重组TACE前肽蛋白对小鼠内毒素血症抑制作用的实验研究

目的 建立小鼠内毒素血症模型,观察肿瘤坏死因子前体转换酶(TACE)前肽结构域对其催化结构域的自身抑制作用,为人工干预炎症过程提供依据和手段.方法 首先利用DNA重组技术分别构建含前肽结构域和催化结构域的重组载体:用PCR方法扩增出TACE的胞外结构域(T1300)和前肽结构域(T391),并克隆至载体pET-28a(+)中,转化至大肠杆菌BL21,经IPTG诱导表达出带有His-tag的目的 蛋白,两者均为包涵体,变性复活后经Ni2+-NTA亲和层析柱对表达的重组蛋白进行纯化.对纯化后的蛋白进行Western印迹分析.构建TACE信号肽+前肽结构域(T648)的真核表达质粒(pEGFP-N1/T648),将其瞬时转染HeLa细胞,通过观察绿色荧光蛋白的表达观察pEGFP-N1/T648转染后在细胞中分布情况.建立小鼠内毒素血症模型组,前肽重组蛋白治疗组和PRS对照组,通过流式细胞术检测小鼠腹腔巨噬细胞mTNF-a的表达,并取肝,肺,肾组织行HE染色,观察纯化的前肽蛋白对炎症的抑制作用.结果 成功构建了pEGFP-N1/T648真核表达载体,在体外转染HeLa细胞,可见荧光主要分布在细胞膜上.流式细胞技术证明重组前肽蛋白可明显对抗LPS诱发的sTNF-a分泌增加,使小鼠腹腔巨噬细胞膜表面mTNF-a增加,常规病理切片HE染色显示该蛋白可降低LPS诱发的小鼠肝,肾,肺组织的炎症反应.结论 TACE前肽重组蛋白降低了sTNF-a的分泌,对炎症有明显的抑制作用,为抗炎药物的设计和改造提供了新的依据和方法.     

肿瘤坏死因子前体转换酶前肽结构域对TACE活性调节作用

目的： 探讨肿瘤坏死因子前体转换酶（TACE）的前肽结构域在TACE成熟过程中所起的作用，为人工干预炎症过程提供依据和手段。方法: 以pIRES2-EGFP质粒为载体，利用DNA重组技术分别构造含信号肽结构域+前肽结构域、全长结构域及缺失前肽结构域的真核表达重组体，根据其碱基数目分别命名为pIRES2-EGFP/T648、pIRES2-EGFP/T2472、 pIRES2-EGFP/T57-T1824；真核转染U937细胞；LPS刺激转染细胞后，用ELISA法及流式细胞技术检测TNF-α。结果: pIRES2-EGFP/T648的真核表达能显著抑制TACE的活性，减少sTNF-α分泌，抑制率达61.09%；pIRES2-EGFP/T57-T1824的真核表达对sTNF-α的分泌无影响；pIRES2-EGFP/T2472的真核表达能显著增加sTNF-α分泌。结论: 前肽结构域在TACE的成熟过程中起了双重作用，TACE抑制剂的研制和开发为抗炎药物的设计和改造提供了新的依据和方法。     

三类TACE抑制剂的作用机理探讨

目的 :以脂多糖 (LPS)刺激的HL 6 0细胞系为天然模型 ,筛选肿瘤坏死因子α转换酶的抑制剂 ,为人工干预炎症过程提供依据和手段。方法 :分时相刺激HL 6 0细胞后 ,用分子生物学和细胞学技术在蛋白质和mRNA水平上研究TACE表达和活性的改变。结果 :中药热毒清 (RDQ)能明显降低LPS诱导的TACEmRNA增加 (与对照组比 ,P <0 .0 1) ;针对TACEmRNA的反义寡核苷酸 (anti ODN)在翻译水平上抑制TACE表达 ,抑制率达 78.9% ;化学合成的针对TACE催化域的小分子底物模拟肽可抑制LPS诱导的TACE表达增加 ,从而抑制sTNFα分泌增加 ,削弱LPS诱发的炎性病理反应强度。结论 :3种不同类型的抑制 (RDQ、anti ODN、底物模拟肽 )在不同层次上调节TACE的表达 ,最终均抑制了sTNFα的分泌 ,为控制炎症提供了明确的治疗靶标。     

