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Cooperative Therapeutic Effects of Herpes Simplex Virus Thymidine Kinase Gene/Ganciclovir System and Chemotherapeutic Agents on Prostate Cancer in vitro
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作者 邢毅飞 肖亚军 +4 位作者 鲁功成 曾甫清 赵军 熊平 冯玮 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2006年第5期610-613,共4页
The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC)... The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) im- munohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC. 展开更多
关键词 prostatic neoplasms herpes simplex virus thymidine kinase gene ganciclovir CHEMOTHERAPY gene therapy
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Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene 被引量:1
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作者 Bao-Jin Li Chao Zhang +3 位作者 Yuan-Xue Yi Ying Hao Xiao-Ping Liu Qing-Jia Ou 《World Journal of Gastroenterology》 SCIE CAS CSCD 2007年第29期4006-4010,共5页
AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS: Recombinant adenovir... AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS: Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (Ⅰ), Ad group (Ⅱ), AdCMV-tk group (Ⅲ) and AdKDR-tk group (Ⅳ). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg·kg^-1·d^-1 (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and alter the last GCV dose.RESULTS: Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to (28.94 ± 5.67)% and (75.45 ± 2.91)% at proper order, respectively (P 〈 0.01), while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to (17.56 ± 2.48)% and (23.15± 5.72)%, respectively (P 〉 0.05). Compared with group I, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group M and Ⅳ (P 〈 0.05). The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 (microvessels/mm^2) in group Ⅰ, Ⅱ, M and IV, respectively. And there was a marked difference between group M and Ⅱ (P 〈 0.05), Ⅳ and Ⅱ (P 〈 0.01), and Ⅳ and M (P 〈 0.01). CONCLUSION: KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV. 展开更多
关键词 ANTI-ANGIOGENIC Vessel-targeted ADENOvirus Hepatocellular carcinoma herpes simplex virus thymidine kinase gene therapy
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Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113
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作者 蓟光辉 邹敬之 +2 位作者 渠乐 岳瑛 蒯建科 《Journal of Medical Colleges of PLA(China)》 CAS 2004年第3期157-160,共4页
Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell ... Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113. 展开更多
