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51.
52.
目的 建立一个在大鼠关节滑膜细胞表达人白细胞介素10(human interleukin10,hIL-10)的重组逆转录病毒载体基因转移系统,为下一步的研究工作做准备。方法 构建表达人白细胞介素10的 转灵病毒重组体的pLX(hIL-10)SN,经PA317细胞包装,G418筛选,NIH3T3细胞进行病毒滴度测定,选滴度最高的克隆(6×10^8集落形成单位/L)作为感染大鼠关节滑膜细胞的感染细胞; 相似文献
53.
构建含小鼠白细胞介素12双亚基及新霉素磷酸转移酶基因多顺反子逆转录病毒载体,并观察其在小鼠肝癌细胞中的表达。方法利用脑心肌炎病毒(EMCV)及脊髓灰质炎(Polio)病毒内核糖体进入位点(IRES),连接mIL-12p40及p35cDNAs和筛选基因新霉素磷酸转移酶(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,通过 相似文献
54.
膀胱灌注逆转录病毒载体HSV-TK基因治疗大鼠膀胱癌 总被引:1,自引:0,他引:1
目的:研究膀胱内灌注逆转录病毒载体单纯疱疹病毒胸腺嘧啶核苷激酶(HSVtk)基因治疗膀胱癌的作用。方法:用N-甲基-亚硝基脲(MNU)膀胱灌注诱导建立大鼠原位膀胱肿瘤模型,该肿瘤与人类膀胱肿瘤病理特点非常相似。随后经尿道膀胱灌注逆转录病毒载体HSV-TK(基因,腹腔内注射羟基无环鸟苷(ganciclovir,GCV),连续6天。结果:经尿道膀胱灌注逆转录病毒HSV-TK基因后48小时RTPCR检测出膀胱肿瘤中HSV-TK基因表达。GCV处理后.肿瘤体积以及膀胱总重量检测表明膀胱肿瘤的生长被显著抑制。肿瘤细胞DNA电泳显示诱导凋亡可能是HSV-TK/GCV系统治疗肿瘤的重要机制。结论:经尿道膀胱灌注重组逆转录病毒载体可以向肿瘤转导HSV—TK基因。HSV-TK/GCV系统能抑制鼠原位膀胱肿瘤的生长。 相似文献
55.
双靶区反义RNA抑制乙型肝炎病毒 总被引:1,自引:0,他引:1
目的:研究双靶区反义RNA的逆转录病毒载体的转染、表达及其抗乙肝病毒(HBV) 的作用.方法:合成互补于HBV X区(1400-1430)的X片段、互补于HBV P区(2375-2405) 的P片段,分别构建表达单靶区反义X或P的重组逆转录病毒载体质粒和表达双靶区正义、反义 X 和 P 重组逆转录病毒载体质粒 ,转染PA317细胞,NIH3T3 细胞扩增法测假病毒颗粒的滴度.用假病毒颗粒感染2.2.15细胞,用酶标法检测2.2.15细胞上清 HBsAg和HBeAg含量.结果:对HBsAg的抑制率在感染后第3、5、7、9天时分别为 pLXSN:5%, 4%, 6%, 4%; pLXSN+Xpos/Ppos:1%, 5%, 5%, 11%; pLXSN+X:16%, 45%, 44%, 38%; pLXSN+P :34%, 59%, 55%,51%; pLXSN+X/P:73%, 83%, 81%, 79%.对HBeAg的抑制率在感染后第3 、5、7、9天时分别为pLXSN:2%, 6%, 4%, 3%; pLXSN+Xpos/Ppos:6%, 0, 0, 0; pLXSN+ X: 13%, 53%, 52%, 45%; pLXSN+P: 21%, 57%, 56%, 49%; pLXSN+X/P :62%, 87%, 84%,79%.结论:细胞内表达HBV反义RNA有明显抗乙肝病毒复制和表达的作用 ,而且双靶区反义RNA抗乙肝病毒的作用较单靶区反义RNA更明显. 相似文献
56.
A. L. David M. Themis T. Cook C. Coutelle C. H. Rodeck 《The Ultrasound Review of Obstetrics & Gynecology》2001,1(1):14-27
Fetal gene therapy has the potential to treat inherited genetic diseases in utero before significant organ damage has occurred. Rapidly expanding stem cell populations may be targeted and the introduction of transgenes to the fetus during development of the immune system could result in immune tolerance and facilitate repeat treatment postnatally. Genetic diseases such as cystic fibrosis, which are life-threatening and for which there are no currently acceptable treatments available, are suggested targets for this therapy.
Ultrasound may be a safe method of delivering a therapeutic gene into the fetus, although most studies have used more invasive techniques, even in large animal models. Viral vectors currently offer the most potential. Adenovirus-based vectors are stable, independent of host cell replication, efficient at tissue infection and have been used as a 'pathfinder' to test routes of administration. Unfortunately, they are also highly immunogenic and other systems based on retrovirus or adeno-associated virus may offer advantages because of their lower immunogenicity and potential for permanent transgene expression.