LPS刺激对TACE基因表达和对TNF-α前体加工的影响

目的 :研究LPS刺激对肿瘤坏死因子转换酶 (TACE)基因转录和表达的影响及其与TNF α前体加工的关系。方法 :采用Dot blotting、Dot ELISA、间接免疫荧光和FACS等方法分别检测HL 6 0细胞在LPS刺激的不同时间段TACE、TNF α基因转录表达和膜分子分布情况。结果 :①HL 6 0细胞构成性高表达有活性TACE ,主要分布于细胞膜和核周边。②LPS刺激TNF α基因转录表达 ,但由于TACE的作用 ,膜TNF α(TM TNF α)下降 ,而分泌型TNF(sTNF α)增加 ;TACE的反义寡核苷酸 (ODN)显著抑制TNF α这种前体转换作用。③LPS同样刺激TACE基因表达增加 ,而且TACE酶解作用增强 ,但其蛋白表达和膜分子分布反而下降 ,推测TACE在发挥其催化膜蛋白作用后 ,其分子结构形式发生改变。结论 :炎症时分泌型TNF α增加不仅是该细胞因子基因表达增高所致 ,更受到TACE基因表达水平和活性状态的约束。     

TNF结合肽和TNFR封闭肽对TNBS诱导的大鼠溃疡性结肠炎的治疗作用

目的 检测TNF-α结合肽和TNFR封闭肽对TNBS诱导的大鼠溃疡性结肠炎的治疗作用.方法 以TNBS诱导的大鼠溃疡性结肠炎为模型,联合给予TNF-α结合肽和TNFR封闭肽[2.5 mg/(k·次)]处理,同时设各种对照;观察大鼠的症状、体重、结肠组织病理改变等;采用一般的生物化学检测方法检测大鼠血清和结肠组织中NO含量、活性氧以及结肠组织中MP0活性等炎性介质;采用RT-PCR技术检测大鼠腹腔MφIL-1β和IL-8等mRNA水平的变化;采用免疫组织化学SP法检测大鼠结肠组织中TNF-α蛋白质表达水平的改变.结果 TNF结合肽和TNFR封闭肽联合多肽组大鼠的体重、结肠组织形态学评分、组织病理学评分及血清和结肠组织中NO含量、MP0活性等指标均显著低于模型组及无关肽组(P＜0.01);其腹腔Mφ的IL-1β和IL-8 mRNA水平和结肠组织中TNF-α蛋白的表达水平及阳性细胞率也显著低于显著低于模型组及无关肽组(P＜0.01).结论 TNF结合肽和TNFR封闭肽联用可显著减轻TNBS诱导大鼠实验性溃疡性结肠炎的病理损伤,改善症状,有效抑制大鼠血清和结肠组织中NO含量以及结肠组织中MP0活性的活性,抑制大鼠腹腔巨噬细胞中Ⅱ-1β、IL-8的mRNA转录和结肠组织中TNF-α蛋白的表达,对TNBS诱导的大鼠溃疡性结肠炎具有治疗作用.本研究为进一步研制和开发拮抗TNF-α的新型肽类药物提供了重要的实验依据.     

利用噬菌体肽库筛选TNF-α表位的相关短肽

目的 从噬菌体肽库筛选TNF－α相关短肽，为临床上治疗肿瘤奠定基础。方法 用可溶性TNF受体Ⅰ（sTNFR Ⅰ）筛选噬菌体随机12肽库，利用细胞毒效应进行生物学效应筛选，再进行单链DNA测序。结果 分别得到若干模拟跨膜型TNF－α（TM－TNF－α）和模拟分泌型TNF－α（S－TNF－α）细胞毒效应的短肽，选择细胞毒效应最好的模拟TM－TNF－α短肽进行人工合成，体外实验显示此肽段对S－TNF－α耐受肿瘤细胞系HL－60具有明显细胞毒效应，且主要引起靶细胞凋亡。另外，利用激光共聚焦显微镜证实该合成肽段不能诱导NF－κB发生核转位。结论 获得模拟TM－TNF－α的短肽，为TM－TNF－α用于临床抗瘤治疗提供新线索。     

