RV／HSV1—tk／GCV基因治疗系统对卵巢癌细胞体外作用

观察逆转录病毒载体／单纯疱疹病毒胸腺嘧啶核苷激酶基因／羟甲无环鸟苷基因治疗系统对卵巢癌细胞的体外作用及其“旁观者效应”,并探索提高携有ＨＳＶ１－１基因的假型逆转录病毒对卵巢癌 感染率的有效途径。     

卵巢癌药物敏感基因治疗实验研究——(Ⅱ)体内实验

为探索将单纯疱疹病毒胸腺嘧啶核苷激酶基因／羟甲基无环鸟苷（ＨＳＶ１－ｔｋ／ＧＣＶ）药物敏感基因治疗系统应用于卵巢癌,本刊［１］已介绍体外实验结果,现将体内实验情况介绍如下。材料与方法１．细胞来源及培养详见文献［１］　ＧＣＶ仍由ＲｏｃｈＳｙｎｔｅｘ公司惠赠;４～６周龄ＢＡＬＢ／ｃ裸小鼠由上海市肿瘤研究所动物室提供并饲养。２．ＡＯ皮下移植瘤治疗　本组１２只裸鼠,每只裸鼠左右侧前背部皮下以５号针头分别接种５×１０６的ＡＯ细胞或ＡＯ／ＨＳＶ１－ｔｋｃ细胞。２周后选取左右两侧背部皮下触摸肿块大小较一致的９…     

卵巢癌药物敏感基因治疗实验研究——(Ⅰ)体外实验

为探索Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因／羟甲基无环鸟苷（ＨＳＶ１－ｔｋ／ＧＣＶ）药物敏感基因治疗系统应用于卵巢癌,本文进行了一系列实验研究,这里先介绍体外实验结果。材料与方法１．卵巢上皮癌细胞系ＡＯ来自中国科学院上海细胞库;病毒生产细胞（ＶＰＣ）...     

动脉灌注GE7系统介导Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因导入大鼠卵巢癌细胞

目的 采用大鼠诱发性卵巢癌模型,观察经肿瘤动脉灌注结合GE7导入系统对Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因(HSV1-tk)转导的效率和靶向性.方法 构建GE7四元复合物,将诱发的荷瘤大鼠9只随机分成A组、B组和C组,A组经卵巢动脉注入四元复合物(8μg/只),B组注入同体积生理盐水,C组经尾静脉注入四元复合物(8μg/只).给药72 h后,分别以逆转录聚合酶链反应(RT-PCR)和Western blot检测大鼠的肿瘤、心、肝、脾、肺及肾脏组织的HSV1-tk mRNA及蛋白的表达.结果 A组肿瘤组织中HSV1-tk mRNA和蛋白均高表达,而其他组织中则无表达或表达极少;B组未见HSV1-tk mRNA及蛋白的表达;C组肿瘤组织中未见HSV1-tkmRNA和蛋白的表达,肝脏、脾脏、肺脏及肾脏组织中则有少量的表达.RT-PCR半定量检测结果显示,A组卵巢肿瘤组织中的HSV1-tk mRNA含量(1.692±0.221)明显高于C组卵巢肿瘤组织(0.012±0.002;P<0.01).结论 动脉灌注结合GE7导入系统对HSV1-tk有较高的转导率和靶向性,为HSV1-tk/GCV治疗系统在卵巢癌基因治疗的应用提供了新的策略.

Abstract：

Objective To observe the gene and protein expression of herpes simplex virus type Ⅰ thymidine kinase (HSV1-tk) in the ovarian tumor tissues and other organs after arterial infusion of HSV1-tk gene mediated by GE7 delivery system.Methods GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20complexes were constructed.Nine rats with induced ovarian tumor were divided into 3 groups, injecting the 4-element complexes or saline buffer through the ovarian artery and complexes through the tail vein,respectively.The ovarian tumors, hearts, livers, spleens, lungs and kidneys were obtained at 72 hours after injection.RT-PCR and Western Blot were preceeded to determine the expression of HSV1-tk gene and protein in the tumor tissues and other organs.Results In the group of arterial injection with 4-element complexes, the HSV1-tk gene and protein were expressed strongly in the tumor tissues, while little or none was detected in other organs.In the group of arterial injection with saline buffer, no HSV1-tk gene and protein was detected in both tumor tissues and other organs.In the group of tail vein injection, none was detected in tumor tissues and only little was found in the livers, spleens, lungs and kidneys.Conclusion High target and gene transfer rates can be obtained when HSV1-tk gene is transferred via the artery route mediated by GE7 delivery system.HSV1-tk protein can be expressed after the gene transfer.The results may provide a new strategy for target killing of HSV1-tk/GCV system in ovarian tumors.     

