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Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro 被引量:5
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作者 Zhao-Shen Li Xue Pan +4 位作者 Guo-Ming Xu Long Cui Guan-Rong Dai Yan-Fang Gong Zhen-Xing Tu the Department of Gastroenterology Department of General Surgery Changhai Hospital, Second Military Medical University, Shanghai 200433, China 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2003年第1期147-151,共5页
OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pA... OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-l were recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added. XTT assay was used to estimate relative numbers of viable cells. RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2×1O^(11) pfu/ml. It was found that significant cytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer. 展开更多
关键词 pancreatic cancer adenovirus verctor cytosine deaminase gene therapy
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Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene 被引量:29
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作者 Li-Zong Shen Wen-Xi Wu Qiang Ding Yi-Bing Hua,Department of General Surgery,The First Affiliated Hospital of Nanjing Medical University,Nanjing,210029,Jiangsu Province,China De-Hua Xu Zhong-Cheng Zheng Xin-Yuan Liu,Shanghai Institute of Biochemistry and Cell Biology,The Chinese Academy of Sciences,Shanghai,200031,China Kun Yao,Department of Microbiology and Immunology,Nanjing Medical University,Nanjing,210029,Jiangsu Province,China 《World Journal of Gastroenterology》 SCIE CAS CSCD 2002年第2期270-275,共6页
AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was c... AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 】15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P【0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P【0.05, P【0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was 】15000 and 214.5+/-31.3 micromol.L(-1), P【0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma. 展开更多
关键词 Gene Therapy Genetic Vectors ADENOVIRIDAE Animals ANTIMETABOLITES Bystander Effect Carcinoembryonic Antigen Cell Line Colorectal Neoplasms cytosine deaminase FLUcytosine Hela Cells Humans Nucleoside deaminases Promoter Regions (Genetics) Research Support Non-U.S. Gov't Tumor Cells Cultured
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N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication 被引量:2
