应用RV-HSV-TK基因转染胰腺癌细胞及转染阳性的细胞对GCV敏感性研究

由缺陷型逆转录病毒介导的TK基因系统（RV－HSV－TK）是众多肿瘤生物治疗方法中一个技术较为成熟的方案，本实验采用该系统在体外成功地转染了人胰腺癌细胞株SW1990并能够稳定传代培养，转染了TK基因的SW1990细胞（SW±K），其生长曲线与未转染的SW1990细胞无差异。10－4～102μg／ml的GCV对SWtk有明显的毒性作用（IC50＝2．5μg／ml），杀伤效应与时间成正比，作用48小时以后开始出现毒性作用。将SW1990细胞与包装细胞共孵育48小时以上，再加入10μg／ml的GCV作用120小时，约有50％的SW1990细胞被杀死。结果表明：采用RV－HSV－TK系统转染胰腺癌细胞，有较高的转染效率，转染了TK基因的胰腺癌细胞对GCV敏感。     

应用RV-HSV-TK基因转染胰腺癌细胞及转染阳性的细胞对GCV敏感性研究

 由缺陷型逆转录病毒介导的TK基因系统(RV－HSV－TK)是众多肿瘤生物治疗方法中一个技术较为成熟的方案, 本实验采用该系统在体外成功地转染了人胰腺癌细胞株SW1990并能够稳定传代培养, 转染了TK基因的SW1990细胞(SW±K), 其生长曲线与未转染的SW1990细胞无差异。 10－4～102μg/ml的GCV对SWtk有明显的毒性作用(IC50＝2.5μg/ml), 杀伤效应与时间成正比, 作用48小时以后开始出现毒性作用。 将SW1990细胞与包装细胞共孵育48小时以上, 再加入10μg/ml的GCV作用120小时, 约有50%的SW1990细胞被杀死。 结果表明:采用RV－HSV－TK系统转染胰腺癌细胞, 有较高的转染效率, 转染了TK基因的胰腺癌细胞对GCV敏感。     

Induction of a bystander effect in HeLa cells by using a bigenic vector carrying viral thymidine kinase and connexin32 genes

We previously showed that gap junction intercellular communication mediates the bystander effect in anticancer gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir. Because most cancer cell lines have lost their ability to communicate through gap junctions, we investigated whether we could induce such a communication by transferring a gene for a gap junction. We transfected a vector carrying the HSV-tk (tk) and gap junction (connexin (Cx) 32) genes (Cx32(+)tk(+)) into noncommunicating HeLa cells. We compared the cytotoxicity of ganciclovir with mixtures of these cells and HeLa cells that expressed (Cx32(+)) or did not express (Cx32(-)) the Cx32 gene. The bystander effect was strong when the two mixed cell types expressed Cx32 (i.e., Cx32(+)tk(+) cells and Cx32(+)tk(-) cells). Only 25% of cells survived in this communicating mixture, even when only 10% of the cells were Cx32(+)tk(+). There was also a moderate bystander effect when the Cx32(+)tk(+) cells were mixed with noncommunicating HeLa cells in a 50% ratio. These results demonstrated that the bystander effect is enhanced by Cx32 and suggested that expression of Cx in only one cell type in a mixture can cause a bystander effect. Mol. Carcinog. 30:176--180, 2001.     

Potent bystander effect in suicide gene therapy using neural stem cells transduced with herpes simplex virus thymidine kinase gene

