双启动子调控的条件复制腺病毒制备及其体外抑瘤作用

目的：构建并制备survivin及hTERT双启动子调控的条件复制腺病毒,探讨其特异性溶瘤作用。方法：PCR方法分别扩增肿瘤特异性survivin及hTERT启动子,分别克隆入腺病毒载体pXCl的两个复制必需基因E1A和E1B序列上游启动子区,构建出双肿瘤特异性启动子调控的条件复制腺病毒载体pXCl-SP—TP;脂质体法与pBHGE3骨架质粒共转染293E细胞进行重组腺病毒包装,稀释法测定腺病毒滴度;应用MTT、活细胞计数等方法观察其对肝癌细胞HepG2的特异性溶瘤作用并以正常人的血管内皮细胞ECV304作为对照。结果：测序及双酶切鉴定结果证实,成功构建了双肿瘤特异性启动子调控复制腺病毒载体;在293E细胞中获得了重组腺病毒Ad—sP—TP,滴度测定显示病毒滴度达到3．9×10^10TCID50／ml;MTT结果显示,Ad—sP—TP可有效抑制肝癌细胞增殖而对正常细胞无增殖抑制作用;活细胞计数及细胞形态观察结果显示,重组腺病毒在肝癌细胞中选择性复制并发挥溶细胞作用。结论：双启动子调控的腺病毒具有显著的溶瘤作用但对正常人血管内皮细胞不发挥溶细胞作用,实验结果为肝癌靶向治疗提供了更为良好的条件复制型病毒载体及新的治疗策略。     

双启动子调控的条件复制腺病毒制备及其体外抑瘤作用

目的:构建并制备survivin及hTERT双启动子调控的条件复制腺病毒,探讨其特异性溶瘤作用.方法:PCR方法分别扩增肿瘤特异性survivin及hTERT启动子,分别克隆入腺病毒载体pXC1的两个复制必需基因E1A和E1B序列上游启动子区,构建出双肿瘤特异性启动子调控的条件复制腺病毒载体pXC1-SP-TP;脂质体法与pBHGE3骨架质粒共转染293E细胞进行重组腺病毒包装,稀释法测定腺病毒滴度;应用MTT、活细胞计数等方法观察其对肝癌细胞HepG2的特异性溶瘤作用并以正常人的血管内皮细胞ECV304作为对照.结果:测序及双酶切鉴定结果证实,成功构建了双肿瘤特异性启动子调控复制腺病毒载体;在293E细胞中获得了重组腺病毒Ad-SP-TP,滴度测定显示病毒滴度达到3.9×1010TCID50/ml;MTT结果显示,Ad-SP-TP可有效抑制肝癌细胞增殖而对正常细胞无增殖抑制作用;活细胞计数及细胞形态观察结果显示,重组腺病毒在肝癌细胞中选择性复制并发挥溶细胞作用.结论:双启动子调控的腺病毒具有显著的溶瘤作用但对正常人血管内皮细胞不发挥溶细胞作用,实验结果为肝癌靶向治疗提供了更为良好的条件复制型病毒载体及新的治疗策略.     

重组腺病毒Ad—hTERTp-HSV—TK／GCV系统的构建及其对人肝癌细胞HepG2的杀伤作用及旁观者效应研究

背景与目的:肝癌的基因治疗已成为肝癌治疗的重要发展方向,本文探讨Ad-hTERTP-HSV-TK/GCV系统对人肝癌细胞株HepG2的杀伤作用;并观察其过程中旁观者效应的作用。方法:用重组腺病毒Ad-hTERTP-HSV-TK系统分别感染肝癌细胞株HepG2和正常肝细胞L-02,加入前体药物更昔洛韦(GCV),四甲基偶氮唑盐比色法检测其对感染后细胞的杀伤作用;将感染后的TK阳性HepG2细胞和未感染的TK阴性HepG2细胞按不同比例浓度(0∶10、1∶9、2∶8、3∶7、4∶6、5∶5、6∶4、7∶3、8∶2、9∶1、10∶0)混合,观察其旁观者效应。结果:肝癌细胞HepG2的存活率随病毒感染复数(MOI)和GCV浓度增加而逐渐降低,L-02细胞无明显变化。旁观者效应表明,在TK阳性HepG2细胞为10%时,细胞抑制率达28.06%,70%时达90.44%。结论:重组腺病Ad-hTERTP-HSV-tk/GCV系统对肝癌细胞HepG2有选择性杀伤作用及明显的旁观者效应。     