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作者 Yan-Chang Lei Yong-Jun Tian +7 位作者 Hong-Hui Ding Bao-Ju Wang Yan Yang You-Hua Hao Xi-Ping Zhao Meng-Ji Lu Fei-Li Gong Dong-Liang Yang 《World Journal of Gastroenterology》 SCIE CAS CSCD 2006年第46期7488-7496,共9页
AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis... AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo. 展开更多
关键词 cytosine deaminase domain Apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G Hepatitis B virus Antiviral therapy
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Migration to gliomas of bone mesenchymal stem cells transfected with cytosine deaminase gene following transplantation in vivo
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作者 Guangchun Ji Fei Song +4 位作者 Qi Xing Jian Liu Kedong Song Daqing Zhang Zihan Wang 《Neural Regeneration Research》 SCIE CAS CSCD 2011年第19期1462-1466,共5页
This experiment sought to observe the migration and distribution of bone mesenchymal stem cells transfected with the cytosine deaminase gone (BMSCs-CD/eGFP) after transplantation in vivo through three pathways. In a... This experiment sought to observe the migration and distribution of bone mesenchymal stem cells transfected with the cytosine deaminase gone (BMSCs-CD/eGFP) after transplantation in vivo through three pathways. In addition, we examined the tropism of these cells to glioma. Intracranial C6 glioma models were established in Sprague-Dawley rats using an intracranial stereotactic inoculation method. When tumors were 7 days old, rats were inoculated with lx106 BMSCs-CD/eGFP cells via the tumor-bearing internal carotid artery, the contralateral hemisphere and the tumor-bearing glioma. Fluorescence microscopy revealed that BMSCs-CD/eGFP exhibited a strong capacity for migration to tumors. BMSCs-CD/eGFP transplanted via the tumor-bearing intemal carotid artery were observed to distribute in glioma tissues. BMSCs-CD/eGFP inoculated via the ipsilateral glioma mainly located within and at the edge of glioma tissues. BMSCs-CD/eGFP inoculated via the contralateral hemisphere mainly distributed at the proximal end of the tumor at the incubation site. 展开更多
关键词 cytosine deaminase gone bone mesenchymal stem cells GLIOMA TRANSDUCTION MIGRATION
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Inhibitory effects of cytosine deaminase gene-transfected bone marrow mesenchymal stem cells on glioma cell proliferation