肿瘤坏死因子α转化酶的性质及其作用

体内成熟的TNF为 17KD ,其由 2 6KD之前体蛋白在Ala76 ～Val77之间断裂产生 ,催化此过程的酶称为肿瘤坏死因子α转化酶 (TumorNecrosisFactorαConvertingEnzyme ,TACE) ,其确切性质及作用意义 ,目前尚未完全明了 ,本文就此作一简要综述。     

014 肿瘤坏死因子α转化酶的性质及其作用

体内成熟的TNF为17KD,其由26KD之前体蛋白在Ala76～Val77之间断裂产生,催化此过程的酶称为肿瘤坏死因子α转化酶(Tumor Necrosis Factor α Converting Enzyme,TACE),其确切性质及作用意义,目前尚未完全明了,本文就此作一简要综述.     

羧胺三唑与小剂量地塞米松合用对A549肺癌移植瘤生长抑制作用的研究

目的观察羧胺三唑（CAI）对A549肺癌移植瘤生长及肿瘤环境中肿瘤坏死因予-α含量和血管生成的影响,研究小剂量地塞米松（DEX）是否能通过降低肿瘤环境中肿瘤坏死因子-α含量和抑制血管生成增强CAI对移植瘤生长的抑制作用。方法向裸鼠腋下接种A549肺癌细胞,建立A549肺癌移植瘤模型,接种第2天随机分5组,并开始给药,分别是PEG400溶剂对照组（每天一次灌胃给药,0．1ml／10g体重）、CAI组（每天一次灌胃给药,20mg／kg）、DEX组（每周两次背部皮下注射给药,1mg／kg）、CAI与DEX合用组（CAI,每大一次灌胃给药,20mg／kg;DEX,每周两次背部皮下注射给药,1mg／kg）及英夫利昔单抗组（每周两次腹腔注射给药,10mg／kg）。40d后剥离瘤组织,拍照并称重;将部分新鲜肿瘤组织速冻后制作冰冻切片,进行CD31荧光染色;另一部分新鲜瘤组织制各匀浆后用ELISA方法对其TNF—α含量进行检测。结果CAI、DEX和TNF—α抗体（英夫利昔单抗）对移植瘤生长均有抑制作用,而CAI与DEX的联合应用抑癌作用最强,PEG、CAI、DEX、COM和TAB组瘤重分别为（0．23±0．04）、（0．21±0．02）、（0．17±0．04）、（0．12±0．03）和（0．17±0．04）g。CD31血管染色显示,CAI和DEX均能减少肿瘤组织内的血管数目,两者合用在极大程度上抑制了血管的生成;CAI能够降低肿瘤组织内TNF-α含量,与DEX合用使TNF-α含量降至更低水平,PEG、CAI、DEX、COM和TAB组肿瘤组织匀浆中TNF-α的含量分别为（933．9±286．8）、（636．1±157．4）、（684．9±170．1）、（364．7±50．1）和（606．8±190．3）pg／mg蛋白。结论CAI不仅能够直接影响肿瘤细胞的增殖和凋亡,还能通过调控肿瘤环境中的TNF-α抑制血管的生成,从而减缓肿瘤的生长,这可能是CAI发挥抗肿瘤作用一个新的机制。小剂量DEX与CAI联合用药对移植瘤生长的抑制作用明显增强,优化了CAI的抗肿瘤作用,可能成为一种新的联合用药方案。     

