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1.
病毒胸苷激酶基因治疗人胃癌的实验研究 总被引:4,自引:0,他引:4
为观察单纯疱疹病毒脱氧胸苷激酶(HSV-TK)/丙氧鸟苷(GCV)基因治疗系统对胃癌细胞的杀伤作用,将HSV-TKcDNA定向克隆入逆转录病毒载体pDOR-neo中,用LIPOFECTAMINE法将该HSV-TK基因转染胃癌细胞系SGC-7901,测定阳性转染细胞(SGC-7901/TK)在体内外对GCV的敏感性。结果示GCV在体外对SGC-7901/TK细胞有明显的杀伤作用,其作用呈现剂量和时间依赖性特点,且同时表现出对周围亲代SGC-7901细胞的杀伤效应(旁观者效应)。体内实验表明GCV可抑制HSV-TK阳性胃癌细胞的生长,使已形成的肿瘤逐渐消失。提示逆转录病毒载体介导的HSV-TK基因转移胃癌细胞,配合以GCV治疗,是胃癌基因治疗的又一途径。 相似文献
2.
携带单纯疱疹病毒tk基因的人肝癌特异性逆转录病毒载体的构建(英文) 总被引:3,自引:0,他引:3
目的构建携带单纯疱疹病毒tk基因(HSVtk)的人肝癌特异性逆转录病毒载体.方法用SalI酶切除逆转录病毒载体pMNSM内部的SV40启动子成为pMNM;从真核表达载体pBPGktk质粒中用BamHI酶游离带有磷酸甘油酸激酶基因(pgk)启动子调控的HSVtk片段,克隆到pMNM的PL位点上,构建成普通型pMNPtk逆转录病毒载体;从pGEM.7ZAFPe质粒中用EcoRI酶游离人甲胎蛋白基因增强子核心序列,克隆到pMNPtk的pgk启动子上游,构建成人肝癌特异型pMNAPtk逆转录病毒载体.结果酶切鉴定pMNPtk和pMNAPtk载体构建正确.结论该载体对肝癌特异性前药转换基因治疗有重要意义. 相似文献
3.
肺癌是目前最常见和最难治的肿瘤之一 ,发病率和病死率都很高 ,易发生转移。肺癌治疗目前主要依靠传统的手术、放射治疗和化学治疗 ,其疗效较差。随着分子生物学技术的发展 ,基因治疗肿瘤成为可能。“自杀基因”的出现因其能彻底杀伤肿瘤细胞 ,因而受到广泛重视 ,被认为是最有希望成为临床肿瘤治疗的有效方法。单纯疱疹病毒胸苷激酶(HSV tk)基因是肿瘤基因治疗中最具前景的“自杀基因”[1] ,实验研究显示 ,导入HSV tk基因的肿瘤细胞联合抗病毒药物更昔洛韦 (GCV)治疗 ,可有效地杀伤肿瘤细胞并可使小鼠体内实体瘤完全消退[2 ] … 相似文献
4.
目的:观察单纯疱疹病毒胸苷激酶(HSV-tk),丙氧鸟苷(GCv)自杀基因治疗系统在体内外对结肠癌的杀伤效应。方法:构建携带HSV-tk基因的逆转录病毒载体,将该重组逆转录病毒感染结肠癌细胞株SW480,筛选稳定表达tk的细胞克隆SW480/tk,测定SW480/tk细胞在体外对GCV的敏感性;SW480/tk细胞在裸鼠皮下成瘤,用GCV治疗并观察疗效。结果:HSV-tk基因整合入SW480细胞并在SW480/tk细胞中稳定表达;GCV在体外对SW480/tk细胞有明显的杀伤作用,其作用呈现剂量和时间依赖性特点和旁杀伤效应,对未转染细胞则无明显毒性。裸鼠ex vivo实验得到相应结果,治疗组肿瘤受到明显抑制。结论 在in vitra和ex vivo水平,表达HSV-tk基因的肿瘤细胞均可被GCV有效杀伤,逆转录病毒介导HSV-tk/GCV自杀基因治疗系统有可能成为结肠癌的有效治疗方法。 相似文献
5.