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作者 Fei Song Qi Xing +6 位作者 Kedong Song Jian Liu Guangchun Ji Yufang Ma Tianqing Liu Minghai Wei Xuehu Ma 《Neural Regeneration Research》 SCIE CAS CSCD 2011年第16期1238-1242,共5页
Bone marrow mesenchymal stem cells were isolated from C57BL mice, transfected with the cytosine deaminase (CD) gene using a lentivirus vector and co-cultured with C6 glioma cells to verify anti-tumor effects of bone... Bone marrow mesenchymal stem cells were isolated from C57BL mice, transfected with the cytosine deaminase (CD) gene using a lentivirus vector and co-cultured with C6 glioma cells to verify anti-tumor effects of bone marrow mesenchymal stem cells carrying CD genes. C57MSC-CD/eGFP cells converted 5-fluorocytosine to 5-fluorouracil and exhibited significant inhibition of proliferation and apoptosis in C6 glioma cells. C57MSC-CD/eGFP cells were then implanted into rat models of brain C6 glioma. Rats were also intraperitoneally injected with 5-fluorocytosine after 7 days. MSC-CD/eGFP cells were irregularly distributed at the margin of the glioma, as well as encased and reduced the volume of the glioma. CD-transfected bone marrow mesenchymal stem cells inhibit the in vivo growth and in vitro proliferation of glioma. 展开更多
关键词 magnetic resonance imaging GLIOMA gene therapy cytosine deaminase gene bone marrow mesenchymal stem cells LENTIVIRUS
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Adenovirus-mediated tissue specific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression level
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作者 Hiroaki Wakimoto Hirofumi Hamada 《中国实验血液学杂志》 CAS CSCD 1997年第3期298-299,共2页
Hepatocellular carcinoma (HCC) is one of the mostcommon cancers in the world, especially in East Asia.There is no standardized or effective strategy could beadapted routinely except of some early diagnosedpatients, an... Hepatocellular carcinoma (HCC) is one of the mostcommon cancers in the world, especially in East Asia.There is no standardized or effective strategy could beadapted routinely except of some early diagnosedpatients, and the prognosis is poor. In recent years, genetherapy has become a standard experimental approach fortreating cancers that have escaped conventionaltherapies. One such an approach is to confer the tumorcells with sensitivity to chemical reagents through 展开更多
关键词 deaminase ADENOVIRUS cytosine standardized prognosis confer NUDE suppressed promoter THYMIDINE