肿瘤坏死因子 α抑制剂对妊娠的影响

 肿瘤坏死因子 α（TNF-α）是类风湿关节炎（RA）发病和维持关节慢性滑膜炎症反应的最重要的致炎性细胞因子之一，它在 T 细胞依赖的炎症性肠病（IBD）发病机制中也起着十分重要的致炎作用。大量临床研究证实，TNF-α抑制剂（TNFAI）可改善 RA 患者关节功能，减少 RA临床活动性，并延缓关节损坏的进展^[1]。各种 TNFAI 也逐渐用于治疗其他风湿性疾病，如银屑病、幼年特发性关节炎、强直性脊柱炎等。某些 TNFAI 被证明对难治性 IBD 有效。目前经美国 FDA 批准上市的 TNFAI 类药物共有 3 种，它们是依那西普（Etanercept，商品名Enbrel），英利西单抗（Infliximab，商品名 Remicade）和阿达木单抗（Adalimumab，商品名Humira）。依那西普是 II 型TNF受体（TNFR-II）与 IgG1-Fc 的融合蛋白，用药方式为皮下注射。英利西单抗是由人 IgG1-Fc 和鼠 Ig 可变区组成的嵌合体 TNF-α 单抗，阿达木单抗是人源 IgG1 型 TNF-α 单抗，这2 种药物均经静脉注射给药[1]。TNFAI的主要不良反应有注射部位反应、感染、肿瘤、淋巴增殖性疾病、神经脱髓鞘病变以及狼疮样综合征^[1]。由于风湿性疾病多累及生育年龄女性，同时以 TNFAI 为代表的生物制剂应用越来越广泛，TNFAI 对妊娠是否有影响日益受到临床关注。我们复习了2007 年 2 月1日以前 PubMed 中关于上述 3 种 TNFAI对妊娠影响的临床观察文献，现综述如下。......     

Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.     

TACE cleaves neogenin to desensitize cortical neurons to the repulsive guidance molecule

Neogenin is a receptor for netrins and proteins of the repulsive guidance molecule (RGM) family. It regulates several key developmental processes within the nervous system. The binding of RGMa to neogenin induces the inhibition of neurite outgrowth and the collapse of the growth cone of neurons. Here, we report that a disintegrin and metalloprotease (ADAM) transmembrane protein regulates the sensitivity of neurons to RGMa, by inducing the shedding of the ectodomain of neogenin. The extracellular domain of neogenin is directly associated with and cleaved off by the tumor necrosis factor-α converting enzyme (TACE), also called ADAM17. TACE is endogenously expressed in embryonic cortical neurons and regulates the cleavage of neogenin, and the inhibition of TACE in turn enhances RGMa-induced inhibition of neurite outgrowth and collapse of the growth cone. Conversely, exogenous expression of TACE abolishes the effect of RGMa. Therefore, TACE may play a role in modulating the RGM-induced repulsive behavior of neurons by regulating the expression of neogenin on the cell surface.     

Independently expressed N-terminal pro-domain of aqualysin I precursor complements the folding of its mature domain to active form in Escherichia coli

Aqualysin I is a subtilisin-type serine protease secreted into the culture medium by Thermus aquaticus YT-1. It is first produced as a large precursor that consists of a signal peptide, an N-terminal pro-domain, the mature protease domain and a C-terminal pro-domain. To investigate whether the N-terminal pro-domain supplied in trans as an independent peptide plays an important role in the folding and secretion of the protease, the N-terminal pro-domain in E. coli has been expressed independent of the mature domain with or without the C-terminal pro-domain using an expression system with separate promoters and signal peptides. Protease assay and SDS-PAGE clearly showed that the N-terminal pro-domain plays an essential role in guiding the proper folding in trans of the enzymatically active conformation of aqualysin I. The N-terminal amino acid sequences of the purified enzymes were identical and had no signal peptides. These results indicate that independently expressed domains are secreted into the periplasmic space before the N-terminal pro-domain-assisted folding of the mature domain.     

