病毒载体。方法针对Egr-1基因设计并合成1条Egr-1过表达序列和4条干扰序列,过表达序列与双酶切Ubi-MCS-3FLAG载体连接,干扰序列与双酶切hU6-MCS-CMV-EGFP连接,DNA测序鉴定重组载体。共转染人脐静脉血管内皮细胞后(OE-RNAi-1,OE-RNAi-2,OE-RNAi-3,OE-RNAi-4)Western blot检测Egr-1蛋白表达,筛选有效干扰靶点,实时定量荧光PCR检测Egr-1mRNA,验证干扰效果。结果测序表明Egr-1过表达及RNAi慢病毒载体构建成功;Western blot结果显示,OE-RNAi-4组Egr-1蛋白表达抑制率最高(P<0.05);实时荧光定量PCR验证结果显示,OE-RNAi-4组Egr-1mRNA表达受到明显抑制(P<0.05)。结论本研究成功构建了靶向Egr-1基因RNAi慢病毒载体,为进一步研究Egr-1在静脉桥血管再狭窄中早期内皮功能失调作用机制及基因治疗提供了实验依据。     

应用腺病毒载体介导RNA干扰技术构建糖皮质激素受体基因阻断的人U937巨噬细胞模型

目的:利用腺病毒载体介导的RNA干扰技术,建立糖皮质激素受体基因阻断的人U937巨噬细胞模型。方法:实验于2004-06/2005-05在解放军第三军医大学新桥医院呼吸研究所实验室完成。将针对糖皮质激素受体发挥RNA干扰效应的合成寡核苷酸序列连入含有绿色荧光蛋白基因的腺病毒穿梭质粒pRNAT-H1.1/Adeno中,在含有pAdEasy-1病毒骨架的BJ5183大肠杆菌内进行同源重组;重组子通过脂质体介导转染293T细胞,并在293T细胞内包装为具有感染能力的病毒颗粒,通过反复感染扩增病毒以达到感染靶细胞的适当滴度,通过绿色荧光表达来监控腺病毒扩增;应用Westernblot法检测U937巨噬细胞感染后糖皮质激素受体蛋白的表达。结果:转染腺病毒载体的293T细胞表达绿色荧光,随着时间逐渐增强,并且出现明显的细胞病变效应,经过3轮扩增,病毒达到合适的滴度。受腺病毒感染的U937巨噬细胞内糖皮质激素受体蛋白表达明显降低。结论:可以成功构建针对糖皮质激素受体基因的RNA干扰腺病毒载体。     

慢病毒介导环氧化酶-2的沉默对食管鳞癌细胞增殖的抑制效应

目的制备环氧化酶-2(COX-2)的RNA干扰(RNAi)重组慢病毒(lentivirus),观察COX-2表达沉默对人食管鳞癌(ESCC)细胞增殖的影响。方法将目的基因短发卡RNA(sh RNA)载体与包装载体共转染293T细胞,收集上清液后浓缩纯化、滴度测定、感染ESCC细胞;应用荧光显微镜观察包装效率及感染效率,实时荧光定量PCR及Western blot评价COX-2基因沉默效应,MTS法检测细胞的增殖。结果成功制备了沉默COX-2的重组慢病毒,该病毒感染ESCC细胞后,COX-2 m RNA和蛋白的表达量分别降低了80%和50%左右,细胞的增殖明显减慢。结论慢病毒介导的sh RNA干扰能有效沉默ESCC细胞COX-2基因,COX-2表达沉默抑制ESCC细胞增殖。     