蜂毒素基因重组腺病毒对肝癌细胞的杀伤作用

目的：构建携蜂毒素基因及甲胎蛋白（AFP）启动子（rAFP)的重组腺病毒载体，探讨rAFP驱动的蜂毒素基因在体外对肝癌细胞的特异性杀伤作用。方法：人工合成蜂毒素基因，置于rAFP之后，用细菌内高效同源重组法将目的基因重组入腺病毒质粒中，转染人胚肾293细胞进行腺病毒包装；采用MTT法测定蜂毒素基因转染对AFP阳性，阴性肝癌细胞及正常肝细胞增殖的影响。结果：携蜂毒素基因重组腺病毒载体构建成功，MTT实验证明携蜂毒素基因重组腺病毒转染后，AFP阳性肝癌细胞的增殖受到明显抑制，而对AFP阴性肝癌细胞及正常肝细胞无明显影响；无蜂毒素基因的重组腺病毒转染，则对各种细胞增殖均无抑制作用。结论：表达rAFP驱动的蜂毒素基因的重组腺病毒在体外可特异性地杀伤AFP阳性肝癌细胞。     

重组腺病毒介导反义c-myc基因对人肝癌细胞系的治疗作用

目的：探讨重组腺病毒介导反义ｃ－ｍｙｃ基因（Ａｄ－ＡＳｍｙｃ）治疗人肝癌细胞的作用。方法：观察Ａｄ－ＡＳｍｙｃ对人肝癌细胞系的转导效率,通过细胞生长曲线、克隆形成实验、ＤＮＡ片段化分析、ＲＴ－ＰＣＲ、裸鼠皮下移植瘤治疗实验,分析Ａｄ－ＡＳｍｙｃ对人肝癌细胞系Ｂｅｌ－７４０２、ＱＳＧ－７７０１、ＳＭＭＣ－７７２１和ＨＣＣ－９２０４细胞生长和ｃ－ｍｙｃ基因表达及裸鼠肿瘤生长的抑制作用。结果：Ａｄ－ＡＳｍｙｃ可高效转导人肝癌细胞系,抑制细胞生长;转染细胞克隆形成能力降低,克隆成活率为对照组的５３．９％～６９．１％,ｃ－ｍｙｃ基因表达下降;Ａｄ－ＡＳｍｙｃ处理肝癌细胞,ＤＮＡ凝胶电泳出现明显的梯形条带,瘤内注射Ａｄ－ＡＳｍｙｃ可抑制裸鼠皮下移植瘤生长。结论：重组腺病毒介导的反义ｃ－ｍｙｃ基因转移,有可能成为肝癌基因治疗     

NT4p53(N15)Ant融合基因重组腺病毒的构建及其体外抑瘤作用

背景与目的:有研究发现P53氨基端15肽与穿膜肽(antennapedia,Ant)的融合肽能迅速穿透细胞膜,直接、快速引起乳腺癌、胰腺癌细胞坏死而非凋亡,但对正常细胞几乎没有毒性。本研究构建缺陷型腺病毒载体表达NT4p53(N15)Ant融合基因,研究其体外抑瘤作用。方法:应用Ad-EasyTM系统,在大肠杆菌同源重组,构建NT4p53(N15)Ant腺病毒表达载体,在HEK-293细胞内成功包装并鉴定后,感染肝癌细胞株HepG2,用倒置相差显微镜、透射电镜观察细胞形态学变化、四甲基偶氮唑蓝(MTT)比色实验及培养基中的乳酸脱氢酶(lactatedehydrogenase,LDH)测定研究其体外抑瘤作用。结果:Ad-NT4p53(N15)Ant对肝癌细胞株HepG2有明显抑制作用,48、72、96h细胞存活率分别为36.67%、20.47%、17.82%,与空病毒处理组比较差异有统计学意义(P<0.05);电镜观察到感染的HepG2细胞胞膜、核膜破坏,胞浆内出现囊泡状物质,染色质聚集。Ad-NT4p53(N15)Ant处理HepG2细胞24、48、72、96h时培养液中LDH释放分别为94、236、267、313U/L,较空病毒处理组明显增加,其差异有统计学意义(P<0.05)。结论:构建的腺病毒载体Ad-NT4p53(N15)Ant感染HepG2细胞后能抑制细胞增殖。     