Inhibition of complement alternative pathway in mice with Fab antibody to recombinant adipsin/factor D

Mouse adipsin is a serine protease secreted mainly by adipocytes. Similarly to factor D of human complement, it cleaves factor B. That adipsin is the equivalent of human factor D in the mouse is further suggested by their structural homology. Specific antisera against recombinant mouse adipsin (r-adipsin) were produced in rabbits. Anti-r-adipsin IgG was shown to bind to radiolabeled r-adipsin and to inhibit its hemolytic activity. In vitro, these antibodies Ab and Fab fragments thereof inhibited the adipsin/factor D hemolytic activity of mouse serum. They also blocked C3 activation induced by cobra venom factor (CVF), but did not interfere with classical pathway function. After intravenous injection of antir-adipsin Fab into BALB/c mice, the adipsin/factor D hemolytic activity of serum was abolished during a 4-h period. The C3 depleting effect of CVF injected intravenously was significantly delayed in BALB/c mice which had been pretreated with anti-r-adipsin Fab. These experiments demonstrate that mouse adipsin is the only form of mouse factor D and that anti-r-adipsin antibody can be used to produce a specific inhibition of the alternative pathway in vivo.     

Inhibition of the intracellular growth of Histoplasma capsulatum by recombinant murine gamma interferon.

Recombinant murine gamma interferon as well as lymphokines prepared from immune splenocytes and concanavalin A-stimulated T-cell hybridoma activated normal mouse peritoneal macrophages to inhibit the intracellular growth of Histoplasma capsulatum. The activities of the lymphokine from immune splenocytes and of recombinant murine gamma interferon were neutralized by rabbit anti-murine gamma interferon antibody. The intracellular yeasts were not killed by the interaction even though growth was completely inhibited.     

Inhibition of Chlamydia trachomatis growth by recombinant tumor necrosis factor.

Purified human recombinant tumor necrosis factor (rTNF) alpha inhibited the growth of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cell cultures. The inhibition of C. trachomatis yield could be achieved even when the rTNF alpha (200 ng/ml) was added up to 12 h after infection. The effect of rTNF alpha on chlamydial infection was synergistic with that of gamma interferon.     

Inhibition of hepatitis B virus replication by recombinant small interfering RNAs

RNA interference (RNAi) is a biological phenomenon in which introduction of a small, double-stranded interfering RNAs (siRNAs) into a cell causes a specific degradation of homologous single-stranded RNA. siRNA can be delivered into the cell by different approaches including synthetic RNA, in vitro transcribed RNA and RNA transcribed from polymerase III-based recombinant vectors. As hepatitis B (HB) represents a worldwide health problem, we attempted to develop a fast and easy approach to generation and screening of specific siRNA-targeted HB virus (HBV) genes. Using PCR amplification, specific siRNA expression cassettes (SECs) were developed and used to generate effective siRNAs against HB virus (HBV) replication and gene expression in mammalian cells. After screening, we identified two SECs that expressed siRNAs which efficiently decreased the level of HBV pre-c/c gene expression in transfected Bel-7402 cells by 81.9% and 87.3%, respectively. In addition, the level of HBV DNA was decreased by 83.5% and 85.2% in HepG2 2.2.15 cells, respectively. This study provides (i) a new effective application of RNA interference to study viral gene function and viral replication and (ii) a new tool for the prevention and treatment of human HBV infection.     

Inhibition of NF-kappaB mediated inflammation by siRNA expressed by recombinant adeno-associated virus

NF-kappaB mediated inflammation is a key process to many diseases. RNA interference (RNAi) is the specific suppression of genes by short double-stranded RNA. It was the aim of the present study to modify NF-kappaBdependent inflammation by small interfering RNA (siRNA) expressed by recombinant adeno-associated virus (rAAV). To study the kinetics of rAAV mediated expression of siRNA, the expression of the luciferase gene was targeted and resulted in a significant decrease of luciferase activity as compared to a control vector in the human 293 cell line. The effect was dose dependent and was detectable 24 h after infection. rAAV coding for siRNA against the p65 subunit of NF-kappaBsignificantly reduced the p65 protein. In a cellular model of TNF-alpha induced inflammation, expression of siRNA against p65 significantly suppressed the secretion of IL-8 from BEAS-2B cells. In conclusion, rAAV vectors coding for siRNA are an useful tool for efficient gene silencing in mammalian cells and can be used to modify NF-kappaB mediated inflammation.