OBJECTIVE: The herpes simplex virus thymidine kinase (HSVtk)/ganciclovir suicide gene therapy system has been considered as one of the most promising therapeutic strategies for malignant gliomas. We have been using HSVtk gene-transduced neural stem cells (NSCtk) that possess an ability to migrate toward a tumor mass for the treatment of experimental brain tumors. In the present study, we evaluated the potency of anti-tumor effect mediated by the bystander effect between NSCtk and C6 glioma cells in the HSVtk/ganciclovir suicide gene therapy system. METHODS: NSCtk and C6 glioma cells were mixed at various ratios (NSCtk:C6 cell ratios of 1:1 to 1:64) and the bystander effect was evaluated both under in vitro and in vivo conditions. RESULTS: In vitro co-culture experiment showed a complete tumor growth inhibition at the NSCtk:C6 ratios as low as 1:16. In vivo co-implantation study in the rat brain showed no visible tumors at the NSCtk:C6 ratios as low as 1:16 and all those rats survived more than 100 days. CONCLUSION: The results clearly demonstrated an extremely potent bystander effect between NSCtk and C6 cells, and the minimum number of NSCtk cells needed for the treatment of tumors was roughly estimated.     

Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase (TK) gene from herpes simplex virus (HSV) after treatment with the antiviral drug, ganciclovir (GCV). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV-TK-expressing transduced cells in the culture resulted in >90% tumor cell death/stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication (GJIC) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell-cell contact of parental and HSV-TK-transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK+/TK- homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK+/TK - cells showed that the level of the bystander killing obtained was characteristic of the TK-bystander cells. The in vitro findings were confirmed in vivo with GCV-treated homologous and chimeric tumors composed of TK+/TK- cells. Day 21 mean tumor volumes (MTVs) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high-GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin-43, in facilitating the bystander effect was demonstrated with the HT29 low-GJIC cell line. When the TK-nontransduced cell population expressed connexin-43, a better bystander kill was achieved compared to the parental counterpart.     

In vivo enhancement of herpes simplex virus thymidine kinase/ganciclovir cancer gene therapy with polyamine biosynthesis inhibition

We have earlier demonstrated that inhibition of polyamine biosynthesis with difluoromethylornithine (DFMO) can be used to enhance the cytotoxicity of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in different tumor cell lines. Here, the utility of this treatment combination was tested in vivo in a nude mouse tumor model. First, the effect of DFMO was verified by treating mice bearing subcutaneous 9L rat glioma tumors with 2% DFMO in drinking water. The drug treatment induced almost complete suppression of ornithine decarboxylase activity, and as a result, a strong decrease in intratumoral putrescine and spermidine concentrations, which were normalized 4 days after drug removal. Consequently, the tumors displayed a significant reduction in the proliferation activity that was increased to 20% higher than the normal level at day 4 and returned to normal level 7 days after DFMO removal. Next, 9L tumors with 30% of TK-GFP fusion gene positive cells were induced and the animals were given DFMO and GCV in 2 treatment schemes, with the drug administration periods overlapping either 5 or 2 days. The analysis of tumor size at the end of the treatment revealed that DFMO can enhance HSV-TK/GCV cytotoxicity when the overlap between DFMO and GCV was 5 days, but the result was not significant. However, the 2-day overlap scheme yielded a significantly (p < 0.05, ANOVA) enhanced antitumor effect. In conclusion, the data here confirms that a novel combination of 2 clinically relevant treatment modalities, polyamine deprivation and HSV-TK/GCV suicide gene therapy, can be used synergistically in vivo.     

Ganciclovir-mediated cell killing and bystander effect is enhanced in cells with two copies of the herpes simplex virus thymidine kinase gene

Delivery and expression of the herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir is currently being evaluated for the treatment of many types of cancer. After initial phosphorylation by HSVtk, cellular kinases generate the toxic triphosphate form of ganciclovir (GCV). To further define the role of GCV metabolism in cells expressing HSVtk, two human tumor cell lines, UMSCC29 and T98G, were transduced with HSVtk and screened for insertion of one or two copies of the viral transgene by Southern blot analysis. Both the relative capacities for incorporating labeled GCV and the levels of GCV metabolites were determined for each of the parental cell lines and their derivatives containing either one or two copies of the HSVtk gene. The efficiency of GCV killing and the magnitude of the bystander effect were compared for the single- and double-copy HSVtk cell lines. Consistently, cells that expressed two copies of HSVtk metabolized GCV more efficiently, were more sensitive to GCV, and demonstrated improved bystander killing relative to single-copy HSVtk cells. The implications of these results for future and current therapies employing HSVtk and GCV are discussed.     

