Tet-On在乳腺癌自杀基因治疗中诱导细胞凋亡的实验研究

目的：构建Tet调节的自杀基因HSVtk重组逆转录病毒，探讨其治疗乳腺癌的作用机制。方法：构建重组逆转录病毒RevTRE／HSVtk与RevTet—On感染乳腺癌细胞株MCF-7。Southern杂交检测细胞中HSVtk基因的整合。应用倒置荧光显微镜、流式细胞术、TUNEL、苔盼蓝排斥试验观察分析在Tet调控下，重组病毒对感染的乳腺癌细胞杀伤作用机制。以MCF-7细胞构建裸鼠乳腺癌模型。观察Tet调控下病毒对肿瘤细胞的杀伤作用．并对瘤体进行病理分析。结果：重组病毒感染乳腺癌细胞株MCF-7后，乳腺癌细胞随着DOX浓度的增高和GCV作用后，细胞死亡增加并呈现细胞凋亡等特征。乳腺癌裸鼠在DOX诱导7天GCV治疗30天后与对照组比较，在Tet调控下乳腺癌裸鼠肿瘤体积减小并可诱导乳腺癌细胞凋亡。结论：重组逆转录病毒在DOX诱导GCV作用下对病毒感染的MCF-7细胞具有诱导细胞凋亡的作用．能有效地杀伤肿瘤细胞。     

可调控自杀基因的SCID小鼠乳腺癌基因治疗的研究

目的：探讨经强力霉素（Dox)诱导后，丙氧鸟苷（GCV）对SCID小鼠乳腺癌的调控性治疗作用。方法：重组逆转录病毒载体pRevTRE/HSVtk与pRevTet-On共转染乳腺癌细胞MCF－7，接种SCID小鼠成瘤后，腹腔内分别注射生理盐水（NS），GCV及Dox GCV治疗15d。观测肿瘤体积和组织病理改变及用RT－PCR分析肿瘤组织有无HSVtk的表达。结果：乳腺癌SCID小鼠Dox GCV治疗15d后，肿瘤体积明显减小，生长受抑，组间比较有显著性差异（P＜0．05）；HE染色发现治疗组肿瘤有局部坏死，炎性细胞浸润。RT－PCR结果显示Dox诱导后肿瘤组织HSVtk表达较明显。结论：在Dox的诱导作用下，GCV对可调控性自杀基因乳腺癌SCID小鼠有显著治疗作用。     

Tet-On调节的自杀基因对人乳腺癌细胞的体外杀伤和旁观者效应研究

目的探讨Tet调控下的自杀基因HSVtk对人乳腺癌细胞的杀伤作用和旁观者效应。方法构建重组逆转录病毒载体pRevTRE／HSVtk，通过磷酸钙共沉淀法，分别将pRevTRE／HSVtk和pRevTet-On导入乳腺癌细胞株MCF-7。以RT—PCR法检测不同Dox浓度下转染乳腺癌细胞中HSVtk基因的表达，MTT法检测细胞生长抑制率，通过检测混合细胞生长克隆来评价Tet调控下的自杀基因的旁观者效应和对乳腺癌细胞的杀伤作用。结果建立了一株稳定的受多西环素（Doxcycline，Dox）诱导、表达HSVtk基因的乳腺癌细胞株MCF／TRE／tk／Tet—On。当Ganciclovir（GCV）及Dox浓度增加时，细胞存活率下降。MCF／TRE／tk／Tet—On在总细胞中的比例〉10％时，有明显的旁观者效应。结论在Tet—On调控下，导入乳腺癌细胞中的HSVtk基因产物能将药物前体GCV转变为毒性代谢物质，并诱发旁观者效应进一步杀灭乳腺癌细胞。     

黄荆子乙酸乙酯提取物对人乳腺癌T47D细胞及其裸鼠移植瘤生长抑制的影响

目的:研究黄荆子乙酸乙酯提取物(EVn-50)对人乳腺癌(T47D)细胞及其裸鼠移植瘤生长抑制的影响.方法:体外培养T47D细胞,溴化标记尿嘧啶(BrdU)掺入法测定细胞核酸合成的抑制作用;平皿克隆形成法测定细胞锚定依赖生长能力的抑制作用;人乳腺癌T47D裸鼠移植瘤模型治疗实验,观察EVn-50在体内对T47D细胞生长抑制作用,HE染色观察乳腺癌组织和细胞形态改变.结果:EVn-50抑制体外培养T47D细胞核酸合成和生长,呈剂量依赖性;EVn-50对裸鼠移植瘤的生长具有显著抑制作用,呈剂量和时间依赖性;EVn-50 80 mg/ kg实验组对移植瘤的瘤重抑制率为51.4% (P<0.001);镜下观察EVn-50 80 mg/ kg实验组肿瘤组织坏死组织较多,细胞异型性较少.结论:黄荆子乙酸乙酯提取物对人乳腺癌有生长抑制作用.     