关键词 herpes simplex virus thymidine kinase INTERLEUKIN-2 internal ribosome entry sites Tca8113 cells
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Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo
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作者 何祥梁 何东华 +4 位作者 郭先健 钱桂生 黄桂君 陈维忠 李淑萍 《Journal of Medical Colleges of PLA(China)》 CAS 2002年第3期227-231,共5页
Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing ... Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05) , and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance. Conclusion: A549-TK cells acquires sensitivity to ACV both in vitro and in vivo, and ACV can inhibit the growth of tumors induced by A549-TK cell inoculation in nude mice. 展开更多
关键词 pulmonary carcinoma herpes simplex virus thymine kinase gene gene therapy ACYCLOVIR nude mice
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An Oncolytic Adenovirus Expressing Herpes Simplex Virus-Thymidine Kinase for Targeting Cancer Therapy:An in vitro Evaluation
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作者 Fei-qun Zheng Yin Xu +2 位作者 Yi-de Qin Ren-jie Yang Jun Han 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2009年第2期90-96,共7页
Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Her... Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods: A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase (hTERT), the coding sequence of E1A gene, an internal ribosome entry site sequence (IRES) and the coding sequence of herpes simplex virus-thymidine kinase (HSV-TK) was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results: Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir (GCV) induced an obvious cell death. Conclusion: An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy. 展开更多
关键词 Conditionally replicative adenovirus Cancer gene therapy herpex simplex virus-thymidine kinase
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HSVTK Gene Therapy for CarcinoembryonicAntigen-Producing Human Lung Cancer Cells
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作者 Xiao Geng-fu Qi Yi-peng +1 位作者 Cheng Xuan-hong Li Ling-yun 《Wuhan University Journal of Natural Sciences》 CAS 1999年第3期367-371,共5页
The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CE... The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells. 展开更多
关键词 carcinoembryonic antigen herpes simplex virus thymidine kinase gene gene therapy
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SPECT imaging of cardiac reporter gene expression in living rabbits