HSV-TK/GCV系统治疗难治性卵巢癌动物试验

目的:运用单纯疱疹病毒腺苷激酶/更昔洛韦(HSV-TK/GCV)自杀基因系统治疗模型动物并观察其疗效。方法:常规培养携带HSV-TK基因的包装细胞PA317,测定病毒滴度。在移植瘤内多点注射PA317细胞,观察更昔洛韦(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。结果:重组逆转录病毒滴度为6×104cfu/ml,当肿瘤体积较小,应用HSV-TK/GCV系统治疗后肿瘤生长受到抑制(P<0.05);当肿瘤体积较大时,则抑制作用不明显;经PCR检测证实,TK基因能在肿瘤组织中转导。结论:HSV-TK/GCV系统对人难治性卵巢癌荷瘤裸鼠具有一定的治疗作用,但当肿瘤体积较大时,疗效不佳。     

HSV-tk/GCV基因治疗在卵巢癌中的研究进展

卵巢癌自杀基因治疗多采用HSV-tk/GCV系统.该系统的旁观者效应可在一定程度上弥补基因转染率低的缺陷,通过缝隙连接胞间通信(GJIC)增强剂可提高该系统的旁观者效应,通过选择构建高效能载体系统、应用靶向性调控机制或与其他方法联合治疗可增强疗效.该系统治疗卵巢癌具有广阔的发展前景.     

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).     

HSV1-tk/GCV系统联合Topotecan治疗人卵巢癌的动物实验研究

目的探讨Topotecan能否增强HSV1-tk/GCV自杀基因系统对卵巢癌的体内治疗作用.方法 先用携带tk基因的重组逆转录病毒上清转染人卵巢癌细胞系SKOV-3,用含G418的培养液筛选抗性克隆(命名为SKOV-3/TK).PCR方法检测tk基因整合情况.用SKOV-3细胞建立荷瘤鼠模型作为对照组和Topotecan组.用SKOV-3与SKOV-3/TK细胞按82比例混合细胞建立者为HSV1-tk/GCV组和HSV1-tk/GCV联合Topotecan组;从用药第1天开始每5天测量肿瘤体积一次,至用药结束后一周,绘制肿瘤生长曲线,计算抑瘤率,并取瘤组织做病理学检查.结果与对照组比较,HSV1-TK/GCV组和Topotecan组、联合用药组抑瘤率分别为38.8%、25.3%和89.7%,差异均有显著性,P＜0.01.组间两两比较差异亦有显著性,P＜0.01.病理显示实验组出现不同程度点、片状坏死,以联合用药组为重.结论HSV1-tk/GCV自杀基因系统具有强大的杀伤肿瘤效应及旁观者效应,联合Topotecan化疗将起到协同作用.     

人端粒酶逆转录酶启动子调控腺病毒介导胸苷激酶基因治疗人膀胱癌的动物实验研究

目的：探讨带有人端粒酶逆转录酶（hTERT）启动子驱动单纯疱疹病毒胸腺嘧啶激酶（HSV—tk）基因的重组腺病毒Ad—hTERT—HSV—tk结合无毒的环氧鸟苷（GCV）对人膀胱癌的治疗作用．方法：建立人膀胱癌细胞株253JBALB／C裸小鼠移植痛模型．采用Ad—hTERT—HSV—tk裸鼠尾静脉注射，腹腔注射GCV治疗并观察结果通过常规病理切片观察肿瘤破坏情况及安全性；通过TUNEL染色进一步观察肿瘤的凋亡情况，免疫组化法测定增值细胞核抗原（PCNA），结果：Ad-hTERT—HSV—tk／GCV治疗组，显示出明显的肿瘤抑制作用，其肿瘤体积、重量显著低于各对照组（P〈0．05）．抑瘤率明显，Ad—hTERT—HSV—tk／GCV治疗组凋亡指数高于其它各组，而增殖指数却明显低于其它各组。结论：Ad—hTERT—HSV—tk／GCV系统对裸鼠移植瘤的生长有明显抑制作用，可以靶向性转入人膀胱癌细胞，治疗效果显著．     

