血管形成抑制因子HIAF-1在昆虫细胞中的高效表达和活性研究

背景与目的：大肠杆菌表达的人抑制血管生成因子－1（human inhibiting angiogenesis factor-1,HIAF-1）可抑制肿瘤血管的形成。本研究在昆虫细胞中表达HIAF－1,并研究其生物活性。方法：用DNA重组技术,构建了重组杆状病毒表达载体pAcuW51-HIAF-1,用Cellfectin将其与线性病毒DNA共转染昆虫细胞sf9,获得重组病毒。用SDS－PAGE电泳和Western blot鉴定重组病毒感染的昆虫细胞中重组HIAF－1重组蛋白的表达。重组蛋白经亲和层析纯化后,在体外用MTT法检测其对内皮细胞的作用,用食管癌移植瘤研究其体内抑瘤活性。结果：在昆虫细胞中高效表达了分子量为26kDa的HIAF－1重组蛋白,其表达量可达昆虫可溶性蛋白的5％－10％。重组HIAF－1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3．1μg/ml,而且显著抑制食管癌移植瘤的生长。结论：在昆虫细胞中高效表达了分子量为26kDa的HIAF－1重组蛋白,其表达量可达昆虫可溶性蛋白的5％－10％。重组HIAF－1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3．1μg/ml,而且显著抑制食管癌移植瘤的生长。结论：在昆虫细胞中高效表达了HIAF－1重组蛋白;HIAF－1重组蛋白的生物活性优于相应的原核重组蛋白。     

人血管抑素克隆表达及抑制血管生成的研究

目的　研究并获得重组人血管抑素 (angiostatin) ,探讨其临床应用价值。方法　采用逆转录 -聚合酶链式反应 (RT PCR )方法 ,从人胚胎肝细胞中扩增出人全长血管抑素cDNA片段。用原核细胞表达载体构建 pBV 2 2 0 /angiostatin重组质粒 ,并将其转化大肠杆菌DH 5α进行表达 ,初步纯化。应用鸡胚尿囊膜血管生成实验及脐静脉内皮细胞增殖实验检测其活性。结果　经SDS PAGE分析 ,表达产物相对分子量约 3 8kD ,薄层扫描显示表达产物可达细菌总蛋白的 3 0 %。活性实验显示 ,重组蛋白能够抑制血管形成。结论　重组人Angiostatin在大肠杆菌中实现高效表达 ,并具有很好的生物学活性     

血管生成抑制素Endostatin研究新进展

１９９７年Ｏ‘Ｒｅｉｌｌ从小鼠血管内皮细胞瘤中分离到一种新的２０ｋＤ具有特异抑制血管内皮细胞生长的抑制因子－－Ｅｎｄｏｓｉａｔｉｎ,它对牛它细血管内皮细胞、牛肺主动脉内皮细胞有特异的抑制增殖作用,而对非血管内皮细胞系细胞如ＮＩＨ３Ｔ３,Ａ１０平滑肌细胞等则无增殖抑制作用,实验证实大肠杆菌生产的复性及未复性的Ｅｎｄｏｓｔａｔｉｎ及杆状病毒生产的重组Ｅｎｄｏｓｔａｔｉｎ均有抑制鸡胚血管生成的作用,而且     

Ⅴ型腺病毒纤维蛋白基因真核表达质粒的构建及表达

目的：克隆VEGF受体sFET-1片段1-3区，并使之在原核中进行表达以探讨其对内皮细胞增殖和血管生成方面的抑制作用。方法：提取脐静脉血管内皮细胞总RNA，克隆sFLT-1基因1-3区，并使之在QIA表达系统进行表达，Ni^2 -Sepha-rose6B亲和层析纯化，复性，应用MTT和鸡胚绒毛尿囊膜血管生成模，探讨重组蛋白对血管生成的抑制作用。结果；该表达系统能表达sFLT-1基因片段，表达量低，纯化后，经复性，sFLT-1具有VEGF特异结合功能，1μg重组sFLT-1蛋白能抑制10ngVEGF诱导的脐静脉内皮细胞增殖和20ngVEGF诱导的鸡胚绒毛尿囊膜血管生成，结论：sFLT-1基因片段能在QIA表达系统中得到表达，复性后重组蛋白具有与VEGF结合和拮抗VEGF生物学活性的功能。     