TIP30基因腺病毒载体的构建及体内外抑癌作用

目的 利用缺陷型腺病毒载体表达TIP30基因,观察其体内外抑瘤作用。方法 用Ad-Easy^TM系统,在大肠杆菌同源重组,构建Ad-TIP30腺病毒载体,经在293细胞内成功包装并鉴定后,感染p53基因型不同的肝癌细胞株HepG2(p53-wt)、PLC／PRL／5(p53-mut)和骨肉瘤细胞株Saos-2(p53-null)。用台盼蓝拒染法检测存活细胞,观察TIP30的体外抑瘤作用;用RT-PCR方法检测HepG2细胞p53基因的表达水平;通过裸鼠皮下肝癌移植瘤模型观察Ad-TIP30的体内抑瘤效果。结果 Ad-TIP30对3种肿瘤细胞的增殖均有抑制作用,但对p53基因野生型的肝癌细胞株HepG2抑制作用最明显。经Ad-TIP30感染后,HepG2细胞p53基因表达水平显著升高。Ad-TIP30可显著抑制裸鼠皮下肿瘤的生长,其抑瘤率达62．8％。结论 腺病毒载体表达TIP30基因可通过p53基因依赖性或非依赖性途径抑制肿瘤细胞的增殖,是一种潜在的肿瘤生物治疗手段。     

溶瘤腺病毒靶向治疗恶性肿瘤研究进展

恶性肿瘤是当今世界严重威胁人类健康的疾病,也是人类生命的主要“杀手”之一。传统的放、化疗及手术治疗存在抗肿瘤能力低、杀伤指数过小、副作用大极易复发等缺点,对很多中晚期肿瘤患者往往缺少合适的治疗手段,因此,开发安全高效的生物治疗方法已成为肿瘤治疗领域关注的热点。基因-病毒治疗即是其中之一,它是将基因通过载体转入患者体内,从而达到治疗和预防肿瘤的目的。溶瘤腺病毒作为基因治疗的一种常用载体,在肿瘤治疗方面有着自生的优点和长处。本文简单对这一领域的研究进展进行综述。     

肝癌靶向性腺病毒载体的构建及其作用的研究

背景与目的:抑癌基因p16是肿瘤基因治疗最常用的治疗基因之一,通过腺病毒载体介导p16基因表达引发肿瘤细胞凋亡在多种p16基因失活的肿瘤细胞系中都得到了证实。研究表明,p16在人原发性肝细胞癌中存在高频率失活。因此,本研究通过观察外源性p16基因经AFP启动子驱动表达后对AFP阳性肝癌细胞的影响,以探讨p16基因在肝癌靶向基因治疗中应用的可行性。方法:将外源性p16基因置于人AFP核心启动子AF0.3下游,构建了携带p16基因的重组复制缺陷型腺病毒Ad-AFP-p16,感染肝癌细胞后,应用MTT比色法、Westernblot、DNA片段化分析、流式细胞术分析等方法在体外研究外源性p16基因表达对细胞生长的影响。小鼠皮下接种感染重组腺病毒的鼠肝癌细胞H22,观察病毒对肝癌细胞体内成瘤性的影响。结果:感染Ad-AFP-p16的肝癌细胞高效表达P16蛋白,MTT结果显示细胞生长受到明显抑制,到第六天Ad-AFP-p16对肝癌细胞Bel-7402和HepG2的生长抑制率分别为(94.42±11.70)%和(94.99±6.74)%,DNA片段化分析和流式细胞术分析证实p16能诱导肝癌细胞发生显著凋亡,感染后48hBel-7402和HepG2的凋亡率分别为39.57%和39.75%。感染Ad-AFP-p16的肝癌细胞体内成瘤受到明显抑制,对照组、Ad-GFP组、Ad-AFP-p16组以及Ad-CMV-p16组瘤体体积分别为(1.54±0.65)cm3、(1.71±1.01)cm3、(0.25±0.39)cm3和(0.25±0.45)cm3。结论:AFP启动子驱动p16基因表达可以诱导肝癌细胞发生凋亡。     