单纯疱疹病毒Ⅰ型胸苷激酶(HSV1-TK)基因是一个药敏基因,转导该基因的细胞对非毒性抗病毒药更昔洛韦(GCV)或阿昔洛韦(ACV)敏感,能被其杀灭。通过探讨该基因转染肺癌细胞后对GCV或ACV的敏感性,为其进入临床治疗肺癌积累实验资料。本文用电穿孔法将含胸苷激酶(TK)基因的逆转录病毒载体导入人肺腺癌细胞A549,在体内外观察转基因细胞对GCV的敏感性。材料与方法合TK基因的逆转录病毒表达载体PLXSN-TK参照文献[1]构建,A549细胞购自上海细胞所。新霉素(G418)、四甲基偶氮唑盐(M… 相似文献
6.
白细胞介素12对小鼠肝癌基因治疗的实验研究 总被引:4,自引:0,他引:4
目的:研究白细胞介素12对小鼠肝癌细胞基因治疗效果。方法:利用脑心肌炎病毒(EMCV)及脊髓灰质炎(Po-lio)病毒内核糖体进入位点(IRES),连接mIL-12 P40及p35 cDNAs和筛选基因新霉术磷酸转移酶(NeoR),克隆至逆转录病毒载体pGCEN中,使三个基因同时受逆转录病毒载体5’端LTR启动子控制,转录至同一mRNA转录本上,从而构建成多顺反子逆转录病毒载体,即pGCEN/mIL-12。在LipofectAMINE介导下将pGCEN/mIL-12转染包装细胞PA317,G418筛选,直至出现阳性克隆(命名为:M45/mIL-12),挑取抗性克隆,扩大培养,收集上清,用小鼠成纤维细胞NIH3T3测定病毒滴度。然后用重组逆转录病毒感染小鼠肝癌细胞MM45T.Li,G418筛选,直至出现阳性克隆,扩大培养,对阳性克隆进行鉴定。将60Co照射(60 Gy)M45/mIL-12细胞对荷瘤小鼠进行瘤内接种,每周一次,连续治疗三次,观察其治疗效果。结果:病毒上清中重组逆转录病毒滴度为5×10~5CFU/ml。 PCR及RT-PCR证明外源基因已整合至小鼠肝癌细胞基因组中,以及外源基因在mRNA水平上的表达。 相似文献
7.
乙型肝炎病毒-S基因重组逆转录病毒载体诱导小鼠体液和细胞免疫反应 总被引:1,自引:3,他引:1
探索逆转录病毒载体在基因治疗乙型肝炎中的应用。方法用DNA重组技术构建HBV-S基因重组逆转录病毒载体,电穿孔转染PA317后,用假病毒颗粒感染HepG2、P815和EL-4细胞,并进行基因免疫。结果HBV-S基因在上述细胞获得高效表达,在免疫动物体内产生高滴度抗一HBS及有效的CTL反应。结论该载体肌肉注射后有效激发对HBsAde异性的体液免疫及细胞免疫反应,在细胞内稳定表达,是一种高效的基因转移系统,适用于基因治疗试验。 相似文献
8.
丙型肝炎病毒核心基因免疫诱生细胞免疫应答研究 总被引:15,自引:0,他引:15
目的 研究丙型肝炎病毒(HCV)核心(C)基因免疫在诱生特异性细胞免疫应答中的作用。方法 将包含HCV C基因片段的重组真核表达质粒pcCNA HCV C,在主宰其可以在小鼠骨髓瘤SP2/0(H-2^d)中表达之后,注射BALB/c小鼠股四头肌。ELISA法检测血清中抗体水平;^3H-TdR掺入法测定免疫小鼠脾细胞特异性增殖能力;^51Cr释放法检测免疫小鼠细胞毒性T淋巴细胞(CTLs)体外杀伤功能。结 相似文献
9.
逆转录病毒介导单纯疱疹病毒-胸苷激酶基因治疗胰腺癌的实验研究 总被引:2,自引:0,他引:2
目的 探讨应用逆转录病毒载体介导单纯疮疹病毒-胸苷激酶(herpes simplex virus type Ⅰthymidine kinase gene,HSV-TK)基因治疗实验性人胰腺癌细胞系8988的价值。方法 HSV-TK被定向克隆入逆转录病毒载体pMNDM的DV40下游。重组逆转录病毒包装细胞PA317细胞,产生的重组病毒将HSV-TK转入人胰腺癌细胞系8988细胞内。结果 Sorthern blot试验及药敏试验均证实HSV-TK基因已整合至细 相似文献
10.