人血管抑制因子Vasostatin短片段(120-180 aa)的克隆、表达及功能研究

目的:利用基因工程技术,克隆并表达人血管抑制因子Vasostatin120-180aa功能区片段,探讨其对鸡胚绒毛尿囊膜新生血管抑制的作用.方法:采用PCR技术扩增人血管抑制因子Vasostatin120-180aa功能区基因,并利用pQE30原核表达系统诱导表达Vasostatin120-180aa,经镍金属螯合层析法纯化,通过鸡胚绒毛尿囊膜实验验证其抑制新生血管生成的作用.结果:PCR扩增出了长度为180 bp的Vasostatin120-180aa功能区基因,后经pQE30原核表达系统表达并纯化出Vasostatin120-180aa,SDS-PAGE显现一条约8 kD的阳性条带,Vasostatin120-180aa可显著抑制鸡胚绒毛尿囊膜新生血管的生成.结论:原核表达的Vasosta-tin120-180aa功能区片段具有生物学活性,可明显抑制鸡胚绒毛尿囊膜新生血管的生成,在一定范围内呈量效依赖性.     

和厚朴酚抗血管生成作用的实验研究

目的：本研究通过检测和厚朴酚在体内对鸡胚尿囊膜（chicken chorilallantoic membranes，CAM）血管生成的抑制作用及体外对肿瘤血管生成的影响，探讨和厚朴酚的抗血管生成作用及其初步机制。方法：（1）用四甲基偶氮唑盐（MTT）法检测和厚朴酚对人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）、人成纤维细胞及人结肠癌RKO细胞增殖的抑制作用；（2）采用鸡胚绒毛尿囊膜模型，观测和厚朴酚对CAM新生血管生成的抑制作用；（3）逆转录聚合酶链反应（RT-RCR）方法检测和厚朴酚对RKO细胞血管内皮生长因子A（VEGF-A）mRNA表达及细胞培养上清液VEGF蛋白分泌的影响。结果：和厚朴酚对血管内皮细胞具有优先抑制作用，对HUVEC的半数抑制剂量（inhibitory concentration 50％，IC50）为14．5μmol/L，而对原代培养的人成纤维细胞的IC50高达150μmol/L，对人结肠癌RKO细胞的IC50为42μmol/L；和厚朴酚0．1μg／只和0．2μg／只剂量时显著抑制CAM新生血管形成，抑制率分别为58％和86％；和厚朴酚在10μmol/L和20μmol/L剂量时显著抑制RKO细胞的VEGF-A mRNA表达及细胞培养上清液中VEGF蛋白的分泌，具有显著性差异。结论：和厚朴酚在非凋亡剂量时具有抗血管形成作用，其机制与抑制血管内皮细胞增殖以及抑制肿瘤细胞表达VEGF有关。     

CXCR4 单克隆抗体对新生血管形成的影响

目的：探讨CXC趋化因子受体4（CXCchemokinereceptor4，CXCR4）单克隆抗体（CXCR4mAb）对新生血管形成的影响及其作用机制。方法：以鸡胚绒毛尿囊膜（chorioallantoicmembrane，CAM）血管抑制实验检测CXCR4单克隆抗体对新生血管形成的影响，WesternBlot免疫印迹法检测血管内皮细胞SDF-1蛋白的表达。结果：CXCR4单克隆抗体可明显抑制新生血管的形成，并且使培养的血管内皮细胞SDF-1蛋白表达明显降低：结论：CXCR4mAb可通过SDF-1／CXCR4生物轴抑制新生血管的形成。     