小鼠α1,3-半乳糖基转移酶RNA干扰重组慢病毒的构建与鉴定

背景:研究表明,通过抑制α1,3-半乳糖基转移酶的生成而减少甚至避免供体Gala(1,3)Gal的生成,是渡过器官移植后超急性排斥反应的一条切实可行的方法.目的:构建小鼠α1,3-半乳糖基转移酶基因的RNA干扰慢病毒载体,有效沉默小鼠血管内皮细胞的α1,3-半乳糖基转移酶基因表达,以期为有效控制异种移植超急性排斥反应提供稳定的转染细胞载体.设计、时间及地点:单一样本观察,于2006-07/2007-05在天津医科大学总医院普外研究所完成.材料:慢病毒载体系统(四质粒)购自Tronolab公司,包装细胞293T细胞株购自中科院上海细胞所.方法:在线软件设计小鼠α1,3-半乳糖基转移酶基因发卡小分子干扰RNA片段.合成的双链DNA片段并将其连接到Tele-sh003/U6.2载体的黏性末端,产生重组质粒Tele-sh003/U6.2-shRNN/α1,3-半乳糖基转移酶.对干扰质粒做测序分析.用磷酸钙法将得到的阳性重组子与慢病毒包装质粒共转化293T细胞,72 h后收集含病毒上清液,超速离心后得到重组慢病毒.用实时定量聚合酶链反应测定病毒滴度.主要观察指标:干扰质粒的测序分析,慢病毒颗粒的包装和滴度测定.结果:小鼠α1,3-半乳糖基转移酶基因小发卡RNA片段被成功克隆进Tele-sh003质粒中,测序结果显示干扰质粒小干扰RNA编码序列与设计片段的序列完全一致.所得慢病毒亦为阳性克隆,经定量聚合酶链反应测定病毒滴度为2×108 Tu/mL.结论:成功构建出小鼠α1,3-半乳糖基转移酶基因的小发卡RNA慢病毒RNA干扰表达载体,为进一步控制异种移植超急性排斥反应提供了稳定的转染细胞的载体.     

Nesprin基因RNAi慢病毒载体的构建

背景：Nesprin蛋白缺失将影响细胞骨架组织和动态平衡,引起细胞骨架刚性丧失或导致细胞过早成熟老化,其对间充质干细胞的作用如何？目的：构建Nesprin蛋白siRNA慢病毒载体,并转染骨髓间充质干细胞.方法：针对Nesprin靶基因序列设计并合成4对miRNA oligo,将4种miRNA干扰质粒转入大鼠血管平滑肌细胞,筛选最有效干扰序列;将最佳干扰序列和pDONR221载体进行重组反应,获得含干扰序列的入门载体,再将入门载体和慢病毒表达目的载体pLenti6/V5-DEST进行重组反应,获得含干扰序列的慢病毒表达载体,转染包装细胞293T细胞,包装慢病毒,以293T细胞GFP蛋白水平测定病毒滴度.慢病毒转染大鼠骨髓间充质干细胞.结果与结论：测序证实合成的4对miRNA oligo正确,RT-PCR和western-blot筛选出最佳干扰miRNA质粒为SR-3,成功构建了Nesprin siRNA的慢病毒载体LV-siNesprin.包装慢病毒,浓缩病毒悬液的活性滴度为106 TU/mL.慢病毒成功了转染骨髓间充质干细胞细胞.     

Nesprin基因RNAi慢病毒载体的构建

背景:Nesprin蛋白缺失将影响细胞骨架组织和动态平衡,引起细胞骨架刚性丧失或导致细胞过早成熟老化,其对间充质干细胞的作用如何?目的:构建Nesprin蛋白siRNA慢病毒载体,并转染骨髓间充质干细胞.方法:针对Nesprin靶基因序列设计并合成4对miRNA oligo,将4种miRNA干扰质粒转入大鼠血管平滑肌细胞,筛选最有效干扰序列;将最佳干扰序列和pDONR221载体进行重组反应,获得含干扰序列的入门载体,再将入门载体和慢病毒表达目的载体pLenti6/V5-DEST进行重组反应,获得含干扰序列的慢病毒表达载体,转染包装细胞293T细胞,包装慢病毒,以293T细胞GFP蛋白水平测定病毒滴度.慢病毒转染大鼠骨髓间充质干细胞.结果与结论:测序证实合成的4对miRNA oligo正确,RT-PCR和western-blot筛选出最佳干扰miRNA质粒为SR-3,成功构建了Nesprin siRNA的慢病毒载体LV-siNesprin.包装慢病毒,浓缩病毒悬液的活性滴度为106 TU/mL.慢病毒成功了转染骨髓间充质干细胞细胞.     

Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay

BACKGROUND: The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment. METHODS: Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1beta (IL-1beta) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes. RESULTS: The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy-Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1beta levels were lower in carriers of the G allele. CONCLUSIONS: This PCR assay is a rapid and reliable technique for TLR4 genotyping.     

Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary--K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2(C95Y), functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.     

Pathways of renal injury in systemic gram-negative sepsis

Acute renal failure is a grave complication of systemic gram-negative sepsis. The pathophysiological mechanisms of sepsis leading to kidney injury result in part from systemic inflammatory and haemodynamic alterations. These are triggered by the interaction of endotoxin with Toll-like receptor 4 (TLR4) on cells of the immune system. Recently, TLR4 and other co-effector molecules were identified on renal tubular and vascular cells. Furthermore, it was demonstrated that systemic endotoxin has direct access to renal sites where these receptors are expressed. Therefore, we review data in support of this novel pathway of renal injury in sepsis, whereby systemic endotoxin causes direct injury through interactions with local epithelial and endothelial TLR4.     

脂多糖刺激前后小鼠肺肝脾组织中Toll样等受体基因表达情况

目的了解脂多糖(LPS)对肺、肝、脾组织中与LPS信号转导有关的受体的基因表达情况的影响。方法LPS刺激前后取小鼠肺、肝、脾组织总RNA，采用逆转录-聚合酶链反应(RT-PCR)半定量测定脂多糖结合蛋白(LBP)、CD14、Toll样受体2(TLR2)、TLR4和肿瘤坏死因子-α(TNF-α)等基因的表达情况。结果正常组织中，肺有TLR2的表达，肝有TLR2、CD14、LBP的表达，脾有TLR2的表达；注射LPS后的5h内，肺组织TLR2、TLR4、CD14和TNF-α，肝组织TLR2、CD14、LPB和TNF-α，脾组织TLR2、TLR4、CD14和TNF-α的基因表达均呈逐渐增加趋势。结论正常情况下TLR2等基因在不同组织中的表达有差异，LPS刺激5h内各基因的表达提高，从而提高机体对LPS等病原体模式识别分子的反应性。     

HMGB1 signals through toll-like receptor (TLR) 4 and TLR2

In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.     

Polymicrobial sepsis and endotoxemia promote microvascular thrombosis via distinct mechanisms

Summary. Background: We reported recently that endotoxemia promotes microvascular thrombosis in cremaster venules of wild‐type mice, but not in mice deficient in toll‐like receptor 4 (TLR4) or von Willebrand factor (VWF). Objective: To determine whether the clinically relevant model of polymicrobial sepsis induced by cecal ligation/perforation (CLP) induces similar responses via the same mechanisms as endotoxemia. Methods: We used a light/dye‐injury model of thrombosis in the cremaster microcirculation of wild‐type mice and mice deficient in toll‐like receptor‐4 (C57BL/10ScNJ), toll‐like receptor 2 (TLR2), or VWF. Mice underwent CLP or sham surgery, or an intraperitoneal injection of endotoxin (LPS) or saline. In the CLP model, we assessed the influence of fluid replacement on thrombotic responses. Results: Both CLP and LPS enhanced thrombotic occlusion in wild‐type mice. In contrast to LPS, CLP enhanced thrombosis in TLR4‐ and VWF‐deficient strains. While TLR2‐deficient mice did not demonstrate enhanced thrombosis following CLP, LPS enhanced thrombosis in these mice. LPS, but not CLP, increased plasma VWF antigen relative to controls. Septic mice, particularly those undergoing CLP, developed significant hemoconcentration. Intravenous fluid replacement with isotonic saline prevented the hemoconcentration and prothrombotic responses to CLP, though fluids did not prevent the prothrombotic response to LPS. Conclusions: Polymicrobial sepsis induced by CLP and endotoxemia promote microvascular thrombosis via distinct mechanisms; enhanced thrombosis induced by CLP requires TLR2 but not TLR4 or VWF. The salutary effects of intravenous fluid replacement on microvascular thrombosis in polymicrobial sepsis remain to be characterized.