Preservation of the bystander cytocidal effect of irradiated herpes simplex virus thymidine kinase (HSV-tk) modified tumor cells

Summary In vitro and animal experiments have demonstrated the potential efficacy of using the bystander effect alone in the treatment of brain tumors. A known problem in some in vitro and in vivo experiments is that a fraction of cells engineered to express the herpes simplex virus thymidine kinase (HSV-tk) gene survive ganciclovir (GCV) treatment and undergo cell division. To prevent the recurrent growth of HSV-tk+ cells in the presence of GCV we examined the potential use of lethal or sublethal irradiation of Walker 256 carcinosarcoma cells selected for expression of the HSV-tk gene (Walker-tk+). Western blot analysis of Walter-tk+ cells showed similar levels of HSV-tk protein expression at 0, 1, 3, 6 and 9 days after lethal gamma-irradiation. In vitro, there was no difference in the bystander effect exerted by non-irradiated, sublethally irradiated or lethally irradiated Walker-tk+ cells on wild-type Walker cells in the presence of GCV. In vivo experiments demonstrated long-term survival (100 days) in rats implanted intrathecally with sublethally or lethally irradiated Walker-tk+ cells with GCV treatments. Intrathecal implantation of irradiated Walker-tk+ cells either pre-mixed with Walker cells or used in in situ treatment of established Walker tumors resulted in prolonged animal survival compared to controls (p < 0.05). These experiments suggest that the bystander tumoricidal effect is preserved despite gamma-irradiation of the HSV-tk modified tumor cells and that irradiation could be an effective method to prevent long-term resistance to GCV in HSV-tk+ tumor cells.     

Cancer gene therapy: Combination with radiation therapy and the role of bystander cell killing in the anti-tumor effect

Current anti-cancer modalities such as surgery, chemo- and radiation therapies have only limited success in cancer treatment. Gene therapy is a promising new tool to improve outcomes. In this review, first we summarize the various strategies to kill tumor cells, and then focus on the bystander effect of gene therapy. A variety of strategies, such as gene-directed enzyme pro-drug therapy, activation of an anti-tumor immune attack, application of replication-competent and oncolytic viral vectors, tumor-specific as well as radiation and hypoxiainduced gene expression, might be applied to target tumor cells. We put special emphasis on the combination of these approaches with local tumor irradiation. Using the available vector systems, only a small portion of cancer cells contains the therapeutic genes under clinical situations. However, cells directly targeted by gene therapy will transfer death signals to neighboring cancer cells. This bystander cell killing improves the efficiency of cancer gene therapy. Death signals are delivered by cell-to-cell communication through gap junction intercellular contacts, release of toxic metabolites into the neighborhood or to larger distances, phagocytosis of apoptotic bodies, and the activation of the immune system. Bystander cell killing can be enhanced by the introduction of gap junction proteins into cells, by further activating the immune system with immune-stimulatory molecules, or by introducing genes that help the transfer of cytotoxic genes and/or metabolites into bystander cells. In conclusion, although bystander cell killing can improve therapeutic effects, there should be additional developments in cancer gene therapy for a more efficient clinical application.(Pathology Oncology Research Vol 12, No 2, 118–124)     

重组腺病毒-胸苷激酶基因对多种肿瘤细胞的杀伤作用

背景与目的:以腺病毒为载体的单纯疱疹病毒胸苷激酶基因(adenovirusvector-mediatedherpessimplexvirus-thymidinekinasegene,ADV-TK)重组体是肿瘤基因治疗应用的主要方法之一,本文旨在鉴定自行构建的ADV-TK重组体的体外抗肿瘤活性。方法:利用腺病毒载体将TK导入体外培养的14种不同组织源性的肿瘤细胞,再加入更昔洛韦(ganciclovir,GCV),用MTT法观察对肿瘤细胞的抑制效应。结果:ADV-TK剂量为每孔1×109病毒颗粒,在100μg/mlGCV底物浓度条件下,它对14种肿瘤细胞中的11种杀伤率达74%以上,而对人喉上皮癌细胞Hep-2、人肝癌细胞Bel-7402和人结肠癌细胞HCT-8敏感性稍差,分别为(55.3±2.0)%、(61.3±2.0)%和(63.7±2.5)%,ADV-TK对肿瘤细胞的抑制率除人喉上皮癌细胞Hep-2外,与相当于组织峰值药物浓度(5μg/ml)的化疗药物顺铂的效率相近。结论:ADV-TK重组体具有明确的体外抗肿瘤活性,有潜在临床应用价值。     