甘油三酯和极低密度脂蛋白对肝星状细胞增殖的影响 总被引:11,自引:3,他引:11
目的探讨甘油三酯(triglyceride,TG)和极低密度脂蛋白(verylow-densitylipoprotion,VLDL)对大鼠肝星状细胞(hepaticstelatecel,HSC)增殖的影响。方法用链霉蛋白酶和胶原酶原位灌流,Nycodenz密度梯度离心分离大鼠HSC,并以MTT比色法观察TG和VLDL对HSC增殖效应。结果TG和VLDL可影响HSC的增殖,TG和VLDL浓度分别在125mg/L和(25~100)mg/L时对HSC增殖有促进作用(P<005或P<001)。结论体外细胞培养表明,TG和VLDL可促进HSC增殖,其与脂肪肝肝纤维化的发生可能有关。 相似文献
11.
目的观察人端粒酶逆转录酶(hTERT)启动子调控的腺病毒介导单纯疱疹病毒胸苷激酶(HSV-tk)基因/丙氧鸟苷(GCV)系统在体外及荷瘤裸鼠体内对人膀胱癌的治疗作用。方法利用hTERT启动子及小鼠巨细胞病毒(CMV)启动子调控的携带HSV-tk基因的重组腺病毒(Ad-hTERT-HSV/tk与Ad-CMV-HSV/tk)分别感染人膀胱癌细胞253 J和人正常肺成纤维细胞MRC-5,加入GCV,MTT法观察受染细胞的存活率。建立人膀胱癌裸鼠移植瘤模型,随机分为4组,A组(Ad-hTERT-HSV/tk+GCV组)、B组(Ad-hTERT-HSV/tk+PBS组)、C组(PBS+GCV组)和D组(PBS对照组),观察各组肿瘤生长状况、重要脏器病理变化及裸鼠存活情况。结果体外实验表明,应用GCV处理后,Ad-CMV-HSV/tk对253 J和MRC-5细胞均有杀伤作用,而Ad-hTERT-HSV/tk只对膀胱癌细胞253 J具有杀伤作用。动物实验显示,A组移植瘤的体积和瘤重低于其他3组(P〈0.01)。A组裸鼠平均存活期长于其他3组(P〈0.01)。结论 hTERT启动子调控的重组腺病毒介导HSV-tk/GCV系统是一种安全、有效且靶向性高的基因疗法。 相似文献
12.
大鼠肝癌细胞HSV-tk/GCV自杀基因系统的构建及其旁观者效应 总被引:6,自引:3,他引:3
13.
Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro 总被引:16,自引:0,他引:16
Krohne TU Shankara S Geissler M Roberts BL Wands JR Blum HE Mohr L 《Hepatology (Baltimore, Md.)》2001,34(3):511-518
For gene therapy of hepatocellular carcinoma (HCC), the Escherichia coli purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may be more useful than the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system as a result of a stronger bystander effect. To analyze the molecular mechanisms involved in PNP/fludarabine-mediated cell death in human HCC cells in comparison with HSV-tk/GCV, we transduced human HCC cells of the cell lines, HepG2 and Hep3B, with PNP or HSV-tk using adenoviral vectors, followed by prodrug incubation. Both systems predominantly induced apoptosis in HepG2 and Hep3B cells. PNP/fludarabine induced strong p53 accumulation and a more rapid onset of apoptosis in p53-positive HepG2 cells as compared with p53-negative Hep3B cells, but efficiency of tumor cell killing was similar in both cell lines. In contrast, HSV-tk/GCV-induced apoptosis was reduced in p53-negative Hep3B cells as compared with p53-positive HepG2 cells. HSV-tk/GCV, but not PNP/fludarabine, caused up-regulation of Fas in p53-positive HepG2 cells and of Fas ligand (FasL) in both HCC cell lines. These results demonstrate cell line-specific differences in response to treatment with PNP/fludarabine and HSV-tk/GCV, respectively, and indicate that PNP/fludarabine may be superior to HSV-tk/GCV for the treatment of human HCC because of its independence from p53 and the Fas/FasL system. 相似文献
14.
The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro. 总被引:16,自引:0,他引:16
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J Fick F G Barker nd P Dazin E M Westphale E C Beyer M A Israel 《Proceedings of the National Academy of Sciences of the United States of America》1995,92(24):11071-11075
Herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) viral-directed enzyme prodrug gene therapy causes potent, tumor-selective cytotoxicity in animal models in which HSV-tk gene transduction is limited to a minority of tumor cells. The passage of toxic molecules from HSV-tk+ cells to neighboring HSV-tk- cells during GCV therapy is one mechanism that may account for this "bystander" cytotoxicity. To investigate whether gap junction-mediated intercellular coupling could mediate this bystander effect, we used a flow cytometry assay to quantitate the extent of heterocellular coupling between HSV-tk+ murine fibroblasts and both rodent and human tumor cell lines. Bystander tumor cytotoxicity during GCV treatment in a coculture assay was highly correlated (P < 0.001) with the extent of gap junction-mediated coupling. These findings show that gap junction-mediated intercellular coupling contributes to the in vitro bystander effect during HSV-tk/GCV therapy and that retroviral transduction of tumor cells is not required for bystander cytotoxicity. 相似文献
15.
Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors 总被引:9,自引:0,他引:9
Gerolami R Uch R Faivre J Garcia S Hardwigsen J Cardoso J Mathieu S Bagnis C Brechot C Mannoni P 《Journal of hepatology》2004,40(2):291-297
BACKGROUND/AIMS: Gene therapy is a promising approach for treatment of hepatocellular carcinoma (HCC). However, transduction of non-tumoral hepatocytes may lead to severe hepatitis when using suicide gene therapy approaches. The aim of our study was to evaluate the gene transfer efficiency into HCC cells and normal hepatocytes using human immunodeficiency virus (HIV)-derived lentiviral vectors in vitro and in vivo. METHODS: Lentiviral vectors encoding for the LacZ gene or the fusion gene HSV-Tk/GFP were tested in vitro in human HCC cells and human hepatocytes in primary culture and in vivo in a chemically induced rat model of HCC. RESULTS: We show that HIV-1-derived lentiviral vectors are efficient in transducing HCC cells in vitro and in vivo. No significant transduction of non-tumorous hepatocytes was observed in vivo whatever the route of administration used. Measurement of tumor growth following direct intratumoral injection of a lentiviral vector containing the HSV-Tk gene and GCV treatment showed a strong antitumoral efficacy in the absence of normal liver toxicity. CONCLUSIONS: These observations suggest that lentiviral vectors allow an antitumoral effect with low liver toxicity when using suicide gene therapy approach and could be efficient tools for HCC gene therapy. 相似文献
16.
Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. 总被引:12,自引:7,他引:12
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S H Chen H D Shine J C Goodman R G Grossman S L Woo 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(8):3054-3057
The therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transduction of rat C6 glioma cells followed by ganciclovir (GCV) administration was studied in tumors generated in the brains of nude mice. C6 glioma cells were efficiently transduced in vitro by a replicative-defective recombinant adenovirus carrying the HSV-tk gene (ADV/RSV-tk) that rendered them sensitive to GCV in a dose-dependent manner. Tumors were generated by stereotaxic intracerebral injection of 1 x 10(4) C6 cells in nude mice. After 8 days of tumor growth, 3 x 10(8) ADV/RSV-tk viral particles were injected into the tumors and the mice subsequently were treated with GCV for 6 days. Tumor size in untreated and treated animals was compared 20 days after tumor implantation. The mean cross-sectional area of the tumors in the treated animals was 23-fold smaller than in control animals and the tumor volume was reduced by > 500-fold. These results demonstrate that the recombinant adenoviral vector can function as an efficient gene delivery vehicle for the treatment of gliomas by in vivo gene therapy. 相似文献
17.
Systemic efficacy of combined suicide/cytokine gene therapy in a murine model of hepatocellular carcinoma 总被引:11,自引:0,他引:11
Stefani AL Barzon L Castagliuolo I Guido M Pacenti M Parolin C Farinati F Palù G 《Journal of hepatology》2005,42(5):728-735
BACKGROUND/AIMS: Gene therapy might be a promising therapeutic approach for advanced hepatocellular carcinoma (HCC) not amenable to any effective traditional treatment. The aim of the study was to evaluate the in vitro and in vivo efficacy of combined gene therapy of HCC with two different MoMLV-derived retroviral vectors, an MFG- and a LXSN-derived vector, both containing HSV-TK and hIL-2. RESULTS: In vitro experiments on HCC cells showed efficient killing of transduced cells and efficient bystander effect after ganciclovir (GCV) treatment, with higher antitumor activity when the MFG-based vector was used. In vivo studies in a murine syngenic model of HCC demonstrated that treatment with GCV led to complete regression of tumors composed of transduced cells and regression of distant non-transduced tumors. Tumor transduction and efficacy of treatment was also demonstrated after in vivo delivery of vectors. Microarray analysis of tumor samples in mice receiving gene therapy showed up-regulation of genes involved in immune response and signal transduction. CONCLUSIONS: We demonstrated the in vitro and in vivo efficacy of combined retroviral-mediated gene therapy for HCC, with significant systemic therapeutic efficacy in vivo. 相似文献
18.