溶瘤腺病毒的研究进展

 引言受某些肿瘤患者在感染病毒后出现自发性肿瘤缓解这一现象的启发,早在上个世纪初便有溶瘤病毒治疗的研究[1] 。腺病毒发现后不久也曾被用来治疗头颈部恶性肿瘤,注射腺病毒后肿瘤有不同程度缩小,但治疗后肿瘤易复发,效果难以持久;直到1996年,Bischoff等[2 ] 首次报道去除部分E1B的重组腺病毒Onyx 0 15能在p5 3异常的肿瘤细胞选择性复制引起肿瘤杀伤作用,溶瘤腺病毒研究才再次受到广泛关注并且发展迅速,出现了许多新型溶瘤腺病毒种类,使溶瘤腺病毒成为恶性肿瘤一种新的治疗平台。1　溶瘤腺病毒的作用机理[1,3- 5]许多种DNA病毒,如腺病毒、SV4 0、HPV 16等都可以改变宿主细胞细胞周期,其原因主要是病毒产生一些蛋白作用于宿主细胞细胞周期调节蛋白,使静止期细胞进入细胞周期以利于病毒DNA的复制。不同病毒影响细胞周期的机制不一,腺病毒主要通过Rb和p5 3细胞信号通路干扰宿主细胞细胞周期。1.1　Rb和p5 3细胞信号通路(见图1) :Rb(眼癌蛋白,retinoblastomaprotein)的作用是与E2F(使细胞从G1期进入S期的一种转录因子)结合,细胞内游离E2F水平下降,细胞由...     

转录hTERT基因shRNA的溶瘤腺病毒的构建及抗肿瘤效应

目的构建转录端粒酶逆转录酶(hTERT)基因小发夹RNA(hTERT-shRNA)的条件增殖腺病毒。方法设计能转录hTERT-shRNA的模板DNA序列,煺火后克隆至pCA13质粒,构建重组质粒pCA13-hTERT。用Bg1Ⅱ从pCA13-hTERT酶切出包含CMV启动子及hTERT-shRNA模板的表达框,将表达框克隆入条件增殖腺病毒质粒pZD55,构建重组质粒pZD55-hTERT。将pZD55-hTERT与腺病毒右臂质粒pBHGE3共转染293细胞,9~12天后出现病毒空斑。提取重组腺病毒的DNA,PCR鉴定正确者即为条件增殖腺病毒ZD55-hTERT。大量扩增,氯化铯梯度离心纯化,测滴度。感染人肝癌细胞株(BEL-7404),通过荧光显微镜、结晶紫染色法观察细胞病作用,MTT法检测细胞存活情况。结果酶切分析、测序鉴定表明pCA13-hTERT构建成功;PCR、酶切分析、测序鉴定表明pZD55-hTERT构建成功;PCR鉴定表明ZD55-hTERT包含目的基因且无野生型腺病毒的污染;ZD55-hTERT滴度为1×1011PFU/m l;ZD55-hTERT在肝癌细胞株BEL-7404中可导致明显细胞病变效应。结论成功构建的ZD55-hTERT为利用hTERT-shRNA靶向肿瘤治疗奠定了基础。     

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector was further modified on a transductional level by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber, which allows the vector to use the alphavbeta3 and alphavbeta5 receptors as alternative receptors. The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines which have low expression level of CAR led to greater degree of tumor regression in vivo than did the intratumoral injection of control adenoviral vectors not so modified.     

Peptide targeting of adenoviral vectors to augment tumor gene transfer

Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild-type adenovirus in those cells. Nevertheless, the incorporation of relatively low affinity peptide ligands into the fiber knob, while effective in vitro, has only minimal targeting efficacy in vivo and highlights the importance of high affinity ligand:receptor interactions to achieve tumor targeting.     