Inducible expression of herpes simplex virus thymidine kinase increases sensitivity to ganciclovir but does not enhance bystander effect in breast cancer cells

Delivery of cancer chemotherapy directly to the cancer cell has great appeal. Previous studies using adenoviral transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) in an ascites model of breast cancer was successful in reducing tumor burden and prolonging life. However, increasing the viral dose resulted in increased toxicity and host mortality emphasizing the need for an improved therapeutic ratio. To test the hypothesis that enhancement of HSV-tk gene expression would lead to increased sensitivity to GCV and improved bystander effect, we created breast cancer cells expressing HSV-tk under the control of the inducible tetracycline promoter. Using this system, we could inducibly increase gene expression and biochemical activation of HSV-tk. These increased levels of HSV-tk decreased the IC₅₀ to GCV nearly 50-fold. However, the bystander effect was not enhanced by increasing HSV-tk gene expression. We conclude that increased HSV-tk gene expression improves sensitivity to CCV. However, additional measures, such as increased gap junction communication, will likely be needed to enhance the bystander effect and the therapeutic efficacy of this strategy.     

靶向非病毒载体一次及持续介导Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因转导卵巢癌细胞的比较

Objective:To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7,a non-viral targeted delivery system,in transfection of thymidine kinase gene of herpes simplex virus(HSV-tk)into ovarian cancer cells.Methods:GE7 was used to prepare recombinants with β-galactosidase(β-gal)and HSV1-tk;the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously.β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system.Ganciclovior(GCV)was introduced into HSV1-tk transfected ovarian cells.Through drawing the cell growth curve and flow cytometry,the killing effects of GCV on once and continuously GE7/HSV1-tk transfected cells were observed.Results:We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%,respectively When 1 μg/mL GCV was used to treat ovarian cell transfected with HSV1-tk gene,growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%,respectively;their apoptosis indices were 15 and 30,respectively.Under same GCV concentration.continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation(P＜0.05).Conclusion:Compared with one time mediation,continuous mediation of transfection with GE7 gene delivery system has higher efficiency.Continuous mediation of GE7/HSV1-tk/GCV therapeutic gene system has more powerful killing effect.     

Non-viral targeted delivery system mediates transfection of thymidine kinase gene of herpes simplex virus into ovarian cancer cells： a comparison between one time and continuous mediation

Objective  To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7, a non-viral targeted delivery system, in transfection of thymidine kinase gene of herpes simplex virus (HSV-tk) into ovarian cancer cells. Methods  GE7 was used to prepare recombinants with β-galactosidase (β-gal) and HSV1-tk; the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously. β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system. Ganciclovior (GCV) was introduced into HSV1-tk transfected ovarian cells. Through drawing the cell growth curve and flow cytometry, the killing effects of GCV on once and continuously GE7/HSV1-tk transfected cells were observed. Results  We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%, respectively. When 1 μg/mL GCV was used to treat ovarian cell transfected with HSV1-tk gene, growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%, respectively; their apoptosis indices were 15 and 30, respectively. Under same GCV concentration, continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation (P < 0.05). Conclusion  Compared with one time mediation, continuous mediation of transfection with GE7 gene delivery system has higher efficiency. Continuous mediation of GE7/HSV1-tk/GCV therapeutic gene system has more powerful killing effect. Supported by a grant from the National Natural Sciences Foundation of China (No. 39800144).     