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Purification and characterization of <i>Aspergillus parasiticus</i>cytosine deaminase for possible deployment in suicide gene therapy
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作者 Hassan Zanna A. J. Nok +1 位作者 S. Ibrahim H. M. Inuwa 《Advances in Biological Chemistry》 2012年第2期152-159,共8页
Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of... Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of 13%. The CD had pH optimum of 7.2, a temperature optimum of 40℃ - 45℃, activation of energy (Ea) of 8.4 KJ/mol and a t1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cyto-sine and 5-fluorocytosine (5-FC) with the KM values of 0.19mM and 0.30 mM respectively. Studies on the effect of pH on KM and Vmax gave pKa1 of 5.8 and pKa2 of 6.3 with enthalpy of ionization of 43.01 KJ/mole suggesting histidine in the active site. The enzyme was strongly inhibited by Ca2+, Co2+, Zn2+ and Hg2+ and completely inhibited by Cu2+ and Fe2+ at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy. 展开更多
关键词 cytosine deaminase cytosine 5-FLUOROcytosine Aspergillus PARASITICUS Activation Energy
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Killing effect of adenoviral mediated cytosine deaminase gene on human pancreatic cancer cell line PaTu8988
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作者 潘雪 李兆申 +4 位作者 许国铭 崔龙 张素贞 龚燕芳 屠振兴 《Journal of Medical Colleges of PLA(China)》 CAS 2001年第4期281-284,共4页
Objective:To evaluatethein vitro killingeffectsof cytosinedeaminasegene mediatedby adenovirusvector on humanpancreaticcarcinoma.Methods:CytosineDeaminase(CD)genewas clonedintopAdTrack-CMV-CD,pAd-Track-CMV-CDandpAdEasy... Objective:To evaluatethein vitro killingeffectsof cytosinedeaminasegene mediatedby adenovirusvector on humanpancreaticcarcinoma.Methods:CytosineDeaminase(CD)genewas clonedintopAdTrack-CMV-CD,pAd-Track-CMV-CDandpAdEasy-1wererecombinedinbacteria,andtheproductscontaininggreenfluorescentprotein(GFP)werepropagatedin293cellsandpurifiedby cesiumchloridegradientcentrifugation.Humanpancreaticcarcinomacellline8988wereinfectedwiththisvirus,then5-FCwasadded;XTTassaywasusedto estimatetherelativenumbersof viable cells.Results:Thepositivecloneswereconfirmedby usingendonucleasedigestion,andthetiterof theviruscontaining CD genewas2×10 11 pfu/ml.Itwasfoundthat5-FCpossessedsignificantcytotoxicactivitiesforCD genetransfected8988cellline,buthadlittleeffectson non-transfectedpancreaticcarcinomacells.Conclusion:CD genemediatedby adenovirus hasa highinfectivityandis efficientforkillingculturedpancreaticcarcinomacells,indicatingsuicidegenemaybe effec-tiveforpancreaticcancerinfuture. 展开更多