血管抑制素基因真核表达载体的构建及转染研究

目的：构建血管抑制素（angiostatin,AG)基因真核细胞表达载体并进行转染研究。方法：根据已发表的血管抑制基因的核苷酸序列,设计并合成一对引物、以鼠的纤溶酶原核酸为模板进行PCR扩增,将其基因克隆到真核表达载体pRC/CMV中,进行序列分析和酶切鉴定后,运用 质体将重组质粒pRC/CMV-AG转入胰腺癌SW1990细胞中,观察其表达情况,以及其对血管内皮细胞生长的作用。结果：PCR扩增出一长1．4kb的片断,酶切和序列分析证明含完整的AG基因序列,与已知的血管抑制素基因的同源性很高（＞96％）。Western blot证实重组体pRC/CMV-AG有较高的表达,对血管内皮细胞的生长有一定的抑制作用。结论：重组体pRC/CMV-AC是一种高效真核表达载体;克隆的AG基因能抑制血管内皮细胞的生长。     

可溶性VEGF受体基因sflt-1与反义VEGF核苷酸对新生血管形成的影响

背景与目的:肿瘤组织中新生血管提供的大量营养物质和生长因子是肿瘤快速生长的关键,因此如何抑制肿瘤组织中新生血管的形成、促使肿瘤组织坏死也是肿瘤治疗中的一条值得探讨的途径。本文拟研究和观察可溶性血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)受体基因sflt-1与反义VEGF对新生血管形成的影响。方法:用Ad-反义VEGF感染肝癌细胞株MM45T.Li后,观察反义VEGF对MM45T.Li分泌VEGF的影响;将人工重组的3'ΔFlt-1蛋白(C末端缺失的Flt-1蛋白)加入条件培养液中,观察3'ΔFlt-1对VEGF刺激的脐静脉血管内皮细胞(humanumbilicalvascularendotheliumcell,HUVEC)增殖的影响;并将Ad-反义VEGF、Ad-sflt-1注射于鸡胚尿囊绒毛膜中,观察sflt-1、反义VEGF对鸡胚新生血管形成的影响。结果:反义VEGF重组腺病毒感染MM45T.Li细胞后,细胞培养上清中VEGF浓度仅为对照组中VEGF浓度的15%(P<0.01);在含有3'ΔFlt-1蛋白的调价培养液中,HUVEC的增殖明显减慢,在一定的范围内与剂量呈负相关;两者都能有效地抑制鸡胚新生血管的形成。结论:sflt-1与反义VEGF均能有效抑制新生血管的形成,两者联合能增强抑制效果,但其作用机制不同。     

软骨抗血管生成组份的分离纯化及性质研究

目的从鲨鱼软骨中分离纯化血管生成抑制剂（CDAI）并对其性质加以研究。方法2MNaCl抽提;CM－SephadexC－50离子交换层析;DEAE－SephadexA－50离子交换层析;SephadexG－50凝胶层析。SDS一聚丙烯酰胺凝胶电泳纯度鉴定。MTT法测定CDAI对1H11内皮细胞的作用。鸡胚绒毛尿囊膜试验（CAM）观察CDAI对血管生成的作用。结果得到电泳纯的CDAI,分子量为36280,CDAI对内皮细胞产生强的抑制作用,0．65mg／L时对1H11内皮细胞的增殖抑制率为50％。鸡胚绒毛尿囊膜试验显示CDAI对血管形成有显著的抑制作用。结论鲨鱼软骨中含有血管生成抑制剂,能抑制血管生长,对具有病态血管形成的疾病具有潜在治疗价值。     

