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1.
逆转录病毒介导单纯疱疹病毒-胸苷激酶基因治疗胰腺癌的实验研究 总被引:2,自引:0,他引:2
目的 探讨应用逆转录病毒载体介导单纯疮疹病毒-胸苷激酶(herpes simplex virus type Ⅰthymidine kinase gene,HSV-TK)基因治疗实验性人胰腺癌细胞系8988的价值。方法 HSV-TK被定向克隆入逆转录病毒载体pMNDM的DV40下游。重组逆转录病毒包装细胞PA317细胞,产生的重组病毒将HSV-TK转入人胰腺癌细胞系8988细胞内。结果 Sorthern blot试验及药敏试验均证实HSV-TK基因已整合至细 相似文献
2.
目的 建立HSV-TK逆转录病毒包装细胞系并获得高产病毒的细胞株.方法 将TK基因与逆转录表达载体PLEGFP-N1连接后转染到包装细胞PA317中,经G418及荧光蛋白双重筛选得到高产病毒的细胞株.结果 经酶切鉴定成功构建PIEGFP-N1-TK重组载体,含HSV-TK基因重组逆转录病毒包装细胞PA317成功建立,经病毒滴度测定获得高产病毒的PA317/TK细胞株.结论 制备的重组病毒具有感染靶细胞的活性,为进一步应用HSV-TK基因进行肺癌的自杀基因治疗研究奠定基础. 相似文献
3.
HSV-TK+GFP/GCV自杀基因系统对小鼠胰腺癌的治疗作用 总被引:1,自引:0,他引:1
目的: 探讨HSV-TK+GFP/GCV自杀基因系统对小鼠胰腺癌细胞系MPC体外及体内杀伤作用及其产生的旁观者效应.方法: 通过RT-PCR从基因组文库中扩增出HSV-TK基因全长CDS序列, 并将其与GFP基因定向克隆到质粒表达载体pcDNA3.1(+), 构建重组质粒pcDNA3.1+/HSV-TK+GFP. 脂质体法将重组质粒转染小鼠胰腺癌细胞株MPC细胞, 得到带有HSV-TK和GFP基因的MPC/HSV-TK+GFP细胞, 并将其分别用于体外和体结果: 重组质粒pcDNA3.1+/HSV-TK+GFP导入小鼠胰腺癌细胞株MPC细胞. 体外实验结果显示, 当MPC/HSV-TK+GFP细胞数占混合细胞10%时, 低浓度(20 mg/L)的GCV就可将50%左右的肿瘤细胞杀死. 体内实验结果显示GCV可明显抑制MPC /HSV-TK+GFP细胞在昆明小鼠体内的肿瘤形成.结论: HSV-TK和GFP基因转入小鼠胰腺癌细胞株MPC细胞并获得稳定表达, HSV-TK+GFP/GCV自杀基因系统在体内外对小鼠胰腺癌均有杀伤作用, 且存在明显的旁观者效应. 相似文献
4.
自杀基因表达产生旁观者效应的初步研究 总被引:1,自引:1,他引:1
目的:构建单纯疱疾病毒胸苷激酶基因(HSV-tk)的重组逆转录病毒载体,初步探讨转染有HSV-tk基因的人肝癌细胞产生旁观者效应的作用机制.方法:EcoRⅠ和PvuⅡ酶切质粒PHSV-106,回收1.9kb HSV-tk基因片段,将其定向克隆人逆转录病毒载体LXSN的EcoRⅠ/HpaⅠ酶切位点.经lipofectin将HSV-tK基因转染入肝癌细胞系,测定转染细胞对GCV或ACV的敏感性.并把转染细胞与非转染相应细胞按不同比例混合,分别在不同细胞密度及特定培养基条件下作用亲本细胞,观察细胞与细胞接触,不接触及GCV作用转染细胞其培养基与旁观者效应的关系.结果:经酶切鉴定,成功构建了含HSV-tk基因的重组逆转录病毒载体,命名为 PLXT;MTT法,~3H-TdR掺入法,流式细胞仪均表明GCV或ACV对SMMC-7721有显著杀伤作用,其作用在一定范围内与剂量和时间呈平行关系;细胞与细胞接触,不接触均有旁观者效应,但前者明显强于后者;GCV作用转染细胞后培养基对本细胞亦有弱的细胞毒作用.结论:阳离子脂质体是转基因的有效载体;经它介导的含有HSV-tk基因的重组逆转录病毒载体赋于人肝癌细胞对GCV或ACV的高敏感性;HSV-tk/GCV或ACV系统的旁观者效应可能是综合因素作用的结果,其中“缝隙连接”可能是主要作用因素. 相似文献
5.