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作者 LIU Ying LAN Xiaoli +3 位作者 ZHANG Liang WU Yao JIANG Rifeng ZHANG Yongxue 《Nuclear Science and Techniques》 SCIE CAS CSCD 2009年第3期170-176,共7页
This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine kinase (HSV1-tk) reporter gene in rabbits myocardium by using the reporter probe 131I-2'-fluoro-2'-deoxy-1-... This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine kinase (HSV1-tk) reporter gene in rabbits myocardium by using the reporter probe 131I-2'-fluoro-2'-deoxy-1-β-D- arabinofuranosyl-5-iodouracil (131I-FIAU) and SPECT. Rabbits of the study group received intramyocardial injection of Ad5-tk and control group received aseptic saline injection. Two sets of experiments were performed on the study group. Rabbits of the 1st set were injected with 131I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1×109, 5×108, 1×108, 5×107 and 1×107 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1×109, 5×108, 1×108, 5×107, 1×107 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131I-FIAU was injected and heart SPECT imaging was performed at 6, 24 and 48 h, before killing them for gamma counting of the hearts. Reverse tran- scription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSV1-tk gene expression. Semi-quantitative analysis derived of region of interest (ROI) of SPECT images and RT-PCR images was performed and the relationship of SPECT images with ex vivo gamma counting and mRNA level were evaluated. SPECT images conformed 131I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images qual- ity was obtained at 24~48 h for different viral titers. The highest radioactivity in the focal myocardium was seen at 6 h, and then declined with time. The threshold was 5×107 pfu of virus titer. The result could be set better in 1~5×108 pfu by SPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECT images correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The HSV1-tk/131I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter gene imaging. The optimal Ad5-tk titer is 1~5×108 pfu and optimal imaging time is 24~48 h after transferred Ad5-tk in rabbit. The imaging of transgene expression in heart might be used for noninvasive imaging of gene therapy in cardiac diseases in human. 展开更多
关键词 SPECT CMV 成像技术 核技术
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Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes
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作者 关菁 马俐君 魏丽惠 《Chinese Medical Journal》 SCIE CAS CSCD 2001年第2期35-39,105-106,共7页
Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retrovira... Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy. 展开更多
关键词 ovarian cancer · gene therapy · herpes simplex virus thymidine kinase · granulocyte macrophage colony stimulating factor
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pLTKcSN/VPC及GCV系统的旁观者效应观察 被引量:7
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作者 张晓鹏 胡加飞 +8 位作者 蔡如珏 王伟民 袁国梁 王驹 许秀兰 卢亦成 张光霁 朱诚 顾健人 《第二军医大学学报》 CAS CSCD 北大核心 1997年第4期325-327,共3页
目的:探讨单纯疱疹病毒Ⅰ型胸苷激酶基因(TK)逆转录病毒载体生产细胞(pLTKcSN/VPC)和更昔洛韦(GCV)系统杀伤恶性胶质瘤细胞过程中的旁观者效应。方法:采用大鼠胶质瘤细胞C6、人恶性胶质瘤U87MG和它们的... 目的:探讨单纯疱疹病毒Ⅰ型胸苷激酶基因(TK)逆转录病毒载体生产细胞(pLTKcSN/VPC)和更昔洛韦(GCV)系统杀伤恶性胶质瘤细胞过程中的旁观者效应。方法:采用大鼠胶质瘤细胞C6、人恶性胶质瘤U87MG和它们的转染TK阳性细胞C6TK,U87TK,将C6与C6TK,U87MG和U87TK按比例(TK阳性细胞占细胞总体的0%~100%,10%梯度)分别混合培养于96孔板中,每种混合比例设6孔。培养24h后,6孔中3孔加入浓度为0.5μg/mlGCV,另外3孔作为空白对照。继续培养72h,直接计数各孔活细胞数并以对照组计数结果为本底计算GCV对各混合比例的抑制率。结果:当TK阳性细胞占10%时,C6和U87MG组的抑制率达到或超过30%,当TK阳性细胞占总体的50%以上时,GCV对各混合比例的抑制率接近100%。结论:pLTKcSN/VPC和GCV系统杀伤恶性胶质瘤细胞过程中存在明显的旁观者效应。 展开更多
关键词 恶性 胶质瘤 基因治疗 逆转录病毒 TK GCV
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逆转录病毒介导的HSV-TK基因系统对人胃癌细胞的转染及杀伤作用 被引量:3
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作者 王毅 胡志前 +3 位作者 徐学俊 王元和 王强 孔宪涛 《第二军医大学学报》 CAS CSCD 北大核心 2001年第4期328-330,共3页
目的 :探讨逆转录病毒介导的单纯疱疹病毒胸苷激酶 (HSV- TK)基因系统对人胃癌细胞的转染及杀伤作用。方法 :应用 PA317细胞包装的逆转录病毒介导的 HSV - TK基因系统 ,在体外以逆转录病毒上清感染法将 TK基因导入 MKN2 8人胃癌细胞 ,... 目的 :探讨逆转录病毒介导的单纯疱疹病毒胸苷激酶 (HSV- TK)基因系统对人胃癌细胞的转染及杀伤作用。方法 :应用 PA317细胞包装的逆转录病毒介导的 HSV - TK基因系统 ,在体外以逆转录病毒上清感染法将 TK基因导入 MKN2 8人胃癌细胞 ,并初步观察更昔洛韦 (GCV )对转染 TK基因的 MKN2 8胃癌细胞的杀伤作用。 结果 :TK基因可成功转导入MKN2 8细胞 ,且对细胞的生长状态无明显影响 ,但对 GCV的敏感性却显著增加 ;同时还观察到显著的“旁观者效应”。结论 :逆转录病毒介导的 HSV- TK系统对人胃癌细胞有较高的转染效率 ,GCV对转染阳性的胃癌细胞有显著的杀伤作用。 展开更多
关键词 胃肿瘤 单纯疱疹病毒 胸苷激酶 更昔洛韦 基因治疗 基因转染 逆转录病毒 HSV-TK基因
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B7联合HSV-TK基因对大鼠乳腺癌的治疗作用 被引量:6
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作者 王烈 卫立辛 +5 位作者 王飞 曹贵松 钱其军 杨广顺 郭亚军 吴孟超 《第二军医大学学报》 CAS CSCD 北大核心 2000年第3期229-232,共4页
目的 :探讨逆转录病毒介导的单纯疱疹病毒胸苷激酶基因 (HSV- TK)和共同刺激因子 B7基因联合使用对乳腺癌动物模型的体内治疗作用。方法 :应用基因重组技术 ,构建分别携带 HSV- TK,B7及 HSV- TK/B7双基因的逆转录病毒载体。以 SHZ- 88... 目的 :探讨逆转录病毒介导的单纯疱疹病毒胸苷激酶基因 (HSV- TK)和共同刺激因子 B7基因联合使用对乳腺癌动物模型的体内治疗作用。方法 :应用基因重组技术 ,构建分别携带 HSV- TK,B7及 HSV- TK/B7双基因的逆转录病毒载体。以 SHZ- 88细胞株建立乳腺癌动物模型 ,随机分为对照组、TK组、B7组及 TK/B7组 ,每组 2 0只。 2周后于瘤体内分别注射转染有空载体及不同的重组载体的乳腺癌细胞 ,3d后于腹腔内连续注射无环鸟苷 (GCV) 15 d(5 0 mg· kg- 1· d- 1 ) ,观察肿瘤体积、荷瘤生存时间的变化和组织病理改变。结果 :B7组、TK/B7组肿瘤内淋巴细胞浸润明显增多。与对照组相比 ,TK组、B7组肿瘤生长减缓 ,体积减小 ,荷瘤生存期延长 ,两组间均有显著差异 (P<0 .0 5 ) ,而 TK /B7组尤为显著 (P<0 .0 1) ;TK组与 B7组相比则无显著差异。 结论 :HSV- TK,B7基因联合治疗乳腺癌动物模型 ,能直接杀伤肿瘤 ,抑制肿瘤生长 ,延长荷瘤动物的生存期。 展开更多