人IER5基因在昆虫杆状病毒表达系统中的表达与鉴定

目的：利用昆虫杆状病毒表达系统表达早期快速反应基因5（IER5）蛋白,为后续蛋白质结晶及探索蛋白质三级结构提供线索。方法：以HeLa细胞cDNA为模板扩增出IER5基因片段,构建重组转移质粒pFastBac1-IER5;重组转移质粒经双酶切及测序鉴定后转化至感受态细胞DH10Bac,以获得重组的穿梭质粒rBacmid-IER5;将重组穿梭质粒感染Sf9细胞,待细胞出现明显病变时收集重组杆状病毒;用间接免疫荧光、SDS-PAGE及Western blot以及对表达产物进行分析鉴定。结果：重组转移质粒pFastBac1-IER5经双酶切后得到与预期相同的2条条带;重组穿梭质粒rBacmid-IER5经PCR鉴定在3 300 bp左右出现一条特异性条带;间接免疫荧光结果提示IER5蛋白在Sf9细胞中得到表达;SDS-PAGE结果显示,表达产物的相对分子质量约为48 k,Western blot结果表明表达产物能与IER5抗体特异性结合。结论：利用昆虫-杆状病毒表达系统成功表达了人IER5蛋白,获得了体外表达重组IER5蛋白的相对分子质量、溶解性等物理化学特性。     

重组人血管抑素的克隆、表达及活性测定

目的：研究血管抑素（ａｎｇｉｏｓｔａｔｉｎ）的临床应用价值,将其开发为多肽类药物。方法：ＲＴ－ＰＣＲ从胎儿肝脏钓取人血管抑素全编码区ｃＤＮＡ,使用Ｐｉｃｈｉａ分泌型酵母表达系统表达重组人血管抑素,重组蛋白经肝素亲和层析纯化后,以鸡胚尿囊膜法血管生成实验及小鼠创伤愈合实验检测活性。结果：重组人血管抑素在酵母系统中得到较高量表达（５ｍｇ／Ｌ）,经肝素亲和层析纯化,ＳＤＳ－ＰＡＧＥ显示分子量为４３ｋＤ。活性实验表明,重组蛋白能够抑制新生管形成、抑制伤口愈合。结论：重组人血管抑素在酵母系统中实现高效表达,并具有很好的生物学活性。     

Her2/ECD-sf162/TM融合杆状病毒表达及鉴定

目的:获得融合表达的Her2/ECD-sf162/TM杆状病毒,为后续基于SIV Gag蛋白的Her2病毒样颗粒疫苗的制备奠定实验基础。方法:将Her2胞外段基因与猴免疫缺陷病毒（simian immunodeficiency virus,SIV）包膜蛋白sf162跨膜区基因融合的Her2/ECD-sf162/TM pFastBac重组表达载体,转座大肠杆菌E.coli DH10Bac菌株,获得重组杆粒,在昆虫细胞中表达获得重组杆状病毒,Western blot方法对该病毒进行鉴定。结果:构建的Her2/ECD-sf162/TM杆状病毒表达载体经PCR鉴定正确,转染昆虫细胞后获得了重组杆状病毒,该融合杆状病毒蛋白可与抗Her2抗体发生免疫反应。结论:成功构建的Her2/ECD-sf162/TM杆状病毒可用于Her2病毒样颗粒疫苗的制备。     

乙型肝炎病毒表面抗原(HBsAg)基因重组杆状病毒的构建及鉴定

目的 利用Bac-to-Bac杆状病毒表达系统，构建乙型肝炎病毒(HBV)表面抗原基因的重组杆状病毒。方法 构建含有HBV S基因的表达截体pFBDHTa—S，转化DH10Bac感受态菌。利用其含有的细菌Tn7转座系统，将该基因重组至杆状病毒穿梭质粒bacmicl中，转染昆虫细胞Sf9，获得含有HBV S基因的重组杆状病毒。结果 构建了的含HBV S基因的重组杆状病毒，感染昆虫细胞后，PCR检测可以扩增出相应大小的片段。结论 利用杆状病毒表达系统，成功地构建了含HBV S基因的重组杆状病毒，为进一步的研究奠定了基础。     

Optimization of expression conditions for production of anti-colorectal cancer monoclonal antibody CO17-1A in baculovirus-insect cell system

The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800?μL), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72?h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400?μL of passage 3 baculovirus (P(3) BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72?h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.     

Recombinant adeno-associated virus 2-mediated antiangiogenic prevention in a mouse model of intraperitoneal ovarian cancer.