目的构建了含人抑瘤素M(HOSM)基因的复制缺陷型重组腺病毒载体AD-HOSM,观察其对人胰腺癌细胞株PC3在体外生长抑制情况。方法通过同源重组方法构建含HOSM基因的缺陷型腺病毒载体AD-HOSM,报告基因AD-GFP检测腺病毒的对胰腺癌细胞系的转染效率;人胰腺癌细胞株PC3转染含HOSM基因的缺陷型腺病毒载体AD-HOSM后,用RT-PCR法检测外源HOSM基因在其中的表达;台盼兰染色、细胞计数法检测AD-HOSM对PC3的体外增殖抑制情况。结果成功构建了重组腺病毒载体AD-HOSM,AD-HOSM对胰腺癌细胞PC3有较高的转染效率,HOSM基因在转染细胞能有效的表达。体外试验示AD-HOSM明显抑制PC3的的生长。结论重组腺病毒介导HOSM表达能有效抑制PC3在体外的增殖,提示重组腺病毒介导的HOSM基因治疗可能成为胰腺癌治疗的候选方案。 相似文献
6.
血管抑制素基因治疗人胰腺癌的实验研究 总被引:3,自引:0,他引:3
目的;探讨应用脂质体介导血管抑制素(angiostatin,AG) 基因治疗实验性人胰腺癌细胞系SW1990的价值。方法:血管抑制素AG基因被定向克隆入真核表达载体pRC/CMV中。运用脂质体将重组体pRC/CMV-AG转入胰腺癌细胞系SW1990中,进行抗肿瘤研究。结果:构建的真核表达载体pRC/CMV-AG经酶切证实正确。Western blot和药敏试验均证实血管抑制素基因已整合到靶细胞DNA中并可分泌表达AG,且可抑制血管内皮细胞的生长。动物模型显示,所构建的载体能在肿瘤内表达,且可有效抑制荷瘤裸鼠人胰腺癌细胞的微血管形成及肿瘤生长。结论:脂质体介导的重组体pRC/CMV-AG有体内治疗胰腺癌的作用,可作为胰腺癌基因治疗的可能方法之一。 相似文献
7.
腺病毒介导CD基因对人胰腺癌细胞株的体外杀伤作用 总被引:4,自引:0,他引:4
目的 探讨腺病毒介导胞嘧啶脱氨酶基因 (CD)对人胰腺癌细胞株体外杀伤作用。方法 构建含CD基因的腺病毒穿梭载体 pAdTrack CMV CD与骨架载体 pAdEasy 1,在细菌内重组为pAd CD ,经 2 93细胞包装、扩增 ,氯化铯密度梯度离心制备纯化高效的含绿色荧光蛋白 (GFP)的CD腺病毒 ,体外转染人胰腺癌细胞株Patu 8988、SW 1990 ,并给予前药 5 氟胞嘧啶 (5 FC) ,观察其体外杀伤效果。结果 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× 10 11PFU/ml,将重组腺病毒转染胰腺癌细胞株Patu 8988、SW 1990后 ,可见 5 FC对转染CD基因的Patu 8988、SW 1990细胞及混育细胞有明显毒性作用 ,而对未导入CD基因的人胰腺癌细胞毒性较低。结论 体外腺病毒介导CD基因 ,不仅转染效果强 ,而且可直接或通过旁观者效应以杀伤胰腺癌细胞 ,可作为胰腺癌基因治疗的有效方法 相似文献
8.