HSV-tk基因治疗靶向载体的构建及特异性表达分析

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。     

Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells

Tumoricidal "bystander effect" observed in the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy was studied between different rat glioma cell lines (9L and C6 cells) under both in vitro and in vivo conditions. For that purpose, mixed populations of wild-type cells (9Lwt and C6wt) and respective HSVtk gene-transduced cells (9Ltk and C6tk) were examined for their sensitivity to GCV. A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Because the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target (wild-type) cells but not in effector (HSVtk gene-bearing) cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells.     

Suicidal gene therapy for pleural metastasis of lung cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene

Pleural metastasis is one of the most common complications in lung cancers. However, no effective therapy for pleural metastasis has been established thus far. We have constructed a metastatic model of non-small cell lung cancer (NSCLC) by injecting human NSCLC cell lines directly into the left pleural cavity of BALB/c nude mice. Because this model is easy to construct and the results are reproducible, we used this model for a preclinical evaluation of gene therapy for pleural metastasis of NSCLC. We took the novel approach of in vivo lipofection of a suicidal gene to lung cancer cells metastasized to the pleural cavity. A human lung cancer cell line, PC14, was inoculated into the pleural cavity of nude mice. After 1 day, a herpes simplex virus thymidine kinase gene expression plasmid was injected intrapleurally as a DNA-liposome complex, and ganciclovir was subsequently administered for 8 days. The survival rates of the ganciclovir-treated group were significantly better than those of the control groups. Flow cytometric analysis using a green fluorescent protein expression plasmid suggested that the transfection efficiency in the pleural cavity was 13.6%. Moreover, due to a bystander effect with PC14 cells, 10% of the gene transfer efficiency was sufficient to eradicate or suppress pleural metastasis. This preclinical study suggests the therapeutic feasibility of an in vivo lipofection-based suicidal gene/prodrug strategy for pleural metastasis of NSCLC.     

Gene therapy of chondrosarcoma using retrovirus vectors encoding the herpes simplex virus thymidine kinase gene.

Human chondrosarcoma cells (HCS-TG) were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ). We investigated the cytotoxicity of human chondrosarcoma bearing an HSV-tk gene after treatment with ganciclovir. Chondrosarcoma cells bearing an HSV-tk gene were more sensitive than non-transduced cells. Coculturing with chondrosarcoma cells bearing both an HSV-tk gene (HCS-TG-tk) and lacZ gene (HCS-TG-Z) in various ratios showed a bystander effect. Chondrosarcoma implanted in nude mice were injected with HCS-TG-tk cells. After 4 weeks, the growth of tumors was significantly prevented.     

Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.     

重组腺病毒胸苷激酶基因构建体联合更昔洛韦治疗移植瘤裸鼠小细胞肺癌的实验研究

目的　观察以腺病毒为载体的单纯疱疹病毒胸苷激酶基因重组构建体 (ADV TK)联合更昔洛韦 (GCV)体内抗肺癌的活性。方法　建立移植瘤裸鼠小细胞肺癌模型 ,肺癌局部注射ADV TK后 ,腹腔注射GCV ,观察肿瘤体积、相对肿瘤体积、瘤重、相对肿瘤增殖率以及肿瘤体积生长变化的时间曲线 ,评价ADV TK的抗肿瘤活性。结果　在作用底物GCV存在条件下 ,ADV TK对人癌裸鼠移植性小细胞肺癌生长具有明显抑制作用 ,对裸鼠移植性小细胞肺癌的体积和瘤重的抑制效应与ADV TK呈剂量依赖性增强 ,且未达量效平台期 ,其中 6 .0× 10 9病毒颗粒 /kg剂量组的肿瘤生长抑制率分别达到 6 4 .6 %和 81.7%。将ADV TK和GCV分别单独应用 ,结果显示对肿瘤生长均有一定的抑制作用 ,但与阴性对照组相比 ,差异无显著性 (P >0 .0 5 )。结论　ADV TK联合GCV对肺癌具有明确的实验性治疗作用 ,值得进一步研究 ,开展临床试验。