关键词 PANCREATIC cancer ADENOVIRUS vector cytosine deaminase GENE therapy
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婴儿双歧杆菌介导的CD和UPRT联合5-FC基因疗法对黑色素瘤的体外治疗实验研究 被引量:12
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作者 安丽娜 李著华 +3 位作者 岳扬 王树人 郭志英 彭克军 《四川大学学报(医学版)》 CAS CSCD 北大核心 2007年第1期27-30,63,共5页
目的采用婴儿双歧杆菌为载体,探讨尿嘧啶磷酸核糖转移酶基因(UPRT)对胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因系统抗瘤作用的增强效应。方法构建原核表达质粒pGEX-UPRT,以电穿孔法将该质粒转化入婴儿双歧杆菌,筛选阳性重组菌并鉴定。... 目的采用婴儿双歧杆菌为载体,探讨尿嘧啶磷酸核糖转移酶基因(UPRT)对胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因系统抗瘤作用的增强效应。方法构建原核表达质粒pGEX-UPRT,以电穿孔法将该质粒转化入婴儿双歧杆菌,筛选阳性重组菌并鉴定。采用M TT法检测表达的U PRT是否可以与CD产生抗瘤协同作用,并观察细胞形态学改变。结果重组婴儿双歧杆菌可以正确表达UPRT。体外M TT检测显示CD+UPRT组细胞存活率低于对照组(P<0.01),且可使B 16-F 10鼠黑色素瘤细胞对5-FC的杀伤敏感性(IC50=0.015μm o l/mL)提高,是CD组(IC50=0.127μm o l/mL)的8.5倍。形态学观察CD+UPRT组肿瘤细胞显示出明显的损伤性改变,细胞生长受到明显抑制,而UPRT组和CD组变化明显不如前者。结论婴儿双歧杆菌联合转导UPRT基因可明显增强CD/5-FC自杀基因系统对鼠黑色素瘤细胞B 16-F 10的杀伤作用。 展开更多
关键词 婴儿双歧杆菌 尿嘧啶磷酸核糖转移酶 胞嘧啶脱氨酶 基因治疗
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组织特异性CD/5-FC系统对大肠癌的原位基因治疗 被引量:5
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作者 黎成金 马庆久 +2 位作者 涂小煌 王烈 李金茂 《肿瘤防治研究》 CAS CSCD 北大核心 2005年第4期206-208,共3页
目的 探讨癌胚抗原(carcinoembryonic antigen,CEA)组织特异性胞嘧啶脱氨基酶基因对不同分泌CEA大肠癌组织的靶向杀伤作用。方法 脂质体法将CEA 组织特异性逆转录病毒载体G1CEACDNa在PA317细胞中进行包装,大肠癌细胞LoVo和SW480分别... 目的 探讨癌胚抗原(carcinoembryonic antigen,CEA)组织特异性胞嘧啶脱氨基酶基因对不同分泌CEA大肠癌组织的靶向杀伤作用。方法 脂质体法将CEA 组织特异性逆转录病毒载体G1CEACDNa在PA317细胞中进行包装,大肠癌细胞LoVo和SW480分别接种到BALB/c裸鼠大腿皮下,成瘤2周后,瘤内多点注射法行原位基因转染,每天腹腔注射500mg/kg的5 FC(5 fluorocytosine),观察治疗效果。结果 病毒滴度为5.6×106CFU/L。经多次注射法转染,目的基因在肿瘤组织中能有效表达,治疗21天后,基因治疗组有明显的抑瘤作用,但对LoVo 细胞肿瘤的抑瘤作用明显大于对SW480细胞肿瘤。结论 CEA组织特异性CD/5 FC系统对LoVo细胞肿瘤的抑瘤作用更明显。 展开更多
关键词 癌胚抗原 胞嘧啶脱氨基酶 5-氟胞嘧啶 大肠癌 基因治疗
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婴儿双歧杆菌介导的CD/5-FC自杀基因系统对黑色素瘤的抑瘤实验 被引量:6
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作者 易成 郭志英 +2 位作者 黄英 王浩毅 王树人 《四川大学学报(医学版)》 CAS CSCD 北大核心 2005年第2期165-168,共4页
目的 观察婴儿双歧杆菌介导的胞嘧啶脱氨酶 / 5 -氟胞嘧啶 (CD/ 5 - FC)自杀基因系统对黑色素瘤的体内、外抑瘤效果。方法 含重组 CD基因的婴儿双歧杆菌中加入 5 - FC,厌氧条件下培养 2 4 h后取其上清液 ,加到黑色素瘤 B16 - F10细胞... 目的 观察婴儿双歧杆菌介导的胞嘧啶脱氨酶 / 5 -氟胞嘧啶 (CD/ 5 - FC)自杀基因系统对黑色素瘤的体内、外抑瘤效果。方法 含重组 CD基因的婴儿双歧杆菌中加入 5 - FC,厌氧条件下培养 2 4 h后取其上清液 ,加到黑色素瘤 B16 - F10细胞上 ,观察其对细胞形态及细胞生长抑制率的影响 ;建立小鼠黑色素瘤动物模型 ,尾静脉注入含重组 CD基因的婴儿双歧杆菌 ,腹腔注入 5 - FC,观察携带重组 CD基因的婴儿双歧杆菌联合 5 - FC对黑色素瘤肿瘤体积及肿瘤体积倍增时间的影响。结果  1体外实验 :实验组肿瘤细胞形态显示出显著的损伤性改变 ,细胞生长受到明显抑制 ,而对照组肿瘤细胞影响不大。 2小鼠黑色素瘤动物模型实验结果显示 ,经重组婴儿双歧杆菌联合 5 -FC治疗 2 1d,实验组肿瘤倍增时间明显长于对照组 ,其肿瘤体积明显小于对照组 ,且随着观察时间的延长 ,这种差异越显著。结论 婴儿双歧杆菌介导的 CD/ 5 - FC自杀基因系统对黑色素瘤具有明显的抑瘤效果。 展开更多