肺腺癌组织特异性自杀基因治疗实验研究

目的 探讨肺腺癌组织特异性自杀基因治疗的安全性及有效性。方法 采用病毒感染法，将癌胚抗原(CEA)基因启动子所驱动的CD基因的组织特异性逆转录病毒载体(G1CEACDNa)，导入分泌CEA的肺腺癌细胞系A549细胞．研究裸鼠体内抑瘤效果；应用重组逆转录病毒裸鼠体内治疗A549肿瘤，观察G1CEACDNa／5-氟胞嘧啶(5-FC)对A549细胞致瘤裸鼠的治疗作用及毒副反应。结果 (1)将转基因的A549细胞和未转基因的A549细胞接种至裸鼠皮下．两者成瘤性无明显差异；(2)在转基因细胞致瘤裸鼠实验中，5-FC对转CEA启动子调控自杀基因的肿瘤生长具有明显的抑制作用；(3)将G1CEACDNa重组逆转录病毒上清直接注射到裸鼠成瘤部位．然后腹腔内注射5-FC同样获得明显的抑瘤效果；(4)与直接注射5-FU相比，组织特异性自杀基因治疗对骨髓的抑制明显降低。结论 组织特异性自杀基因治疗可能成为肿瘤治疗个体化的重要方法之一。     

逆转录病毒介导的胸苷激酶基因治疗难治性卵巢癌裸鼠模型的观察

目的:运用HSV-TK/GCV系统治疗卵巢癌裸鼠移植肿瘤模型,比较不同治疗方式的疗效.方法:常规培养包装细胞PA317,PA317细胞含有携带HSV-TK基因的逆转录病毒,测定病毒滴度.在移植瘤内多点注射PA317细胞以及浓缩病毒,现察GCV治疗后的肿瘤体积变化,对移植肿瘤进行组织病理分析,PCR检测TK基因在移植肿瘤组织的转导情况.比较两种不同治疗方式的疗效.结果:常规细胞培养上清重组逆转录病毒滴度为6×104 cfu/mL;注射PA317细胞后肿瘤生长受到抑制,P<0.05;注射浓缩病毒后肿瘤生长抑制不明显,P>0.05;PCR检测证实TK基因在肿瘤组织中的转导;病理检测见中、重度治疗反应.结论:HSV-TK/GCV系统对人难治性卵巢癌裸鼠移植肿瘤有治疗作用,不同治疗方式疗效不同.     

CXCR4单克隆抗体抑制乳腺癌MCF-7细胞裸鼠移植瘤的实验研究

目的 研究CXC趋化因子受体4(CXC chemokine receptor 4,CXCR4)单克隆抗体(CXCR4 mAb)对人乳腺癌MCF-7细胞裸鼠皮下移植瘤生长的影响,并初步探讨CXCR4 mAb抗肿瘤的作用机制.方法 采用8周龄Balb/c雌性裸鼠,建立乳腺癌MCF-7细胞裸鼠皮下移植瘤模型.运用CXCR4 mAb进行干预,从整体水平观察CXCR4 mAb对肿瘤生长的影响,采用免疫组织化学法检测肿瘤组织中增殖细胞核抗原(PCNA)、半胱氨酸天冬氨酸酶3(Caspase-3)和血管内皮细胞生长因子(VEGF)的表达情况.结果 CXCR4 mAb可明显抑制移植瘤的生长,瘤体抑制率达到71.4%;CXCR4 mAb治疗后的肿瘤组织中PCNA和VEGF表达明显下降,而Caspase-3表达上升.结论 CXCR4 mAb可能是通过抑制肿瘤细胞增殖、促进肿瘤细胞凋亡及抑制肿瘤血管形成而发挥抗肿瘤生长的作用.     