构建携带胸苷激酶(tk)与增强型绿色荧光蛋白(EGFP)融合基因穿梭质粒,并观察其在肝癌细胞系HepG2细胞中的表达。应用基因重组技术,构建EGFP与tk的融合表达穿梭质粒载体,经限制酶切鉴定和测序分析,脂质体转染将其导入HepG2中,48h后用荧光显微镜观察荧光的表达,RT-PCR法检测融合蛋白tk和EGFP的mRNA表达,四甲基偶氮唑蓝(MTT)实验检测转染融合蛋白载体后不同浓度的更昔洛韦(GCV)对HepG2细胞的细胞毒作用。酶切鉴定和测序分析证实重组质粒中插入融合目的基因片断及载体DNA大小、方向和插入位点正确,在转染此穿梭质粒的HepG2细胞中检测到绿色荧光蛋白的表达,RT-PCR检测到融合蛋白tk和EGFP的mRNA表达;GCV对转染了携带tk与EGFP融合基因穿梭质粒载体有明显的细胞毒作用。成功构建携带tk与EGFP融合基因穿梭质粒载体,转染人肝癌细胞株HepG2中tk和EGFP的独立表达没有受到影响,该载体可利用绿色荧光蛋白作为报告基因监测tk的表达并可用于肝癌的自杀基因治疗。 相似文献
19.
转铁蛋白受体介导的自杀基因转移系统的建立及其体外抗瘤效应 总被引:1,自引:0,他引:1
目的 构建转铁蛋白受体介导的自杀基因系统并观察其对肝癌细胞的体外杀伤效应。方法 SPDP法制备抗转铁蛋白受体与多聚赖氯酸(PLL)的复合物并用分子筛层析纯化。根据DNA阻断试验结果,pEBAF/tk重组质粒与Ab-PLL按1:6混合使二者结合形成PEBAF/tk—Ab PLL复合物。将此复合物转入人肝癌细胞株SMMC7721、HepG2和肺癌细胞株A549,并以脂质体转移为对照。加入不同浓度的更昔洛韦(GCV)以观察细胞的自杀效应。结果 加入GCV后HepG2/tk的增殖受抑制最明显。100mg/L和1mg/L时抑制率分别为60.5%和24.3%。SMMC7721/tk受抑制较低,而A549的增殖不受抑制。结论 本基因治疗体系具仃很好的靶向性和杀肿瘤效果。 相似文献
20.
Retrovirus-mediated in vivo gene therapy using the herpes simplex virus thymidine kinase gene against carcinomatous peritonitis 总被引:2,自引:0,他引:2
Toyokawa Y Kuriyama S Tsujinoue H Nakatani T Mitoro A Yoshiji H Fukui H 《Scandinavian journal of gastroenterology》2000,35(8):852-860
BACKGROUND: Carcinomatous peritonitis is characterized by massive malignant ascites, while peritoneally disseminated carcinomatosis is characterized by a large number of metastatic solid tumors in the peritoneal cavity. Although both are fatal end-stage manifestations of malignancies derived from the digestive system, the former is usually more serious than the latter due to massive malignant ascites. Although the effectiveness of gene therapy against peritoneally disseminated carcinomatosis has been shown in animal experiments, its effectiveness against carcinomatous peritonitis remains to be examined. METHODS: A carcinomatous peritonitis model was made by inoculating murine hepatocellular carcinoma cells, MH134, into the peritoneal cavity of syngeneic C3H/He mice, resulting in production of massive malignant ascites without development of intraperitoneal solid tumors. Model animals were injected intraperitoneally with retroviruses carrying the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) treatment. RESULTS: Retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system was shown to have a significant impact on survival of animals with carcinomatous peritonitis not only at an early stage, but also at an advanced stage. Furthermore, repeated injections of HSV-tk-carrying retroviruses significantly prolonged the survival of animals with carcinomatous peritonitis compared with a single injection protocol. When intraperitoneal administration of recombinant interleukin-2 (IL-2) was added to the HSV-tk/GCV system, levels of IL-1beta and IL-2 in malignant ascites were significantly increased, resulting in significantly reduced ascite volume and prolonged survival. CONCLUSIONS: Our results indicate the feasibility of retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system plus IL-2 treatment against carcinomatous peritonitis. 相似文献