腺病毒介导HSV—tk自杀基因联合野生型p53基因对直肠癌细胞的杀伤作用

目的 观察重组腺病毒介导单纯疱疹病毒胸苷激酶（HSV－tk)基因联合人野生型p53基因共转染,对直肠癌细胞杀伤作用。方法 构建重组腺病毒质粒pAdCMV-Link1(tk/p53)、pAdCMV-Link(tik),pAdCMV-Link(p53),分别感染p53炎变的人直肠癌细胞SW837。进行细胞集落形成实验、细胞存活率的测定和裸鼠移植瘤治疗实验,观察HSV－tk/GCV系统与野生p53基因联合对肿瘤细胞的杀伤作用。结果 应用pAdCMV-Link1(tk/p53),pAdCMV-Link(-),pAdCMV-Link(tk),pAdCMV-Link(p53)重组腺病毒感染SW837细胞,加入GCV,各组细胞集落数分别为8、95、40、70。pAdCMV-Link(CD/53)组肿瘤细胞集落形成减少、细胞存活率显著下降（P＜0．01）。裸鼠移植肿瘤生长抑制率分别为76．5％、0．8％、55．8％、23．2％,pACMV-Link(tk/p53)重组腺病毒对肿瘤的抑制作用最强。结论 HSV－tk自杀基因与野生型p53基因共转染,对肿瘤细胞有更强的杀伤作用。     

替莫唑胺联合HSV1-tk/ GCV系统治疗人脑胶质瘤细胞的实验

 目的探讨替莫唑胺联合HSV1-tk/GCV自杀基因系统对人胶质瘤细胞的体外杀伤作用及其作用机制。方法用携带tk基因的重组逆转录病毒转染人胶质瘤细胞系U251细胞,并筛选、鉴定。转染与未转染tk基因的U251细胞按1∶9混合。实验分3组：对照组、 GCV组、GCV+TMZ组。GCV组以5种不同浓度（2、5、10、20、40μM）作用于混合细胞;GCV+TMZ组在上述基础上各加入TMZ 50μM;对照组细胞不做任何处理。各组细胞培养72h后,MTT法检测各组细胞的活力,流式细胞仪检测细胞凋亡及细胞周期分布的变化。结果（1）U251/tk细胞的活力随GCV组浓度的增加逐渐减弱,呈现良好的剂量效应关系;（2）GCV组、TMZ +GCV组的IC50分别为17.3μM、8.1μM（两组相差2.14倍）;（3）GCV+TMZ组的总体抑制率显著高于GCV组（P<0.01）。GCV+TMZ组生存曲线明显左移;（4）流式细胞仪检测显示两组的凋亡率均明显增加（P<0.01）;细胞多被阻滞于G2~M期。结论HSV1-tk/GCV自杀基因系统有一定的肿瘤杀伤效应及旁观者效应;替莫唑胺与HSV1-tk/GCV自杀基因系统两者之间有明显的协同作用;其作用机制可能通过改变细胞周期的分布及促凋亡增加GCV的旁观效应。     

体内实验研究“旁观者”效应机理

近来发现遗传变异而表达单纯疱疹病毒胸苷激酶基因(HSV—tk)的肿瘤细胞在小鼠体内有杀伤肿瘤细胞效果。据证实,这些细胞在接触丙氧鸟苷(GCV)后可能对未经处理过之“旁观者”(Bystander)肿瘤细胞有杀伤效应,而且HSV—tk基因阳性肿瘤细胞对未经转导过之肿瘤细胞之杀伤作用取决于小鼠体内完整的免疫系统。目前正在进行的研究分析了在注射HSV—tk基因转导细胞进入小鼠体内后发生的宿主抗肿瘤免疫反应。该研究用BALB/C小鼠,在第0天,通过向其     

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．     

重组人肿瘤坏死因子α衍生物治疗鼠脑胶质瘤

 目的　探讨重组人肿瘤坏死因子α衍生物 (rhTNF α)治疗鼠脑胶质瘤的有效性。方法　通过改造人肿瘤坏死因子α形成rhTNF α ,体外培养鼠脑胶质瘤细胞 ,加入不同浓度rhTNF α,MTT法测定细胞存活率 ,颅内立体定向种植C6细胞制成鼠脑胶质瘤模型 ,10天后MRI增强测量肿瘤大小 ,腹腔注射rhTNF α ,治疗后第 8天、15天、2 1天分别用MRI增强测量肿瘤大小。结果　体外实验 :加入rhTNF α 5天后 ,大部分C6细胞死亡 ,细胞存活率随rhTNF α的递增而降低。体内实验 ,治疗 8天后肿瘤即开始缩小 ,治疗 2 1天后 ,MRI未见明显肿瘤强化灶。结论　rhTNF α能有效治疗鼠脑胶质瘤。     