双效逆转录载体的构建和受控自杀基因在乳腺癌细胞中表达的研究

目的：构建含有Tet-On基因调节系统及自杀基因HSVtk的重组逆病毒载体，研究其在乳腺癌细胞中HSVtk基因的受控表达。方法：用PCR方法扩增Tet-On基因，将其插入pRevTRE／HSVtk，形成重组载体pRevTRE／HSVtk／Tet-On。用脂质体介导法将pRevTRE／HSVtk／Tet-On导入乳腺癌细胞株(MCF-7)。经过HygromycinB筛选建立了一株稳定的受四环素衍生物强力霉素(Doxcycline，Dox)调控表达HSVtk基因的乳腺癌细胞株MCF／TRE／HSVtk／Tet-On。用RT-PCR的方法检测不同Dox浓度下HSVtk基因的表达，以MTT法检测不同Dox浓度下Ganciclovir(GCV)对乳腺癌细胞的杀伤作用。结果：成功构建了pRevTRE／HSVtk／Tet-On逆病毒载体。筛选出MCF／TRE／HSVtk／Tet-On细胞株，该细胞能在Dox的诱导下表达HSVtk基因并被GCV杀伤。结论：HSVtk基因产物可以将无毒性的Ganciclovir(GCV)转变成一种有毒的代谢产物，杀死乳腺癌细胞。而该基因的表达受到Tet-On调控，由强力霉素调节HSVtk基因表达。     

靶向p65基因的miRNA对人三阴性乳腺癌细胞裸鼠移植瘤生长的抑制作用

背景与目的:三阴性乳腺癌(triple-negative breast cancer,TNBC)是乳腺癌中预后较差的一个亚型,如何防治TNBC的快速生长成为近几年临床研究的热点之一。NF-κB信号通路在肿瘤发生、发展的各个环节中扮演重要角色,有望成为肿瘤基因治疗新的方向。本研究通过建立人TNBC裸鼠移植瘤模型,观察靶向沉默NF-κB p65亚基的微小RNA(microRNA,miRNA)治疗对TNBC裸鼠移植瘤生长及凋亡的影响,并初步探讨其可能的作用机制。方法:建立人TNBC细胞株MDA-MB-231裸鼠移植瘤动物模型,瘤旁注射p65miRNA质粒(p65miRNA组),同时以注射Neg-miRNA质粒和PBS作为Neg-miRNA对照组和空白对照组。监测肿瘤生长变化,测量肿瘤质量。流式细胞术(flow cytometry,FCM)检测肿瘤细胞凋亡的变化。免疫组化法检测肿瘤组织中p65的表达。Western blot法检测肿瘤组织中Bcl-2和Bax蛋白的表达水平。结果:经p65miRNA处理后,裸鼠肿瘤的生长受到明显抑制。FCM结果表明,p65miRNA组肿瘤细胞凋亡率为(31.08±3.52)%,明显高于Neg-miRNA组(5.76±1.02)%和空白对照组(4.29±0.86)%(P<0.05)。此外,p65miRNA组裸鼠肿瘤组织p65和Bcl-2的蛋白表达明显下调,Bax的蛋白表达显著上调。结论:p65miRNA能抑制人TNBC裸鼠皮下移植瘤的生长,且在体内可诱导肿瘤细胞凋亡。     

乏氧射线双调控的TK腺病毒载体联合放疗抑制乳腺癌裸鼠移植瘤的生长

目的观察乏氧射线双调控的TK腺病毒载体-Ad.HRE.CArG.HSV-TK联合放疗对乳腺癌细胞Bcap37裸鼠移植瘤生长的影响。方法皮下接种细胞于BALB/C(nu/nu) 裸鼠建立移植瘤模型。当肿瘤直径达8~10mm时,将裸鼠分为对照组（control）、载体组(Ad)、照射组（RT）和载体合并照射组(Ad+RT)。治疗开始为第1天,于第1、5天瘤内多点注射0.2ml表达载体,给药次日起腹腔注射GCV(40mg/kg),每日1次,连续14天。第2、4、6天给予2Gy X线照射。每3天测量肿瘤长短径,计算肿瘤体积。治疗后30天处死裸鼠,剥离瘤块称重,计算抑瘤率。对肿瘤组织行HE染色和AnnexinV法检测凋亡。结果实验组平均肿瘤体积和瘤重均显著小于对照组（P<0.05）。HE染色可见肿瘤细胞变性坏死和凋亡细胞,AnnexinV检测显示实验组凋亡率明显高于对照组（P<0.05）。结论腺病毒载体联合放疗对Bcap37裸鼠移植瘤生长具显著抑制作用并可介导凋亡,为乳腺癌进一步的放射基因治疗研究奠定了基础。     