目的 探讨以Survivin为靶基因的胰腺癌基因治疗的可能性,为胰腺癌基因治疗提供依据.方法 采用化学合成的小十扰RNA(siRNA)和pGCSi载体中的小发夹RNA(shRNA)抑制胰腺癌细胞系PaTu8988的Sunrivin基因表达,通过观察胰腺癌细胞株Survivin基因表达的下调以及细胞形态、细胞凋亡、细胞活力、凋亡信号途径等的改变评价Survivin作为靶基因的治疗效果.结果 不同序列的siRNA和shRNA抑制胰腺癌细胞Survivin的表达后,Survivin mRNA和蛋白表达水平明显下降(P<0.05);碘化吡啶(PI)染色法观察发现RNA干扰(RNAi)后细胞出现核皱缩、细胞凋亡率>20%;流式细胞仪检测发现RNAi后在G0/G1期前出现了亚二倍体峰(P<0.05);Western blot法检测发现RNAi后Casptrqe-3被激活(P<0.05).结论 通过抑制胰腺癌细胞PaTu8988的Survivin表达可以诱导肿瘤细胞启动凋亡程序,加速肿瘤细胞凋亡,由此可望提高胰腺癌的治疗效果. 相似文献
9.
人Tum-5基因逆转录病毒载体及包装细胞株的构建 总被引:1,自引:0,他引:1
目的 构建携带Tum-5基因的逆转录病毒载体并对其进行包装,获得稳定的产毒细胞系.方法 采用RT-PCR法从胎肾组织中扩增Tum-5基因片断,并将其定向克隆到逆转录病毒载体pLXSN中进行PCR、双酶切和测序鉴定.利用电穿孔方法 将获得的重组质粒转染PA317细胞,经G418 筛选抗性克隆,收集病毒上清后感染NIH3T3 细胞测定病毒滴度.结果 PCR、酶切证实Tum-5基因克隆至逆转录病毒载体pLXSN,Tum-5基因测序结果 和原始序列相同.重组逆转录病毒载体转染PA317 包装细胞,RT-PCR证实转染后的PA317细胞上清液中存在携带人Tum-5基因的病毒RNA,病毒滴度为2.05×104cfu/ml.结论 成功的构建了携带人Tum-5基因的逆转录病毒载体,获得了稳定的产毒细胞系. 相似文献
10.
血管内皮生长因子反义核酸治疗裸鼠皮下种植胰腺癌 总被引:3,自引:2,他引:1
目的:探讨抗血管生成基因转染治疗胰腺癌的可行性. 方法:构建反向插入VEGF165cDNA的复制缺陷型腺病毒载体,体外转染SW1990细胞,MTT法检测重组腺病毒转染对细胞生长的影响.Northern blot和ELISA检测转染前后SW1990细胞VEGF的mRNA水平和蛋白表达.裸鼠皮下种植瘤瘤体内注射重组腺病毒,CD31染色观察反义VEGF重组腺病毒转染对裸鼠种植瘤微血管密度和肿瘤生长速度的影响. 结果:重组腺病毒体外转染并不影响SW1990细胞的生长速度.Northern blot和ELISA检测在mRNA水平和蛋白水平证实反义VEGF重组腺病毒转染对体外培养的SW1990细胞内源性VEGF表达有明显的下调作用.体内实验表明反义VEGF重组腺病毒转染可减少肿瘤内微血管数量,肿瘤生长受到抑制. 结论:反义VEGF165重组腺病毒可以抑制胰腺癌的血管生成和肿瘤的生长,为抗血管生成的基因治疗奠定了基础. 相似文献
11.