重组腺病毒介导自杀基因HSV1-tk转染内皮祖细胞的实验研究

[目的]构建含有自杀基因单纯疱疹病毒胸腺激酶基因( HSV1-tk)的重组腺病毒载体,并感染大鼠骨髓来源的内皮祖细胞(EPCs),以期用于抗肿瘤血管生成的基因治疗及核素报告基因显像.[方法]从大鼠骨髓中分离、培养EPCs,利用流式细胞术进行鉴定.构建重组腺病毒载体Ad5-HSV1-tk-EGFP,并感染EPCs,以腺病毒Ad5-EGFP为对照.利用RT-PCR法、Westernblot免疫印迹法检测HSV1-tk的表达,利用MTT法检测更昔洛韦(GCV)对病毒感染后EPCs的杀伤作用.[结果]流式细胞术结果显示从大鼠骨髓中分离出的EPCs阳性表达CD34(80.09％)和CD 133(81.75％),RT-PCR及Western blot免疫印迹结果显示HSV1-tk基因在转录及蛋白水平可以正确表达,MTT结果显示HSV-tk/GCV自杀基因系统对EPCs细胞具有明显的杀伤作用.[结论]重组腺病毒Ad5-HSV1-tk-EGFP感染EPCs后可以在细胞中成功表达,并且感染后自杀基因具有生物学活性,从而为利用EPCs作为载体进行肿瘤靶向基因治疗、报告基因显像提供实验依据.     

ATRA联合HSV1-TK/GCV系统治疗卵巢癌的实验研究

目的 探讨全反式维甲酸（all-trans retinoic acid,ATRA）能否提高Ⅰ型单纯疱疹病毒胸苷激酶（herpes simplex virus thymidine kinase,HSV1-TK）/丙氧鸟苷（ganciclovir,GCV）系统对卵巢癌的体内治疗作用。方法 用携带tk基因的重组逆转录病毒上清转染人卵巢癌细胞系SKOV-3,用含G418的培养液筛选获得抗性克隆（命名为SKOV-3/TK）。采用PCR方法 检测HSV1-tk基因整合情况。分别用SKOV-3细胞建立皮下移植瘤裸鼠模型作为对照组和ATRA组,用SKOV-3细胞与SKOV-3/TK细胞按8∶2比例混合细胞建立的动物模型作为HSV1-tk/GCV组和HSV1-tk/GCV联合AT-RA组。于接种细胞后15天开始实验用药,从用药第1天开始每5天测量肿瘤体积1次,至用药结束后2周,绘制肿瘤生长曲线,计算抑瘤率,并取瘤组织做病理学检查。结果 与对照组比较,ATRA组、HSV1-tk/GCV组抑瘤率分别为25.5%、38.8%,联合用药组抑瘤率高达67.8%,有统计学意义,P〈0.05。病理结果 显示实验组出现不同程度点、片状坏死,以联合用药组为重。结论 HSV1-TK/GCV系统对卵巢癌有体内杀伤作用;ARTA可以提高HSV1-TK/GCV系统对卵巢癌的体内杀伤作用。     