PURPOSE: In the present study, we sought to determine the potential of sustained transgene expression by a single i.m. administration of recombinant adeno-associated virus 2 (rAAV) encoding angiostatin and endostatin in inhibiting i.p. ovarian cancer growth and dissemination in a preclinical mouse model. EXPERIMENTAL DESIGN: Cohorts of female athymic nude mice received either no virus or 1.2 x 10(11) particles of rAAV encoding green fluorescence protein or endostatin plus angiostatin, i.m. Three weeks later, the mice were i.p. injected with 10(6) human epithelial ovarian cancer cell line SKOV3.ip1. As a measure of effectiveness of the therapy, tumor weight, abdominal distension, ascites volume and vascular endothelial growth factor level, and tumor weight were determined. Immunohistochemistry was done to determine tumor cell apoptosis and endothelial cell proliferation following the therapy. Tumor-free survival was recorded as the end point. RESULTS: Results indicated a significant tumor-free survival (P < 0.003) following therapy with rAAV encoding endostatin and angiostatin compared with untreated or rAAV-green fluorescence protein-treated mice. Ascites volume in rAAV endostatin and angiostatin-treated mice was significantly lower than naive mice and contained less hemorrhage and tumor conglomerates. The level of vascular endothelial growth factor in the ascites of antiangiogenic vector treated mice was also significantly less compared with the untreated mice. Immunohistochemical analyses indicated increased tumor cell apoptosis and decreased blood vasculature following rAAV endostatin and angiostatin treatment. CONCLUSION: The results indicate that antiangiogenic genetic prevention from stable systemic levels of angiostatin and endostatin by i.m. administration of rAAV can be used for the treatment of i.p. ovarian cancer growth and dissemination.     

Baculovirus vectors for antiangiogenesis-based cancer gene therapy

Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors.     

Development of lentiviral vectors for antiangiogenic gene delivery.

Growth and metastasis of malignant tumors requires angiogenesis. Inhibition of tumor-induced angiogenesis may represent an effective cytostatic strategy. We have constructed recombinant self-inactivating lentiviral vectors expressing angiostatin and endostatin, and have tested their antiangiogenic activities. As VSV-G-pseudotyped lentiviral vectors showed low relative transduction titers on bovine aortic and human umbilical vein endothelial cells, it was difficult to achieve significant inhibition of endothelial cell growth by lentivirus-mediated antiangiogenic gene transfer directly to endothelial cells without concomitant vector-associated cytotoxicity. However, lentivirus vectors could efficiently and stably transduce T24 human bladder cancer cells that are relatively resistant to adenovirus infection due to loss of coxsackievirus-adenovirus receptor expression. Long-term expression and secretion of angiostatin and endostatin from lentivirus-transduced T24 cells resulted in significant inhibition of cellular proliferation on coculture with endothelial cells. This report represents the first use of lentivirus-based vectors to deliver the antiangiogenic factors, angiostatin and endostatin, and suggests the potential utility of antiangiogenic gene therapy with lentiviral vectors for the treatment of cancer.     

Simplified production of a recombinant human angiostatin derivative that suppresses intracerebral glial tumor growth.

Angiostatin is an endogenous inhibitor of tumor neovascularization that inhibits the proliferation of endothelial cells. Production of sufficient quantities of biologically active angiostatin by the enzymatic cleavage of plasminogen has proven difficult in that it has delayed clinical testing. We have cloned, expressed, and purified a recombinant human angiostatin derivative (K1-3) using a mammalian expression system. Through the addition of a secretory signal and polyhistidine sequence tag, K1-3 can be purified from post-culture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability to bind to lysine-Sepharose. In vitro, recombinant K1-3 significantly suppressed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-competent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011). Growth suppression was accompanied by a 32% decrease (P = 0.01) in tumor neovascularization. This study demonstrates a simple method to produce a biologically active recombinant angiostatin derivative. The ability to suppress intracerebral tumor growth after systemic administration suggests that K1-3 is likely to have therapeutic value in the treatment of malignant glial tumors.