OBJECTIVE : To study the value of the herpes simplex virus type I thymidine kinase (HSV‐TK) gene mediated by a retroviral vector in the treatment of human pancreatic cancer cell line 8988. METHODS : The HSV‐TK gene was directionally cloned into a site following the SV40 promoter that was adjacent to the 5′ LTR (long terminal repeats) and the neomycin phosphotransferase gene in the retroviral vector pMNSM. This enabled the TK gene to integrate into the chromatin of the host cell. The recombinant plasmid pMNS‐TK‐M was then transfected into the retrovirus‐packaging cell Pa317. Finally, the HSV‐TK gene was transfected into pancreatic cancer cell line 8988 by the recombinant retrovirus after selection with G418. RESULTS : The HSV‐TK gene was stably integrated into the host cell and its expression confirmed by Southern blotting and drug‐sensitivity tests. In vitro studies showed that the acyclovir (ACV) sensitivity level in the 8988/TK+ cells was higher than that of the parent cells. The cells that were transfected with the TK gene were significantly susceptible to ACV. In vivo studies in nude mice showed that intraperitoneal injection of ACV might postpone the formation of implanted tumors and produce a treatment effect on the tumors. CONCLUSIONS : This study demonstrated that the HSV‐TK/ACV retroviral system could be used in vivo to treat pancreatic cancer. 相似文献
12.
Herpes simplex virus thymidine kinase and ganciclovir suicide gene therapy for human pancreatic cancer 总被引:9,自引:1,他引:9
AIM: To investigate the in vitro effects of suicide gene therapy system of herpes simplex virus thymidine kinase gene (HSV-TK) in combination with the treatment of nucleotide analog-ganciclovir (GCV) on human pancreatic cancer, and to provide a novel clinical therapeutic method for human pancreatic cancer. METHODS: We used a replication defective recombinant retrovirus vector GINaTK (bearing HSV-TK gene) to make packaging cell PA317 produce progeny virions. We then transferred the HSV-TK gene to target cells SW1990 using these progeny virions, and treated these gene-modified tumor cells with GCV to study the sensitivity of the cells to GCV and their bystander effects by routine MTT-method. RESULTS: Packaging cell PA317/TK was successfully constructed, and we acquired SW1990/TK through virus progeny infection. These gene-modified pancreatic cancer cells were sensitive to the treatment of GCV compared with unmodified tumor cells (t=4.15, n=10, P<0.0025). We also observed a remarkable bystander effect by mixing two kinds of cells at different ratio. CONCLUSION: Our data demonstrate that HSV-TK/GCV suicide gene therapy system is effective for treating experimental human pancreatic cancer, which is largely resistant to the common therapies, so the suicide gene therapy system may be a potential treatment approach for pancreatic cancer. 相似文献
13.
《International Hepatology Communications》1993,1(5):253-259
We previously demonstrated that a foreign gene transferred by means of a retroviral vector can be expressed selectively in hepatoma cells when a liver-specific promoter was used to direct its expression. We now describe an approach for the treatment of hepatoma by the introduction of herpes simplex virus thymidine kinase (HSV-TK) gene. Expression of HSV TK gene in hepatoma cells was evaluated as an elimination system for a potential use in therapies. A murine retroviral vector was constructed in which the HSV-TK gene was expressed under control of the murine albumin enhancer and promoter elements. Replication-defective vector viral particles were obtained by transfer of the vector DNA into the ecotropic packaging cell line psi2 and were used to infect murine hepatoma cells. The introduction of the HSV-TK gene into hepatoma cells by infection of the recombinant retrovirus did not affect their proliferation at all. The sensitivity of those infected cells to the toxic effects of the nucleoside analog ganciclovir was found to be significantly increased by transfer of the HSV-TK gene. The difference in sensitivity between infected and uninfected cells to ganciclovir concentrations should give the utility for a clinical application indicating the feasibility of gene therapy toward hepatoma by the retroviral-mediated HSV-TK gene transfer. 相似文献
14.