ATRA增强HSV1-TK/GCV系统联合topotecan治疗卵巢癌的动物实验

目的：探讨全反式维甲酸（all-trans retinoic acid，ATRA）提高Ⅰ型单纯疱疹病毒胸苷激酶（herpes simplex virus thymidine kinase，HSV1-TK）／丙氧乌苷（ganciclovir，GCV）系统联合拓扑替康（topotecan，TPT）对卵巢癌的体内治疗作用。方法：首先用携带HSV1-TK基因的重组逆转录病毒上清转染人卵巢癌细胞系SKOV-3，在含有G418的培养液中筛选获得抗性克隆细胞（命名为SKOV-3／TK）。PCR方法检测HSV1-TK基因整合情况；用SKOV-3细胞与SKOV-3／TK细胞按8：2比例混合建立荷瘤鼠模型，作为HSV1-TK／GCV组、HSV1-TK／GCV＋TPT组、ATRA联合HSV1-TK／GCV组及ATRA联合HSV1-TK／GCV＋TPT组4个实验组，再单用SKOV-3细胞建立荷瘤鼠模型作为对照组；从用药第1天开始每5d测量肿瘤体积1次，至用药结束后1周，绘制肿瘤生长曲线，计算抑瘤率，并取瘤组织做病理学检查。结果：各组肿瘤体积与对照组比较抑瘤率分别为38．8％、53．2％、62．3％及95．7％，差异均有显著性（P〈0．01），而且联合ATRA组与未用ATRA组比较差异亦有显著性（P〈0．01）。病理结果显示实验组肿瘤组织均出现不同程度的点、片状坏死，以ATRA联合HSV1-TK／GCV＋TPT组为重。结论：HSV1-TK／GCV系统联合TPT治疗卵巢癌可起到协同作用；ARTA可以提高HSV1-TK／GCV系统联合TPT对卵巢癌的体内杀伤作用。     

THY1基因在卵巢上皮性癌组织中的表达

目的 检测人正常卵巢组织、卵巢浆液性囊腺瘤和浆液性囊腺癌组织中THY1蛋白的表达,探讨其表达变化与卵巢浆液性囊腺癌患者临床病理因素间的关系.方法 应用免疫组化法,检测25例人正常卵巢组织、25例卵巢浆液性囊腺瘤组织和53例浆液性囊腺癌组织中THY1蛋白的表达.结果 THY1蛋白在正常卵巢、卵巢浆液性囊腺瘤和浆液性囊腺癌组织中的阳性表达率分别为60.0%(15/25)、72.0%(18/25)和34.0%(18/53),IOD值分别为288 449.2±60 087.3、271 655.6±66 588.7和252 087.6±45 559.4.卵巢浆液性囊腺癌中,THY1蛋白的阳性表达率明显低于正常卵巢和卵巢浆液性囊腺瘤组织(P<0.05),且THY1蛋白的表达量与卵巢浆液性囊腺癌的手术-病理分期、组织学分级及淋巴结转移呈显著负相关(均P<0.05).结论 THY1基因的缺失可能与卵巢浆液性囊腺癌的发生、发展相关.     

HSV-tk/GCV系统杀伤ACC-M细胞的机理研究

目的 为探索ＨＳＶ-tk/ＧＣＶ（单包疹病毒胸腺嘧啶核激酶基因/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（４００rmol/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４００umol/ＬＧＣＶ作用下有明显的亚     

In vivo Adenovirus-mediated Prodrug Gene Therapy for Carcinoembryonic Antigen-producing Pancreatic Cancer

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)-ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus-carrying HSVtk gene under the control of a carci-noembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA-producing Su.86.86, BxPC-3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA-nonproducing HeLa, were used for analysis of GCV sensitivity induced by adeno-viral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA-positive pancreatic cancer in vivo , a subcutaneously implanted tumor-bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA-production. In contrast, when the cells were transduced with a CEA promoter, the cell-killing effect of GCV was increased in only CEA-producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus-mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA-producing pancreatic cancer cells in vitro and in vivo . This strategy may provide a useful tool for treating pancreatic cancer, especially CEA-producing tumor cells.     

腺病毒介导HSV-TK/GCV系统联合放射治疗口腔鳞癌的研究

目的 探讨单纯疱疹病毒胸苷激酶基因（HSV－TK）／丙氧鸟苷（GCV）自杀基因系统对放射治疗的增敏作用。方法 口腔鳞癌细胞（Tca8113细胞系）经HSV－TK／GCV系统治疗后给予放射治疗，采用LQ和单击多靶（SHMT）模型分析细胞存活曲线参数。结果 细胞存活曲线分析显示：单纯放射治疗组其α为0.1074,β为0.0158,D0为2.2576,Dq为3.5413；与单纯放射治疗组比较，HSV－TK／GCV治疗组α（0.2127)和α：β（9.496)值大，D0（1.4526)和Dq(2.2257)值小，其放射增敏率（SER）为1.55。结论 HSV－TK／GCV系统具有放射增敏作用，可提高放射治疗对口腔鳞癌的治疗疗效。     