关键词 婴儿双歧杆菌 胞嘧啶脱氨酶 5-氟胞嘧啶 基因治疗
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脂质体介导的CD/5-FC自杀基因体系协同干扰素体内抑瘤及旁观者效应的观察 被引量:4
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作者 罗琪 卢毅卓 +1 位作者 刘国彦 张颂恩 《南方医科大学学报》 CAS CSCD 北大核心 2008年第9期1621-1625,共5页
目的观察阳离子脂质体介导的CD/5-FC自杀基因体系协同γ-干扰素活体内对肿瘤的抑制效应及远端旁观者效应。方法采用阳离子脂质体介导CD基因瞬时转染小鼠肝癌H22及未转染的H22细胞,分别接种于昆明小鼠双侧前腋下皮下;向小鼠体内注射5-FC... 目的观察阳离子脂质体介导的CD/5-FC自杀基因体系协同γ-干扰素活体内对肿瘤的抑制效应及远端旁观者效应。方法采用阳离子脂质体介导CD基因瞬时转染小鼠肝癌H22及未转染的H22细胞,分别接种于昆明小鼠双侧前腋下皮下;向小鼠体内注射5-FC及γ-干扰素,观察在γ-干扰素协同作用下5-FC对转染瘤体的抑制作用和远处无转染瘤体的抑制即远端旁观者效应。结果5-FC对转染CD基因侧瘤体抑制明显,抑制率为79.39%(P<0.05);在γ-干扰素的协同下CD/5-FC的抑瘤作用得到了增强,抑制率为93.47%,与无γ-干扰素协同相比,对癌细胞抑制作用明显增强(P<0.05);存在远端旁观者效应,远端未转染瘤体的抑制率为54.42%(P<0.05);在γ-干扰素的协同下远端旁观者效应得到显著增强,抑制率达到88.43%。同无γ-干扰素相比有差异有显著性(P<0.05)。结论体内CD/5-FC自杀基因体系联合γ-干扰素对肝癌细胞有更好的抑制效应,对肝癌的治疗有很好的应用前景。 展开更多
关键词 基因疗法 自杀基因 旁观者效应 胞嘧啶脱氨酶基因 干扰素
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YCD基因修饰对小鼠P388白血病的体内治疗作用研究 被引量:3
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作者 江千里 王健民 +4 位作者 温丽敏 张雨生 江汕 胡晓霞 周虹 《中国肿瘤生物治疗杂志》 CAS CSCD 2003年第2期84-87,共4页
目的 :以P388/DBA小鼠白血病模型 ,探讨新型自杀基因YCD的体内治疗作用。方法 :用逆转录病毒载体将YCD基因导入 ,筛选P388 YCD eGFP细胞克隆 ,以P388 eGFP和野生性 (wt)P388细胞为对照。 (1) 3组细胞腹腔接种给DBA小鼠 (n =5 ) ,5×... 目的 :以P388/DBA小鼠白血病模型 ,探讨新型自杀基因YCD的体内治疗作用。方法 :用逆转录病毒载体将YCD基因导入 ,筛选P388 YCD eGFP细胞克隆 ,以P388 eGFP和野生性 (wt)P388细胞为对照。 (1) 3组细胞腹腔接种给DBA小鼠 (n =5 ) ,5× 10 6/只 (下同 ) ,观察致病性 ;(2 ) 3组细胞腹腔接种给DBA小鼠 (n =5 ) ,分别以 5 FC治疗 2周 ,观察疗效 ;(3) 2组× 5只小鼠腹腔接种P388 YCD eGFP ,5 FC 5 μmol/L·d-1× 2d或PBS治疗 ,根据小鼠存活时间推算杀伤效率。以流式细胞仪、冰冻切片和HE切片检测各脏器中白血病细胞的分布和变化。结果 :基因导入对细胞的生物学特性影响不显著 ;5 FC治疗 2周 ,YCD组小鼠存活 (17.8± 1.89)d ,而eGFP组和wtP388组分别存活 (7.8± 1.10 )d和 (7.7± 1.15 )d (P <0 0 5 ) ;5 FC治疗 2d的杀伤率达 99.6 %。结论 :YCD转基因细胞在体内可被 5 FC有效杀灭 ,该系统具有应用前景。 展开更多
关键词 Ycd基因 修饰 小鼠 P388 白血病 体内治疗作用 研究
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腺病毒介导的大肠杆菌CD自杀基因体内外对肿瘤杀伤作用的研究 被引量:4
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作者 王宝梅 孔令非 +5 位作者 银平章 曹雪涛 鞠佃文 万涛 陶群 于益芝 《中国肿瘤生物治疗杂志》 CAS CSCD 1998年第1期28-31,共4页
表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,... 表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长. 展开更多
关键词 基因治疗 胞嘧啶脱胺酶 腺病毒 黑色素瘤
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婴儿双歧杆菌介导的双自杀基因CD/UPRT对鼠黑色素瘤细胞的杀伤效应 被引量:5
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作者 安丽娜 李著华 +4 位作者 岳扬 王树人 郭志英 彭克军 韩晓群 《华西药学杂志》 CAS CSCD 北大核心 2006年第2期121-123,共3页