肿瘤坏死因子基因转导肿瘤浸润淋巴细胞的初步研究及应用

用单纯疤疹病毒(HSV)的缺陷株作为载体,感染肿瘤细胞治疗脑瘤的研究已取得了不少进展.本文报道将HSV—tk基因转移联合抗瘤药物GCV可使荷脑瘤大鼠长期存活.9L神经纤维肉瘤细胞用HSV载体hrR3转染,hrR3缺乏RNA还原酶,只能在肿瘤细胞中复制而不能在神经细胞中复制存活,同时还携带病毒胸苷激酶(tk)基因,RH105无tk基因,转染hrR3的9L细胞体外培养,加入GCV1μg/ml共孵育,结果发现9L细胞被杀死的数目比单纯感染hrR3的细胞死亡数目增加23%.GCV加到不表达tk基因的RH105载体转染的9L细胞体系中没有杀伤作用.将神经纤维肉瘤9L细胞4×10~4接种到Fischer 344CD大鼠脑前叶,5天后将hrR3载体2×10~7PFU/10μl直接定位注射至脑瘤部位.结果发现hrR3载体治疗可使20%的大鼠存活时间超过5个月,而生理盐水组全部死亡.接种病毒后同时用GCV7.5mg/kg治疗7天,可使48%的荷9T肿瘤细胞的大鼠存活时间超过5个月PH105感染的治疗组大鼠全部死亡,组织化学检查证明外源性的tk基因在肿瘤细     

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.     

丹参酮Ⅱ A增强HSV-tk/GCV旁观者效应及其与Cx43 mRNA表达的关系

目的　研究丹参酮ⅡA(TanshinoneⅡA ,Tan)对HSV tk/GCV旁观者效应的增强作用及其与间隙连接蛋白Cx4 3转录表达的关系。方法　应用polybrene转染、荧光定量RT PCR等技术 ,观察Tan对宫颈癌细胞ME180 (ME)、ME/TK转化细胞旁观者效应及诱导Cx4 3mRNA表达的作用。结果　在HSV tk/GCV系统中 ,Tan显著地提高了ME/TK细胞对GCV的敏感性。在 2 μg/mlGCV作用下 ,以1.3× 10 9mol/LTan与不加Tan的作用相比较 ,在ME与ME/TK不同比例混合细胞的各组中 ,细胞的存活率明显降低 ,差异均有显著性 (P <0 .0 5 )。并观察到Tan对GCV旁观者效应的增强作用 ,在一定范围内为 1.3× 10 -8mol/L和 1.3× 10 -9mol/L。RT PCR结果表明 ,经 1.3× 10 -8mol/L和 1.3× 10 -9mol/LTan处理的ME细胞 ,其Cx4 3mRNA的相对拷贝数比值增高约 8.83倍及 8.4 7倍。结论　在宫颈癌ME180细胞中 ,Tan在 1.3× 10 -8mol/L和 1.3× 10 -9mol/L范围内具有明显增强HSV tk/GCV旁观者效应的作用。Tan在转录水平诱导Cx4 3mRNA表达上调 ,与旁观者效应的增强作用密切相关。     

肿瘤HSV—tk／GCV自杀基因治疗系统的增效策略

HSV—tk／GCV系统是目前应用最广泛的自杀基因治疗系统之一,其重要性日益上升。然而,许多缺陷严重限制了其应用广度和治疗效率。为满足更多领域的研究需要并实现临床治疗,科研人员投入了大量精力改善HSV—tk／GCV系统,增强其治疗效率。增效策略主要包括加强亲和性、提高表达水平、双自杀基因系统联合、诱发免疫系统协同作用、激活细胞周期、与其他肿瘤治疗方法相结合等。本文在阐明HSV—tk／GCV系统的自杀机理的基础上,分析了多种增效策略及效果,列举了应用领域并对未来发展作探讨。     

tk基因治疗脑胶质瘤时的免疫反应与旁观者效应的关系

 目的　探讨tk基因 (thymidinekinasegene)治疗脑胶质瘤时的免疫反应与旁观者效应的关系。方法　将C6 tk+与C6 tk 按 0 %tk+,10 %tk+,30 %tk+,5 0 %tk+,10 0 %tk+5种比例混合 ,接种于SD鼠右侧顶叶 ,7d后腹腔注射GCV(更昔洛韦 ) ,剂量为 30mg/kg/d ,共 10d。细胞接种 3周后 ,处死动物 ,计算成瘤率 ,作病理检查 ,并作CD4 +、CD8+的免疫组化染色。结果　 5 0 %及 10 0 %C6 tk+组的成瘤率明显小于其余3组 (P <0 .0 5 ) ,病理检查发现 5 0 %及 10 0 %tk+组有淋巴细胞及浆细胞浸润。免疫组化显示每一组之间均有差异 ,提示有免疫反应存在。结论　tk基因治疗脑胶质瘤时 ,可激起机体内的免疫反应 ,增强抗肿瘤作用 ,它是旁观者效应的作用机制之一     