AIM: Cancer gene therapy has received more and more attentions in the recent decade. Various systems of gene therapy for cancer have been developed. One of the most promising choices is the suicide gene. The product of thymidine kinase (TK) gene can convert ganciclovir (GCV) to phosphorylated GCV, which inhibits the synthesis of cell DNA, and then induces the cells to death. Cytokines play an important role in anti-tumor immunity. This experiment was designed to combine the TK gene and mIL-2/mGM-CSF genes to treat gastric cancer, and was expected to produce a marked anti-tumor effect. METHODS: TK gene was constructed into the retroviral vector pLxSN, and the mIL-2 and mGM-CSF genes were inserted into the eukaryotic expressing vector pIRES. The gastric cancer cells were transfected by retroviral serum that was harvested from the package cells. 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 experiment, retroviral serum and cytokines plasmid were transfected into tumor-bearing mice, to observe the changes of tumor volumes and survival of the mice. RESULTS: In vitro experiment, 20 % TK gene transduced cells could cause 70-80 % of total cells to death. In vivo results showed that there was no treatment effect in control group and TK/GCV could inhibit the tumor growth. The strongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis of the cancer in the treated groups. CONCLUSION: TK/GCV can kill tumor cells and inhibit the tumor growth in vivo. IL-2 and GM-CSF strongly enhance the anti-tumor effect. Through the retrovirus and liposome methods, the suicide gene and cytokine genes are all expressed in the tissues. 相似文献
15.
Gene therapy of thyroid cancer via retrovirally-driven combined expression of human interleukin-2 and herpes simplex virus thymidine kinase 总被引:10,自引:0,他引:10
Barzon L Bonaguro R Castagliuolo I Chilosi M Franchin E Del Vecchio C Giaretta I Boscaro M Palù G 《European journal of endocrinology / European Federation of Endocrine Societies》2003,148(1):73-80
OBJECTIVE AND DESIGN: Based on our clinical experience with combined gene therapy of glioblastoma, we developed a retroviral vector expressing two therapeutic genes (i.e. thymidine kinase of herpes simplex virus, HSV-TK, and interleukin-2, IL-2) and evaluated its efficiency in vitro and in vivo. METHODS: Expression of therapeutic genes in transduced thyroid carcinoma cells was analyzed by real-time RT-PCR. Ganciclovir sensitivity of infected cells was assessed in vitro in thyroid carcinoma cell lines and in vivo in nude mice bearing xenografted thyroid cancers. The combined effect of IL-2/HSV-TK was compared with the effect of IL-2 alone. RESULTS: Expression of therapeutic genes was higher in differentiated than in anaplastic thyroid carcinoma cells. Ganciclovir treatment led to dose- and time-dependent killing of transduced cells in vitro. A bystander effect was demonstrated by using mixtures of infected and non-infected cells. In vivo studies showed a significant reduction of growth and the presence of an inflammatory infiltrate in transduced thyroid tumors expressing IL-2 alone, as compared with non-infected tumors. By using the retroviral vector expressing IL-2/HSV-TK, treatment with ganciclovir led to complete eradication of anaplastic tumors and a >80% reduction of the size of differentiated thyroid carcinomas. Histological analysis of tumor specimens showed extensive necrosis and inflammatory cell infiltrates. The combination of IL-2/HSV-TK plus ganciclovir was significantly more efficient than IL-2 alone in eradicating tumor masses. The bystander effect was also obtained in vivo. CONCLUSIONS: These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas. 相似文献
16.
目的构建以人midkine(MK)启动子调控的TK基因的重组复制缺陷型腺病毒。方法以Adeno-XTM表达试剂盒为基础,应用分子克隆技术,将穿梭质粒pShuttle的CMV启动子替换为MK启动子,并将由pHSV-106获取HSV-TK基因的编码序列亚克隆至其下游,酶切鉴定阳性重组穿梭质粒pShuttle-MK-TK。通过I-CeuⅠ和PI-SceⅠ两个稀有酶切位点.将目的基因与腺病毒质粒DNA(pAdeno-X)进行体外连接,获得含目的基因的重组腺病毒质粒DNA,后者经限制性内切酶PacⅠ切割后,两端露出反向末端重复序列(ITR),利用脂质体转染293细胞,获得含有目的基因重组腺病毒上清,PCR检测。结果酶切结果显示.MK启动子与TK基因均正向插入pShuttle中.TK位于MK启动子的下游。PCR检测显示重组腺病毒中含有MK启动子及TK基因片段。结论体外连接法可成功构建以人MK启动子调控的TK基因的重组腺病毒。 相似文献
17.