目的探讨尿嘧啶磷酸核糖转移酶(UPRT)基因对胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因系统抗瘤作用的增强效应。方法PCR扩增大肠杆菌UPRT基因,构建原核表达质粒pGEX-UPRT;以电穿孔法将该质粒转入婴儿双歧杆菌,筛选阳性重组菌并鉴定;采... 目的探讨尿嘧啶磷酸核糖转移酶(UPRT)基因对胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因系统抗瘤作用的增强效应。方法PCR扩增大肠杆菌UPRT基因,构建原核表达质粒pGEX-UPRT;以电穿孔法将该质粒转入婴儿双歧杆菌,筛选阳性重组菌并鉴定;采用MTT法检测表达的UPRT可否与CD产生抗瘤协同作用。结果重组婴儿双歧杆菌可以正确表达UPRT。体外MTT检测显示CD+UPRT组细胞存活率明显低于对照组(P<0.01),且可使B16-F10鼠黑色素瘤细胞对5-FC杀伤敏感性(IC50=0.015μmol.ml-1)显著提高,是CD组(IC50=0.127μmol.ml-1)的8.5倍。结论婴儿双歧杆菌联合转导UP-RT基因可显著增强CD/5-FC自杀基因系统对鼠黑色素瘤细胞B16-F10的杀伤作用。 展开更多
关键词 婴儿双歧杆菌 尿嘧啶磷酸核糖转移酶 胞嘧啶脱氨酶 基因治疗 黑色素瘤 杀伤作用
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CD/5-FC自杀基因系统治疗胰腺癌的实验研究 被引量:2
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作者 潘雪 李兆申 +4 位作者 许国铭 崔龙 戴观荣 龚燕芳 屠振兴 《解放军医学杂志》 CAS CSCD 北大核心 2002年第10期878-880,T002,共4页
为探讨自杀基因CD/5-FC系统对胰腺癌的杀伤作用及作用机制,应用细菌内同源重组法构建含大肠杆菌胞嘧啶脱氨酶(cytosine deaminase, CD)基因的腺病毒载体,经293细胞包装、扩增,氯化铯密度梯度离心制备纯化CD腺病毒液,体外转染人胰腺癌... 为探讨自杀基因CD/5-FC系统对胰腺癌的杀伤作用及作用机制,应用细菌内同源重组法构建含大肠杆菌胞嘧啶脱氨酶(cytosine deaminase, CD)基因的腺病毒载体,经293细胞包装、扩增,氯化铯密度梯度离心制备纯化CD腺病毒液,体外转染人胰腺癌细胞,并给予前药5-FC,观察其体外杀伤效果;并建立胰腺癌裸鼠皮下移植瘤模型,瘤内直接注入CD腺病毒液,随后腹腔内注入5-FC,观察CD基因的原位治疗效应。含CD基因腺病毒载体经酶切鉴定正确,包装纯化后,检测病毒滴度为2×1011pfu/ml,将重组腺病毒转染胰腺癌细胞株后,可见5-FC对转导入CD基因的胰腺癌细胞有明显细胞毒性作用,而对未导入CD基因的人胰腺癌细胞毒性较低,体内实验显示CD基因原位转导对裸鼠胰腺癌疗效较明显。腺病毒介导CD基因,不仅转染效果强,而且加用5-FC后,可直接或通过旁观者效应杀伤胰腺癌细胞或抑制移植瘤的生长,可作为胰腺癌基因治疗的有效方法。 展开更多
关键词 胰腺肿瘤 腺病毒 胞嘧啶脱氨酶 基因疗法 动物模型
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大肠杆菌CD自杀基因转染诱导肿细胞凋亡及旁观者效应的研究 被引量:3
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作者 王宝梅 曹雪涛 +5 位作者 鞠佃文 银平章 孔令非 万涛 陶群 于益芝 《中国肿瘤生物治疗杂志》 CAS CSCD 1998年第2期126-129,共4页
以重组腺病毒AdCD为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因体外传染小鼠红白血病细胞FBL3,结果显示,转染了CD基因的FBL3细胞对5-氟胞嘧啶(5-FC)的敏感性显著提高,进一步研究发现,AdCD/5-FC系统可以诱导肿瘤细胞发生凋亡;将经AdCD/5-FC处... 以重组腺病毒AdCD为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因体外传染小鼠红白血病细胞FBL3,结果显示,转染了CD基因的FBL3细胞对5-氟胞嘧啶(5-FC)的敏感性显著提高,进一步研究发现,AdCD/5-FC系统可以诱导肿瘤细胞发生凋亡;将经AdCD/5-FC处理过的FBL3细胞上清倍比稀释后,加入到野生型FBL3细胞中,发现当上清仅占6.25%时,即对野生型FBL3细胞发挥明显的杀伤作用,提示旁观者效应在AdCD介导的细胞毒作用中起着重要的作用。本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠局部注射AdCD并连续10天给予5-FC(300mg/kg)治疗后,小鼠皮下肿瘤结节的生长受到明显抑制。 展开更多
关键词 胞嘧啶脱氨酶 旁观者效应 自杀基因 肿瘤
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CD/5-FC自杀基因治疗系统体外杀伤恶性人脑胶质瘤细胞作用的研究 被引量:2
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作者 吕胜青 刘运生 +2 位作者 杨辉 陈祖林 黄河 《中南大学学报(医学版)》 CAS CSCD 北大核心 2004年第2期174-176,180,共4页