Our group is developing the fetal sheep model for the investigation of ultrasound-guided gene therapy in utero. This model is suitable since the sheep fetus is tolerant to manipulations, has a consistent gestation period and shows many similarities with human pregnancy. We have demonstrated significant transfection of the fetal liver and adrenal cortex after ultrasound-guided percutaneous injection of the umbilical vein with adenoviral vectors in the late gestation sheep. We are investigating and here discuss alternative routes of administration to target the fetus in early gestation via ultrasound-guided minimally invasive techniques. 相似文献
Ultrasound may be a safe method of delivering a therapeutic gene into the fetus, although most studies have used more invasive techniques, even in large animal models. Viral vectors currently offer the most potential. Adenovirus-based vectors are stable, independent of host cell replication, efficient at tissue infection and have been used as a 'pathfinder' to test routes of administration. Unfortunately, they are also highly immunogenic and other systems based on retrovirus or adeno-associated virus may offer advantages because of their lower immunogenicity and potential for permanent transgene expression.
Our group is developing the fetal sheep model for the investigation of ultrasound-guided gene therapy in utero. This model is suitable since the sheep fetus is tolerant to manipulations, has a consistent gestation period and shows many similarities with human pregnancy. We have demonstrated significant transfection of the fetal liver and adrenal cortex after ultrasound-guided percutaneous injection of the umbilical vein with adenoviral vectors in the late gestation sheep. We are investigating and here discuss alternative routes of administration to target the fetus in early gestation via ultrasound-guided minimally invasive techniques. 相似文献
57.
目的:探讨内皮抑素(ES)基因转移在乳腺癌抗血管新生中的作用。 方法: 通过建立逆转录病毒介导的ES基因转移系统,用ES病毒转染人乳腺癌细胞系MDA-MB-231。以聚合酶链反应(PCR)、MTT法和裸鼠成瘤实验分析ES的生物学特性及其功能。 结果: 脂质体转染与交互感染策略获得ES病毒生产细胞;以ES病毒转染MDA-MB-231细胞后,经PCR分析显示其内有ES基因整合并持续表达,其分泌的ES能明显抑制内皮细胞EA.hy926的增殖(P<0.05),但对肿瘤细胞的离体生长无明显影响(P>0.05);裸鼠皮下移植瘤模型表明,ES基因表达可明显抑制MDA-MB-231细胞的生长(P<0.01);实验组的肿瘤微血管密度(MVD)和血管内皮生长因子(VEGF)表达低于对照组(P<0.05)。 结论: 逆转录病毒载体介导的ES基因在乳腺癌细胞中可有效表达,能明显抑制血管内皮细胞生长,并通过旁分泌方式抑制血管新生,具有显著的抗肿瘤生长的作用。 相似文献
58.
丙型肝炎病毒与荧光素酶融合基因细胞模型的初步建立 总被引:2,自引:0,他引:2
Objective To establish HCV cell culture model which is easy to
measure. Methods Controllable retroviral vector with fusion gene of
hepatitis C virus (HCV) cDNA and luciferase (luc) reporter gene was constructed by
molecular cloning technique, the transfection of this retroviral vector in a human hepatic
carcinoma cell (HHCC) line was performed by lipofectAMINE and then luciferase activity in
the cellular lysate was measured by scintillation counter. Results ① Fusion gene of the
HCV 5′ NCR-C region
and luciferase reporter gene identified by restriction endonuclease cleavage have been
cloned into pBPSTR1 vector. ② The luciferase activity could maintain up to 20 days at least, and could be
increased by puromycin treatment and regulated by tetracycline. Conclusion A cell model for
expression of HCV C-E1 and luciferase genes was established for gene therapy studies
against HCV C-E1 sequences. 相似文献
59.
反转录病毒介导的HSV-tk基因治疗大鼠脑胶质瘤的实验研究 总被引:5,自引:0,他引:5
目的:进行C6大鼠脑胶质瘤的基因治疗实验研究。方法:采用反转录病毒介导的基因转移和ACV体内治疗方法进行研究。结果:构建了带有HSV-tk基因的反转录病毒载体GINaTK,应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317,成为产病毒细胞PA317TK,用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞,命名为C6TK细胞,对GCV和ACV的敏感性分别高于亲本450倍和10倍;成功地建立了大鼠脑胶质瘤模型,并证实转染细胞C6TK的成瘤效应未改变,存活期约为15天;而经ACV治疗后,含有C6TK细胞的肿瘤生长明显被抑制,大鼠生存期延长为57.8±8.07天,尤其是采用PA317TK细胞混和治疗组和原位治疗组,肿瘤基本消失,大鼠生存期显著延长,混和治疗组存活120天以上,原位治疗组存活至71.4±36.1天。t检验,P值均小于0.001。结论:HSV-tk/ACV系统基因治疗大鼠脑胶质瘤疗效显著。 相似文献
60.
Summary A new animal model for human rheumatoid arthritis is described, and the unsolved questions regarding the mechanism of primary joint destruction are discussed. Following an analysis of the types of cells and antibodies found in joints affected by rheumatoid arthritis, it is concluded that both expression of oncogenes and the presence of retroviral sequences detectable by monoclonal antibodies to HTLV I p19 and p24 sequences are associated with early abnormal proliferation of apparently transformed cells at the site of initial cartilage and/or bone destruction. 相似文献