《International Hepatology Communications》1995,3(4):223-231
A Moloney murine leukemia virus-derived retroviral vector carrying the herpes simplex virus (HSV) thymidine kinase (TK) gene was constructed and subsequently transfected into Ψ2 packaging cells, with resultant retrovims being transduced into XC rat hepatoma cells. Using HSV-TK gene-transduced XC (XCtkn) cells, we studied the effects of treatment with GCV, as well as with GCV and anticancer drugs or chemical modulators of nuclear proteins. GCV treatment was also examined in co-cultures of XCtkn cells and genetically unmodified XC, PLC/PRF/5 and Huh1 hepatoma cells, respectively. The growth of HSV-TK gene-transduced XC (XCtkn) cells either in vivo or in vitro was suppressed by treating with ganciclovir (GCV). In addition, the proliferation of genetically unmodified XC, PLC/PRF/5 and Huhl hepatoma cells was inhibited on co-culturing in vitro with the XCtkn cells and GCV, indicating that bystander effect operated in the present experimental system. Anticancer drugs and chemical modulators of nuclear proteins acted additively with GCV in inhibiting the proliferation of XCtkn cells. These suggest that the use of HSV-TK/GCV system in combination with other drugs may be a promising therapy for hepatocellular carcinoma. 相似文献
18.
AIM: To evaluate the synergistic antitumor effects of herpes simplex virus thymidine kinase (HSV-TK) together with tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2) gene expression on gastric cancer cell line SGC7901. METHODS: Recombinant vectors pL(TT)SN and pL(TI)SN,which express TK-IRES-TNF-α and TK-IRES-IL-2 genes separately, as well as the control plasmids pL(TK)SN and pLXSN were employed to transfect PA317 cells respectively to generate the viruses that can stably express the objective genes through G418 selection. The gastric cancer cells were then transfected by the retroviral serum from the package cells and maintained in culture to determine the cell growth and apoptosis. The cytotoxic effects of HSV-TK together with TNF-α or IL-2 gene expression on the transfected cancer cells were evaluated by the cell viability and bystander effects in the presence of GCV supplemented in the cultural medium. RESULTS: Expression of recombinant proteins including TNF-α and IL-2 by stable transfectants was confirmed by Western blotting. The percentage of cell apoptosis in the SGC/0, SGC/TK-TNF-α SGC/TK-IL-2 and SGC/TK done was 2.3%, 12.3%, 11.1% and 10.9% respectively at 24 h posttransfection. Cell growth status among all the experimental groups as judged by cell absorbance (A) at 570nm did not exhibit any significant difference (P>0.05); although it was noted to be slightly lower in the SGC/TT group. Cell survival rate in SGC/TI, SGC/TT and SGC/TK group was significantly decreased in a dose-dependent manner of GCV compared with that of the SGC/0 group (P<0.05-0.01). Among all studied cells, the SGC/TT was shown most sensitive to GCV with a half lethal dose of 0.5 mg.L^-1. In contrast, the survival rate of SGC/0 cells was not affected by the presence of GCV with the doses less than 10 mg-L^-1 The half lethal dose of GCV for SGC/0 cells was more than 100 mg-L^-1. Marked bystander effect induced by SGC/TI, SGC/TT and SGC/TK cells was confirmed by the fact that 20% of these stable transfectants could kill 50% of the co-cultured cells, in which the most prominent bystander effect was found in the circumstance of SGC/TT presence. However, no significant difference of these variables was found among SGC/TI,SGC/TT and SGC/TK cells (P>0.05). CONCLUSION: The synergistic antitumor effects produced by the co-expression of HSV-TK with TNF-α or IL-2 geneswere not present in the transfected SGC7901 cells. The mechanism underlying these phenomena was not known. 相似文献
19.
Yong-Mei Shen Xiao-Chun Yang Miao-Li Song Chen-Hao Qin Chen Yang Yi-Hui Sun Radioimmunoassay Center & Clinical Laboratory 《Hepatobiliary & Pancreatic Diseases International》2010,(1)
BACKGROUND:Survivin is known to be overexpressed in various human malignancies,including pancreatic cancer,and mediates cancer cell proliferation and tumor growth,so the regulation of this molecule could be a new strategy for treating pancreatic cancer.In this study,short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo.METHODS:Three kinds of shRNA sp... 相似文献