Retrovirus-mediated tk gene therapy of implanted human breast cancer in nude mice under the regulation of Tet-On

Tight regulation of the therapeutic gene expression is critical in gene therapy. In this report, a doxycycline (Dox)-regulated retrovirus-mediated gene expression system was used to study the effects of suicide gene therapy on human breast cancer cell line MCF-7 and the nude mice model of implanted human breast cancer. To render the expression of suicide gene under control, we used two pseudoviruses simultaneously, RevTRE/HSVtk and RevTet-On, to infect MCF-7 cells or xenografts of nude mice. When infected by the pseudoviruses and followed by Dox and Ganciclovir (GCV) treatment, MCF-7 cells were arrested at S phase and the growth was suppressed. We then evaluated the antitumor efficiency of this system in vivo through studying the mice bearing human breast cancer xenografts. Compared with control groups, the HSVtk mRNA level increased significantly in tumor tissues, mass of the tumors shrank remarkably, and tumor necrosis features occurred after treatment with Dox and GCV. These data suggest that suicide gene therapy using the Dox-induced Tet-On-controlled HSVtk gene expression system is a feasible method to treat human breast cancer.     

Enhanced efficacy of radiation-induced gene therapy in mice bearing lung adenocarcinoma xenografts using hypoxia responsive elements

The aim of the present study was to investigate whether the hypoxia responsive element (HRE) could be used to enhance suicide gene (HSV-tk) expression and tumoricidal activity in radiation-controlled gene therapy of human lung adenocarcinoma xenografts. A chimeric promoter, HRE-Egr, was generated by directly linking a 0.3-kb fragment of HRE to a 0.6-kb human Egr-1 promoter. Retroviral vectors containing luciferase or the HSV-tk gene driven by Egr-1 or HRE-Egr were constructed. A human adenocarcinoma cell line (A549) was stably transfected with the above vectors using the lipofectamine method. The sensitivity of transfected cells to prodrug ganciclovir (GCV) and cell survival rates were analyzed after exposure to a dose of 2 Gy radiation and hypoxia (1%). In vivo, tumor xenografts in BALB/c mice were transfected with the constructed retroviruses and irradiated to a total dose of 6 Gy, followed by GCV treatment (20 mg/kg for 14 days). When the HSV-tk gene controlled by the HRE-Egr promoter was introduced into A549 cells by a retroviral vector, the exposure to 1% O(2) and 2 Gy radiation induced significant enhancement of GCV cytotoxicity to the cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors infected with the hybrid promoter-containing virus gradually disappeared after GCV administration and radiation. These results indicate that HRE can enhance transgene expression and tumoricidal activity in HSV-tk gene therapy controlled by ionizing radiation in hypoxic human lung adenocarcinoma.     

Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine

Purpose The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro and in vivo.Methods Drug sensitivity was determined using the MTT assay. The in vivo effect of Tet was investigated using nude mice grafted with sensitive and resistant KB human epidermoid cancer cells. Doxorubicin (Dox) accumulation was analyzed by fluorospectrophotometry and the protein and mRNA levels of P-glycoprotein (P-gp) were determined by immunocytochemistry and RT-PCR, respectively.Results BrTet at 0.25, 0.5 and 1 M reversed Dox resistance in MDR human breast cancer MCF-7/Dox cells dose-dependently and its potency was greater than that of Tet at the same concentrations. BrTet reversed vincristine (VCR), Dox and paclitaxel resistance in MDR human oral epidermoid carcinoma KBv200 cells as well as innate VCR and Dox resistance in human hepatocellular carcinoma Bel₇₄₀₂ cells. However, BrTet showed no effect on the IC₅₀ values of the above-mentioned anticancer drugs in sensitive MCF-7 and KB cells. No reversal effect of BrTet on the cytotoxicity of 5-fluorouracil and cisplatin, non-P-gp substrates, was observed. In nude mice bearing KBv200 xenografts on the left flank and KB xenografts on the right flank, i.p. injection of 5 mg/kg and 10 mg/kg BrTet significantly enhanced the antitumor activity of Dox against KBv200 xenografts with inhibitory rates of 33.0% and 39.2%, while Dox alone inhibited the growth of KBv200 xenografts by only 11.6%. No enhancement by BrTet was seen in KB xenografts. Moreover, BrTet at 5 mg/kg reversed paclitaxel resistance in KBv200 xenografts. Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of Dox in MCF-7/Dox cells in a dose-dependent manner. BrTet also inhibited the overexpression of P-gp in MCF-7/Dox cells, but had no effect on mdr1 expression.Conclusions BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs. BrTet may be a promising MDR modulator for eventual assessment in the clinic.     

Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo.

To examine the immunological mechanisms involved in cancer gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV), murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, were transduced retrovirally with the HSV-tk gene. HSV-tk-transduced cells exhibited a more than 2,000-fold higher sensitivity to GCV compared with untransduced parental cells. When HSV-tk-transduced HCC cells were mixed with parental cells at a 50% ratio and implanted subcutaneously into immunocompetent syngeneic mice, complete inhibition of tumor formation was achieved by GCV treatment. Conversely, no significant inhibitory effects on tumor formation were observed in athymic nude mice. When established solid tumors in immunocompetent mice containing HSV-tk-transduced cells at an only 5% ratio were treated with GCV, marked infiltration by lymphocytes including CD4(+) and CD8(+) ones, and apoptotic death of tumor cells were induced, and significant reduction or even complete regression of tumors was achieved. Furthermore, such cured mice rejected rechallenge with parental HCC cells into the contraflank regions. Our results indicate that cancer gene therapy with the HSV-tk/GCV system can indeed induce efficient antitumor effects and protective immunity in immunocompetent mice but not in nude mice, indicating that T-cell-mediated immune responses may be a critical factor for achieving successful gene therapy against cancer using the HSV-tk/GCV system.     

利用荧光素酶标记的肿瘤细胞建立活体成像动物模型

目的建立稳定表达荧光素酶的人乳腺癌细胞株并构建适用于小动物活体成像系统观察的裸鼠皮下移植瘤模型。方法采用脂质体将携带荧光素酶基因的质粒转染到人乳腺癌细胞株MCF-7中,G418筛选出稳定表达荧光素酶的单克隆细胞株。扩增后接种于裸鼠,建立裸鼠皮下移植瘤模型,通过活体动物成像系统监测肿瘤的生长过程。结果获得了高水平稳定表达荧光素酶的乳腺癌单克隆细胞株,该单克隆细胞株与母细胞系MCF-7具有相似的生长特性。将稳定表达荧光素酶的克隆接种于裸鼠皮下可成瘤,小动物活体成像系统能准确监测肿瘤细胞在体内的生长过程。结论成功建立了稳定表达荧光素酶的乳腺癌单克隆细胞株。采用活体动物成像系统构建的裸鼠皮下移植瘤模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

缺氧/辐射双敏感性启动子调控HSV-TK表达对肺癌移植瘤的作用

背景与目的放射-基因治疗是近年来国际上肿瘤治疗的新策略,由于实体瘤常处于缺氧状态而对放射敏感性低,放射-基因治疗尚未达理想疗效。本研究拟构建缺氧/辐射双敏感性启动子,增强缺氧条件下放射诱导的HSV-TK表达水平,提高肺癌放射-基因治疗效果。方法利用基因重组构建HRE-Egr启动子及其调控的HSV-TK表达载体;脂质体介导重组质粒转染肺癌A549细胞,分为对照组、放射组(6Gy)、缺氧组(1%氧浓度)和放射合并缺氧组,Northernblot法检测转染细胞中HSV-TK表达,四甲基偶氮唑蓝(MTT)法检测放射、缺氧和GCV处理后的细胞存活率。建立BALB/c裸鼠肺癌移植瘤模型,检测放射合并质粒转染后移植瘤体积变化并计算抑瘤率。结果对照组细胞仅可检测到HyTK的低水平表达(21U),放射组和缺氧组HyTK基因表达水平均显著升高(分别为227U和94U),放射合并缺氧组(769U)显著高于放射组。在缺氧条件下,放射合并GCV处理后细胞存活率为(7.2±1.8)%,显著低于常氧组的(32.7±4.6)%。放射联合GCV可明显抑制HRE-Egr启动子转染肺癌移植瘤,抑瘤率达91.2%。结论HRE-Egr启动子具有辐射/缺氧双重敏感性,并使放射后的HSV-TK表达水平在缺氧下得到显著增强。放射联合HRE-Egr启动子可显著抑制肺癌移植瘤的生长。     

Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer

The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the anti-tumor activity, we probed into whether recombinant retroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The pL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.     

Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer

The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the antitumor activity, we probed into whether recombinant ritroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The PL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.