关键词 基因治疗 乳腺癌 B7基因 HSV-TK基因
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mtHSP70/HSV-tk重组沙门菌抗小鼠黑色素瘤的作用 被引量:8
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作者 曾曙光 刘启才 +3 位作者 王素文 彭细毛 章锦才 张积仁 《南方医科大学学报》 CAS CSCD 北大核心 2012年第1期101-105,共5页
目的研究携带结核杆菌热休克蛋白70(mtHSP70)、人单纯疱疹病毒一胸苷激酶(HSV-TK)双基因的重组沙门菌瘤内注射抗小鼠黑色素瘤的抑瘤效应。方法构建重组沙门菌SL7207/PCMV-mtHSP70-IRES-TK,建立小鼠黑色素瘤动物模型,瘤内注射重组沙门菌... 目的研究携带结核杆菌热休克蛋白70(mtHSP70)、人单纯疱疹病毒一胸苷激酶(HSV-TK)双基因的重组沙门菌瘤内注射抗小鼠黑色素瘤的抑瘤效应。方法构建重组沙门菌SL7207/PCMV-mtHSP70-IRES-TK,建立小鼠黑色素瘤动物模型,瘤内注射重组沙门菌观察其抑瘤效应、荷瘤鼠的生存期并进行安全性评估。结果瘤内注射重组沙门菌,实验组抑瘤率显著高于对照组,延长荷瘤鼠生存期,重组菌治疗后荷瘤小鼠的生存状态良好,无腹泻,治疗期间体质量无明显改变。结论减毒沙门菌携带的mtHSP70/HSV-TK双基因真核共表达质粒瘤内注射对B16肿瘤细胞具有显著抑制作用。 展开更多
关键词 重组DNA 黑色素瘤 沙门氏菌 结核杆菌热休克蛋白70 人单纯疱疹病毒一胸苷激酶
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HSV-tk基因治疗靶向载体的构建及特异性表达分析 被引量:11
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作者 刘晓平 李宝金 张超 《癌症》 SCIE CAS CSCD 北大核心 2006年第2期179-184,共6页
背景与目的:阻断肿瘤组织内血管生成和血管化是一个很有发展前景的灭瘤途径之一。胸苷激酶自杀基因系统(herpessimplexvirus-thymidinekinase,HSV-tk/GCV)能有效杀伤血管内皮细胞,目前多采用巨细胞病毒(cytomegalovirus,CMV)作为启动子... 背景与目的:阻断肿瘤组织内血管生成和血管化是一个很有发展前景的灭瘤途径之一。胸苷激酶自杀基因系统(herpessimplexvirus-thymidinekinase,HSV-tk/GCV)能有效杀伤血管内皮细胞,目前多采用巨细胞病毒(cytomegalovirus,CMV)作为启动子,但其杀伤缺乏特异性。KDR(kinasedomaininsertcontainingreceptor)是血管内皮生长因子的两种受体之一,它在肿瘤血管内皮细胞中高表达,而在正常组织中呈低表达。本研究是构建KDR启动子介导的HSV-tk重组腺病毒、并对其内皮细胞特异性表达作用进行分析。方法:采用pAdeasy系统,按定向克隆方法将KDR-tk片段正向插入表达载体的多克隆位点间,构建受KDR启动子调控并可表达HSV-tk基因的AdKDR-tk,在293细胞中包装、扩增后,体外感染人脐静脉血管内皮细胞系(humanumbilicalvenousendothelialcells,HUVEC)和不表达KDR的肝癌细胞系HepG2。用更昔洛韦(ganciclovir,GCV)处理受染细胞,并以MTT法检测其细胞增殖情况。结果:经限制性酶切分析,RT-PCR及PCR方法鉴定,插入基因大小、位置、方向正确。病毒滴度为1×1010pfu/ml。在感染复数(multiplicityofinfection,MOI)为100的条件下,GCV浓度由0增至50μg/ml时,感染含AdKDR-tk的HUVEC细胞和HepG2细胞存活率由100%分别下降至(28.94±5.67)%和(75.45±2.91)%(P<0.01)。结论:构建的含KDR-tk重组腺病毒可在血管内皮细胞中特异性地表达HSV-tk。 展开更多
关键词 KDR启动子 单纯疱疹病毒胸苷激酶 靶向基因治疗 腺病毒 血管内皮细胞 HEPG2细胞
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含HSV-tk基因的人大肠癌特异性逆转录病毒载体的构建 被引量:5
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作者 崔龙 曹广文 +5 位作者 王元和 屠岳 孟荣贵 高军 仇小芳 吴宗娣 《第二军医大学学报》 CAS CSCD 北大核心 1996年第3期216-218,共3页
目的:为使自杀基因在大肠癌细胞中特异性表达,以达到靶向基因治疗的目的,首先构建以CEA基因转录调控序列驱动的重组逆转录病毒载体。方法:将目的基因片段HSV-tk定向克隆入pUC118质粒载体中,以相应的内切酶取出合适... 目的:为使自杀基因在大肠癌细胞中特异性表达,以达到靶向基因治疗的目的,首先构建以CEA基因转录调控序列驱动的重组逆转录病毒载体。方法:将目的基因片段HSV-tk定向克隆入pUC118质粒载体中,以相应的内切酶取出合适的酶切片段后,与来自pCEA424/2CAT质粒的CEA5'转录调控序列相连接,克隆入逆转录病毒载体中。结果:经鉴定及筛选,构建成重组逆转录病毒载体G1CEAtkNa。结论:G1CEAtkNa的成功构建,为进一步研究大肠癌组织特异性基因治疗奠定了基础。 展开更多