Comparison of HSV-1 thymidine kinase-dependent and -independent inhibition of replication-competent adenoviral vectors by a panel of drugs

Replication-competent adenoviral vectors hold the promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, but they are also associated with a higher risk for adverse effects, especially in light of the fact that there is no established effective therapy for serious, disseminated adenovirus infection. To assess whether the therapeutic options to inhibit adenoviral replication can be enhanced by expressing a suicide gene, we examined the antiadenoviral effects of 15 drugs against wild-type adenovirus type 5 (Ad5) and an Ad5-based replication-competent vector expressing herpes simplex virus-1 thymidine kinase (HSV-tk) (Ad.OW34) using a real-time polymerase chain reaction -based assay and flow cytometry. Ad5 and Ad.OW34 were highly susceptible to the fluorinated pyrimidine analogs 5-fluoro-2'-deoxyuridine (FUdR), 5-fluorouridine (FUR), and trifluorothymidine (TFT), with a mean 50% inhibitory concentration (IC(50)) ranging from 0.12 to 0.32 microM. The mean IC(50) of ribavirin and cidofovir (CDV) for Ad5, the most frequently used drugs to treat adenovirus disease, was 6.87 and 3.19 microM, respectively. In contrast to Ad5, the Ad.OW34 vector was susceptible to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU, IC(50) 0.09 microM), ganciclovir (GCV, IC(50) 0.19 microM), and acyclovir (ACV, IC(50) 32.04 microM). Additionally, we demonstrated in an animal model that Ad.OW34 vector replication can be inhibited significantly by GCV, CDV, and TFT by 35.2, 7.7, and 3.7-fold, respectively, compared to untreated animals. The observed antiadenoviral effects were primarily not through cell killing, since the in vitro 50% cytotoxic concentrations (CC(50)) were more than 1000 times higher than the antiadenoviral IC(50) of the drugs examined, even in cells stably expressing HSV-tk. Since for HSV-tk-dependent inhibition of adenoviral vectors, stability of HSV-tk expression is crucial, we examined Ad.OW34 vector stability, by passaging the vector 10 times serially in the presence of 10 microM GCV. The HSV-tk/GCV system neither changed the susceptibility of Ad.OW34 to GCV significantly nor detectable vector rearrangements occurred, suggesting that the system might be suitable as a fail-safe mechanism to stop adenoviral vector replication.     

Bcl-XL小发夹RNA腺病毒载体的构建及其抗肿瘤作用

目的:构建表达Bcl-XL小发夹RNA的腺病毒载体(Ad/Bcl-XL shRNA)并探讨其抗肿瘤作用。方法:构建、纯化重组腺病毒Ad/Bcl-XL shRNA。通过Western blotting、MTT分析验证它对Bcl-XL的下调及其杀伤肿瘤细胞的作用,并检测其处理后细胞凋亡信号的活化情况;在裸鼠皮下荷瘤模型中验证其体内抗肿瘤作用。结果:成功构建和纯化了Ad/Bcl-XLshRNA,它能显著下调结肠癌DLD1细胞Bcl-XL蛋白的表达;与Ad/GFP、PBS组相比,Ad/Bcl-XL shRNA组明显抑制人结肠癌细胞DLD1的生长[1 000 MOI时(60.6±4.8)%vs(99.0±2.6)%、100%;2 000 MOI时,(37.3±6.9)%vs(99.0±2.1)%、100%,P<0.01],但对正常人成纤维细胞无明显抑制作用(P>0.05);Ad/Bcl-XL shRNA组能有效诱导结肠癌细胞中凋亡信号casepase-9、casepase-3、PARP的活化。在裸鼠荷瘤模型中,与Ad/GFP、PBS组相比,Ad/Bcl-XL shRNA组显著抑制DLD1来源皮下肿瘤的生长[第29天时,(250.1±185.7)vs(880.0±286.1)、(911.0±389.1)mm3;P<0.01]。结论:Ad/Bcl-XLshRNA能显著抑制结肠癌细胞在体内外的生长,其在结肠癌治疗中具有潜在的应用价值。     

Optimization of radiation controlled gene expression by adenoviral vectors in vitro

The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0-6 Gy. Gene expression was determined by FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3% (6 Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs. Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity by a factor of 1.6-2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.