目的 :探讨胞嘧啶脱氨酶 / 5 氟胞嘧啶 (CD/ 5 FC)自杀基因治疗系统对恶性人脑胶质瘤细胞的杀伤作用。方法 :采用Lipofectamine2 0 0 0脂质体介导法将CD基因转染U2 5 1恶性人脑胶质瘤细胞 ,G4 1 8筛选获得抗性克隆 (取名为U2 5 1 /CD... 目的 :探讨胞嘧啶脱氨酶 / 5 氟胞嘧啶 (CD/ 5 FC)自杀基因治疗系统对恶性人脑胶质瘤细胞的杀伤作用。方法 :采用Lipofectamine2 0 0 0脂质体介导法将CD基因转染U2 5 1恶性人脑胶质瘤细胞 ,G4 1 8筛选获得抗性克隆 (取名为U2 5 1 /CD细胞 ) ,使用不同浓度的 5 FC作用于U2 5 1 /CD细胞 ,MTT法测定活性细胞比率。采用高效液相色谱法 (HPLC)检测 5 FC培养液内 5 FU的浓度。结果 :U2 5 1细胞获得了质粒的成功转染。基因转染使G4 1 8抗性细胞 (U2 5 1 /CD细胞 )对 5 FC高度敏感。未转染的U2 5 1细胞对 5 FC不敏感 ,IC50 约为 6 5 0 0 μmol/L ,而转染基因后IC50 约为 1 0 μmol/L。并且加入不同浓度的 5 FC后 ,U2 5 1 /CD细胞培养液内均能检测到 5 FU。结论 :5 FC对CD基因修饰U2 5 1细胞具有杀伤作用 ; 展开更多
关键词 胞嘧啶脱氨酶 5-氟胞嘧啶 基因治疗 恶性脑胶质瘤 细胞杀伤作用
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含血管内皮生长因子启动子驱动CD自杀基因重组腺病毒的制备及其体外杀伤作用观察 被引量:5
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作者 陈建发 黄宗海 +2 位作者 俞金龙 厉周 车小燕 《第一军医大学学报》 CSCD 北大核心 2004年第6期623-627,共5页
目的应用AdEasier-1系统高效制备含血管内皮生长因子(VEGF)启动子驱动CD自杀基因重组腺病毒并观察其体外肿瘤杀伤作用。方法以JM109细菌基因组为模板,PCR扩增出含HindⅢ/BamHⅠ酶切位点的CD基因,亚克隆到pREP8,继而用HindⅢ/XbaⅠ切出... 目的应用AdEasier-1系统高效制备含血管内皮生长因子(VEGF)启动子驱动CD自杀基因重组腺病毒并观察其体外肿瘤杀伤作用。方法以JM109细菌基因组为模板,PCR扩增出含HindⅢ/BamHⅠ酶切位点的CD基因,亚克隆到pREP8,继而用HindⅢ/XbaⅠ切出含pA加尾信号的CD基因,插入到预先构建的含VEGF启动子的转移质粒pAdtrack-VEGFP构建pAdtrack-VEGFP-CD。经PmeⅠ线性化后转化AdEasier-1细菌,用25 μg/ml卡那霉素筛选阳性克隆,先后进行琼脂糖电泳和PacⅠ酶切鉴定,筛选出正确重组腺病毒质粒pAdEasy-VEGFP-CD。经293细胞包装、扩增获得重组腺病毒Ad-VEGFP-CD,行PCR鉴定。通过测定细胞克隆形成率和细胞存活率观察重组腺病毒对体外培养的LoVo细胞的细胞毒作用。结果构建重组腺病毒质粒的成功率为70%(7/10)。包装扩增后重组腺病毒的滴度为4.8×1012 CFU/ml,PCR扩增结果与预期相符。在前药5-氟胞嘧啶存在下,重组腺病毒对LoVo具有明显杀伤作用,细胞克隆形成率和细胞存活率都明显下降。结论应用AdEasier-1系统可以成功制备含VEGF启动子驱动CD自杀基因的重组腺病毒,该重组体具有明显的肿瘤杀伤作用。 展开更多
关键词 腺病毒 胞嘧啶脱氨酶 基因治疗 血管内皮细胞生长因子 同源重组
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HSV-tk/CD融合基因杀伤人胆管癌细胞QBC939的体内实验 被引量:4
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作者 李梅生 梁力建 +2 位作者 黄洁夫 胡文洁 甄作均 《中国普外基础与临床杂志》 CAS 2005年第6期581-584,共4页
目的研究单纯疱疹病毒胸苷激酶(HSV-tk)/胞嘧啶脱氨酶(CD)融合基因在动物体内对人胆管癌的杀瘤作用。方法构建人胆管癌的裸鼠皮下移植瘤动物模型,按排序法随机分为4组,每组8只。对照组,每只裸鼠腋部瘤体内注入不含自杀基因的腺病毒液0.1... 目的研究单纯疱疹病毒胸苷激酶(HSV-tk)/胞嘧啶脱氨酶(CD)融合基因在动物体内对人胆管癌的杀瘤作用。方法构建人胆管癌的裸鼠皮下移植瘤动物模型,按排序法随机分为4组,每组8只。对照组,每只裸鼠腋部瘤体内注入不含自杀基因的腺病毒液0.1 ml,CD基因组t、k基因组和HSV-tk/CD融合基因组,在每只裸鼠腋部瘤体内分别注入CD基因t、k基因及HSV-tk/CD融合基因的重组腺病毒液0.1 ml。注射后24 h每只裸鼠腹腔内注射5-氟胞嘧啶(5-flurocytosine,5-FC)和丙氧鸟苷(ganciclovir,GCV),观察肿瘤的生长情况。结果CD基因组t、k基因组及HSV-tk/CD融合基因组肿瘤的生长均受到不同程度的抑制,与对照组比较,抑制率分别为41.2%、55.7%和70.7%,P均<0.05;病理组织学检查显示,对照组肿瘤生长良好,细胞分裂相多见;其余各组出现不同程度的肿瘤坏死,这种现象以HSV-tk/CD融合基因组最为明显。结论HSV-tk/CD融合自杀基因在胆管癌细胞体内也具有较强的杀瘤活性,但作用不够彻底,可能与旁观者效应(bystander effect,BSE)的效应强度有关。 展开更多
关键词 胆管癌 胞嘧啶脱氨酶 胸苷激酶 基因疗法
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