关键词 单纯疱疹病毒 大肠肿瘤 逆转录病毒 病毒载体
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间隙连接细胞间通讯与胸苷激酶系统“旁观者效应”的关系及其调控 被引量:5
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作者 邢毅飞 鲁功成 +4 位作者 肖亚军 赵军 曾甫清 熊平 冯玮 《细胞生物学杂志》 CAS CSCD 北大核心 2004年第2期162-166,共5页
增强“旁观者效应”是提高单纯疱疹胸苷激酶/更昔洛韦(HSV-TK/GCV)基因系统治疗效果的关键手段。旨在探讨间隙连接细胞间通讯(GJIC)在HSV-TK/GCV系统“旁观者效应”中的作用及其化学调控。通过采用划痕标记染料示踪技术(SLDT)比较ACHN、... 增强“旁观者效应”是提高单纯疱疹胸苷激酶/更昔洛韦(HSV-TK/GCV)基因系统治疗效果的关键手段。旨在探讨间隙连接细胞间通讯(GJIC)在HSV-TK/GCV系统“旁观者效应”中的作用及其化学调控。通过采用划痕标记染料示踪技术(SLDT)比较ACHN、HeLa、NIH-3T3、Cos-7及L-02等五种不同细胞系GJIC功能状况,四甲基偶氮唑盐酶反应比色法(MTT法)检测HSV-TK/GCV对其杀伤效应和“旁观者效应”,并比较GJIC上调剂apigenin和抑制剂18-α-glycyrrhetinic acid(AGA)对GJIC和“旁观者效应”的影响。结果表明,GJIC功能强大的NIH-3T3、Cos-7及L-02细胞对HSV-TK/GCV敏感性及“旁观者效应”程度远较GJIC功能低下的ACHN和HeLa细胞高(P<0.001)。Apigenin可显著提高ACHN细胞GJIC功能,对HeLa细胞则无影响;相反,AGA可显著抑制NIH-3T3、Cos-7和L-02等三种靶细胞的GJIC功能。相应地,apigenin可明显提高ACHN细胞的“旁观者效应”而对HeLa细胞的“旁观者效应”则无影响,AGA则可明显降低上述三种细胞的“旁观者效应”。因此认为,细胞内在的GJIC功能与靶细胞对HSV-TK/GCV的敏感性和“旁观者效应”程度有正向关系,人为调控细胞GJIC后,“旁观者效应”程度亦发生相应改变。 展开更多
关键词 间隙连接细胞间通讯 胸苷激酶系统 “旁观者效应” 单纯疱疹胸苷激酶/更昔洛韦
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腺病毒介导的VEGF启动子-胸苷激酶表达系统对肝癌细胞的选择性杀伤作用 被引量:4
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作者 王孟龙 殷正丰 +2 位作者 吴宗娣 王率 吴孟超 《第二军医大学学报》 CAS CSCD 北大核心 2002年第1期12-14,共3页
目的 :构建携带受血管内皮生长因子 (VEGF)启动子调控的单纯疱疹病毒胸苷激酶基因 (HSV- tk)的重组腺病毒载体 ,并探讨该重组腺病毒对体外培养的肝癌细胞的特异性杀伤活性。方法 :采用 Ad Easy System构建携带受 VEGF启动子或巨细胞病毒... 目的 :构建携带受血管内皮生长因子 (VEGF)启动子调控的单纯疱疹病毒胸苷激酶基因 (HSV- tk)的重组腺病毒载体 ,并探讨该重组腺病毒对体外培养的肝癌细胞的特异性杀伤活性。方法 :采用 Ad Easy System构建携带受 VEGF启动子或巨细胞病毒 (CMV)启动子调控 tk基因表达的 Ad VEGF- tk和 Ad CMV- tk,这两种重组腺病毒载体在 2 93细胞中包装、扩增后 ,体外感染正常肝细胞系 L 0 2和肝癌细胞系 Hep G2 ,并用不同浓度的丙氧鸟苷 (GCV)处理后以 MTT法检测受染细胞的增殖情况。 结果 :Ad VEGF- tk的病毒滴度为 1× 10 1 0 pfu/ ml,Ad CMV- tk为 5× 10 1 0 pfu/ ml。 L 0 2细胞感染 Ad VEGF- tk后对GCV的敏感性无明显改变 ,而 Hep G2细胞感染 Ad VEGF- tk后对 GCV的敏感性明显增加 ,在感染复数 (MOI)为 10 0并用 5 0μg/ m l GCV处理时 ,则有 90 %以上 Hep G2细胞被杀死 ;而 Ad CMV- tk可杀死 95 %Hep G2细胞和 80 %以上 L 0 2细胞。 结论 :VEGF启动子调控的 HSV- tk基因表达可特异性地杀死肝癌细胞 ,同时对正常肝细胞几无影响。 展开更多
关键词 血管内皮生长因子 单纯疱疹病毒胸苷激酶 腺病毒 肝癌 细胞 VEGF 启动子
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阳离子脂质体介导的HSV-tk自杀基因疗法的体内外抗肝癌作用 被引量:4
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作者 杨淑英 段芳龄 +1 位作者 鲁凤民 盛剑秋 《中国肿瘤生物治疗杂志》 CAS CSCD 1998年第2期112-115,共4页