胸苷激酶基因对乳腺癌细胞的杀伤性研究

目的　探讨逆转录病毒 (RV )载体介导的单纯疱疹病毒胸苷激酶 (HSV -TK )基因转染小鼠乳腺癌细胞 ,联合抗病毒药物丙氧鸟苷 (GCV )对小鼠乳腺癌细胞系MA 782 /5S -810 2体外杀伤作用及其产生的旁观者效应。方法　采用脂质体转染法将GINaTK载体转入包装细胞PA3 17、G 418筛选抗性克隆 ,采用PCR方法检测HSV -TK基因整合情况。取滴度最高的病毒上清液感染小鼠乳腺癌细胞MA 782 /5S -810 2 ,经G 418筛选后 ,得到带有HSV -TK基因的MA 782 /5S -810 2 /TK细胞 ;将MA 782 /5S -810 2和MA 782 /5S -810 2 /TK细胞按不同比例混合培养后 ,给予 10 μg/mlGCV ,4天后采用MTT法计算细胞成活率 (SR ) ,观察旁观者效应。结果　GINaTK载体成功转入包装细胞PA 3 17,病毒滴度为 4.0× 10 4cfu/ml。用病毒上清液感染MA 782 /5S -810 2 ,成功得到了表达HSV -TK基因的MA 782 /5S -810 2 /TK细胞。混合培养结果显示 ,当MA782 /5S -810 2 /TK细胞数占混合细胞数 10 %时 ,低浓度 (10 μg/ml)的GCV就可将 5 0 %左右的肿瘤细胞杀死。 结论　逆转录病毒可介导HSV -TK基因转入小鼠乳腺癌细胞MA782 /5S -810 2并获稳定表达 ,RV -HSV -TK /GCV系统杀伤癌细胞存在旁观者效应。     

HSVTK/GCV自杀基因系统治疗乳腺癌的研究

 目的　探讨HSV TK/GCV自杀基因系统对小鼠乳腺癌细胞系MA782 / 5S 810 2体外及体内杀伤作用及其产生的旁观者效应。方法　采用脂质体转染法将GINaTK载体转入包装细胞PA317。取病毒上清液感染小鼠乳腺癌细胞MA782 / 5S 810 2 ,得到带有HSV TK基因的MA782 / 5S 810 2 /TK细胞 ,并将其分别用于体外和体内实验。结果　载体HSV TK导入了PA317细胞。体外实验结果显示 ,当MA782 / 5S 810 2 /TK细胞数占混合细胞 10 %时 ,低浓度 (10 μg/ml)的GCV就可将 5 0 %左右的肿瘤细胞杀死。体内实验结果显示GCV可明显抑制MA782 / 5S 810 2 /TK细胞在BALB/C小鼠体内的肿瘤形成。实验组肿瘤组织与对照组相比存在明显的病理学改变。结论　逆转录病毒可介导HSV TK基因转入小鼠乳腺癌细胞MA782 / 5S 810 2并获稳定表达 ,HSV TK/GCV自杀基因系统在体内外对乳腺癌细胞均有杀伤作用 ,且存在明显的旁观者效应。     

Oncolytic Viral Therapy for Human Prostate Cancer by Conditionally Replicating Herpes Simplex Virus 1 Vector G207

Over the last few years, a conditionally replicating herpes simplex virus 1 (HSV-1) vector, G207 has been used for the treatment of several malignant tumors. In this article we evaluate the antitumoral effect of G207 against prostate cancer in vitro and in vivo . The susceptibility of the human prostate cancer cell lines, DU145 and PC3 to G207 at a multiplicity of infection (MOI) of 0.1 was examined. In addition, the growth characteristics of G207 were assessed. Athymic mice with s.c. tumors were inoculated in vivo intraneoplastically with 1x10⁷ plaque-forming units (PFU) of G207. For the pathological analyses, s.c. tumors were stained with X-gal. DU145 and PC3 were efficiently destroyed by G207 within 7 days. The viral yields of G207 increased time-dependently. In vivo , the intraneoplastic inoculation of G207 induced a significant inhibition of the tumor growth. The mean tumor growth ratio was significantly inhibited in the G207-treated tumors (DU145, P < 0.0001; PC3, P < 0.001 versus controls). In a pathological study, many lacZ -positive cells were diffusely present in the G207-treated tumors. G207 showed a significant antitumoral effect against human prostate cancer cell lines, and thus may be considered a useful agent for the treatment of prostate cancer.