为研究阳离子脂质体介导HSV-tk自杀基因系统的体内外抗肝癌作用,本课题用基因重组技术构建了含HSV-tk基因的重组逆转录病毒载体,命名为pLXT。用Lipofectin介导转染人肝癌细胞系SMMC-7721,经G418筛选抗性克隆及流式细胞仪分析,获得了持... 为研究阳离子脂质体介导HSV-tk自杀基因系统的体内外抗肝癌作用,本课题用基因重组技术构建了含HSV-tk基因的重组逆转录病毒载体,命名为pLXT。用Lipofectin介导转染人肝癌细胞系SMMC-7721,经G418筛选抗性克隆及流式细胞仪分析,获得了持续表达HSV-tk的克隆细胞SM/tk。~3H-TdR掺入法测定表明HSV-tk/ACV系统对SM/tk细胞具有强杀伤力。将Lipofectin-pLXT复合物直接注射于鼠肝癌细胞(H22)实体瘤内,ACV治疗后监测抑瘤率,发现脂质体介导pLXT、ACV治疗组及裸pLXT注射、ACV治疗组平均瘤体积均显著小于其它六组对照组(P【0.001,P【0.05),前两者比较亦有显著差异(P【0.02)。空载体转染组和阴性对照组平均瘤体积及平均瘤重均无显著差异(P】0.05)。这些结果表明阳离子脂质体介导重组逆转录病毒载体基因转染的方法简便、安全、有效,且可使目的基因获得持续表达;在体内HSV-tk/ACV系统具有强的细胞毒作用,提示存在着强的旁观者效应。此外,瘤体内直接注射裸HSV-tk基因的治疗也有效。 展开更多
关键词 基因治疗 肝肿瘤 单纯疹疹病毒 脂质体
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HSV-tk与p53基因对涎腺多形性腺瘤细胞的杀伤作用 被引量:3
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作者 王旭 王洁 +2 位作者 董福生 董玉英 侯亚丽 《华西口腔医学杂志》 CAS CSCD 北大核心 2005年第1期65-68,共4页
目的 观察腺病毒介导的单纯疱疹病毒胸苷激酶(HSV_tk)基因、野生型p53基因(wt_p53)联合杀伤人涎腺 多形性腺瘤细胞的效果。方法 采用腺病毒介导的HSV_tk基因、wt_p53基因联合转染人涎腺多形性腺瘤细胞,逆 转录聚合酶链式反应(RT... 目的 观察腺病毒介导的单纯疱疹病毒胸苷激酶(HSV_tk)基因、野生型p53基因(wt_p53)联合杀伤人涎腺 多形性腺瘤细胞的效果。方法 采用腺病毒介导的HSV_tk基因、wt_p53基因联合转染人涎腺多形性腺瘤细胞,逆 转录聚合酶链式反应(RT_PCR)检测转染后的基因表达;四唑盐比色(MTT)法测定tk与p53基因治疗后对肿瘤细 胞的杀伤作用及旁观者效应,并采用相差显微镜观察基因治疗后肿瘤细胞的形态学变化。结果 单独转染wt_p53 及HSV_tk GCV基因5d后,涎腺多形性腺瘤细胞的存活率分别为54%和38%;两者联合转染5d后,细胞的存活率 降至20%,两者差异有显著性(P<0.05)。结论 HSV_tk、wt_p53基因联合应用对涎腺多形性腺瘤的杀伤作用强于 此两基因单独应用的杀伤作用。 展开更多
关键词 腺瘤 涎腺 基因治疗 野生型P53 单纯疱疹病毒胸苷激酶基因
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腺病毒为载体的单纯疱疹病毒胸苷激酶/更昔洛韦系统联合光动力治疗口腔恶性肿瘤的初步观察 被引量:3
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作者 陈世璋 张燕升 +5 位作者 范忠 李维弟 郭莹 王潇 陈剑飞 李宁 《华西口腔医学杂志》 CAS CSCD 北大核心 2011年第6期610-613,617,共5页
目的应用腺病毒为载体的单纯疱疹病毒胸苷激酶(ADV-TK)/更昔洛韦(GCV)系统联合光动力疗法对口腔恶性肿瘤复发患者进行治疗,探讨此法治疗口腔恶性肿瘤的临床效果。方法选取10名口腔不同部位的恶性肿瘤复发患者,用光敏剂——血卟啉衍生物(... 目的应用腺病毒为载体的单纯疱疹病毒胸苷激酶(ADV-TK)/更昔洛韦(GCV)系统联合光动力疗法对口腔恶性肿瘤复发患者进行治疗,探讨此法治疗口腔恶性肿瘤的临床效果。方法选取10名口腔不同部位的恶性肿瘤复发患者,用光敏剂——血卟啉衍生物(HPD)静脉给药,选择相应激光输出功率定时照射,以ADV-TK基因行瘤体及周边注射,经GCV静脉输入治疗后,通过影像学及瘤体血流动力学分析进行临床疗效评估。结果经联合法治疗后,患者肿瘤明显缩小(甚至完全消失),瘤体内血流量降低,影像结果对比改变明显,治疗效果理想。结论将ADV-TK/GCV系统联合光动力治疗口腔恶性肿瘤具有显著的抗肿瘤效应,具备副作用轻微、临床安全性高的特点,为系统性治疗相关肿瘤提供了一种安全可靠的治疗手段。 展开更多
关键词 腺病毒为载体的单纯疱疹病毒胸苷激酶/更昔洛韦系统 光动力 血卟啉衍生物 恶性肿瘤
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HSV-tk/GCV系统杀伤ACC-M细胞的机理研究 被引量:3
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作者 孙春晓 何荣根 +3 位作者 张志愿 刘兴坤 周晓健 陈诗书 《肿瘤》 CAS CSCD 北大核心 2000年第1期16-18,共3页
目的 为探索HSV-tk/GCV(单纯疱疹病毒胸腺嘧啶核苷激酶基因/羟甲基无环鸟苷)基因治疗系统杀伤人高转移性腺样囊性癌细胞ACC-M 的机理。方法 采用荧光染色荧光显微镜观察、细胞DNA 电泳、流式细胞仪检测等方法。结果 研究发现,荧光染... 目的 为探索HSV-tk/GCV(单纯疱疹病毒胸腺嘧啶核苷激酶基因/羟甲基无环鸟苷)基因治疗系统杀伤人高转移性腺样囊性癌细胞ACC-M 的机理。方法 采用荧光染色荧光显微镜观察、细胞DNA 电泳、流式细胞仪检测等方法。结果 研究发现,荧光染色有明显的细胞凋亡小体和染色质边聚现象;在GCV浓度较大(400 μm ol/L)作用时间较长(4 天)时有较明显的DNA 梯状现象;而FCM 发现在400 μm ol/LGCV 作用下有极明显的亚G1 峰。结论 HSV-tk/GCV基因治疗系统杀伤ACC-M 展开更多
关键词 HSV-TK/GCV 腺样囊性癌 基因治疗
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