HSV—tk／GCV系统对神经胶质瘤的自杀基因治疗研究

目的在ｉｎｖｉｔｒｏ和ｉｎｖｉｖｏ水平建立致死神经胶质瘤的ＨＳＶ－ｔｋ／ＧＣＶ自杀基因系统,并验证该系统的有效性。方法用携带单纯疱疹病毒胸苷激酶（ＨＳＶ－ｔｋ）基因的重组逆转录病毒,感染大鼠神经胶质瘤细胞Ｃ６,筛选稳定表达ｔｋ的克隆细胞Ｃ６／ｔｋ;经生长抑制试验,比较Ｃ６／ｔｋ细胞对三种核苷类似物ＧＣＶ、ＢＶｄＵ和ＡＣＶ的敏感性;Ｃ６／ｔｋ细胞在裸鼠皮下成瘤,用ＧＣＶ进行治疗并观察疗效。结果ｔｋ基因整合入Ｃ６细胞并在Ｃ６／ｔｋ细胞中稳定表达;生长抑制试验表明,ＧＣＶ是最为有效的原药,Ｃ６／ｔｋ细胞对ＧＣＶ高度敏感,ＩＣ５０＜０．２μｍｏｌ／Ｌ,而野生型Ｃ６细胞和转染空载病毒载体的Ｃ６／０细胞对ＧＣＶ的ＩＣ５０≥１００μｍｏｌ／Ｌ,相差５００多倍。裸鼠ｉｎｖｉｖｏ实验得到相应结果,治疗组肿瘤受到明显抑制和杀伤。结论在ｉｎｖｉｔｒｏ和ｉｎｖｉｖｏ水平,表达ｔｋ基因的肿瘤细胞均被ＧＣＶ有效杀伤,这表明ＨＳＶ－ｔｋ／ＧＣＶ自杀基因系统有可能成为基因治疗脑瘤的有效方法。     

自杀基因治疗恶性胶质瘤的研究

目的：单纯疱疹病毒Ⅰ型胸苷激酶（ＨＳＶ－ｔｋ）基因治疗恶性胶质瘤体内外试验。方法：分子克隆及真核细胞基因转染技术构建逆转录病毒（ＲＶ）载体ｐＭＶ７（ｔｋ）及ＰＡ３１７ｔｋ包装细胞系;体外不同比例混合鼠Ｃ６胶质瘤细胞与ＰＡ３１７ｔｋ细胞,在ＧＣＶ（Ｇａｎｃｉｃｌｏｖｉｒ）作用下观察细胞存活率;建立ＳＤ大鼠颅内Ｃ６胶质瘤模型（种植５×１０５Ｃ６细胞）,治疗组第３天原位注射５×１０６ＰＡ３１７ｔｋ细胞,５天后腹腔给予ＧＣＶ（３０ｍｇ／ｋｇ．ｄ）,ＭＲＩ全程监测肿瘤消长,观察病症及存活期,并行病理检查。结果：在ＧＣＶ０．１～１０１μｇ／ｍｌ浓度范围内,Ｃ６细胞存活率随ＰＡ３１７ｔｋ细胞混入比例增加而逐渐减低（Ｐ＜０．００１）,并具有ＧＣＶ剂量依赖性（Ｐ＜０．０１）;体内试验治疗组病症轻,生存期延长,ＭＲＩ表明治疗组肿瘤体积较对照组明显减小（Ｐ＜０．０１）,１个月时病理检查见肿瘤细胞消失,代之以小胶质细胞增生并形成坏死囊。结论：应用本实验室构建的ＲＶ载体ｐＭＶ７（ｔｋ）及其ＰＡ３１７ｔｋ包装细胞系治疗恶性胶质瘤是一种有效及具有前途的治疗方法。     

RETROVIRAL-MEDIATED SUICIDE GENE THERAPY OF EXPERIMENTAL GLIOMA

Ｍａｌｉｇｎａｎｔｇｌｉｏｍａ，ｔｈｅｍｏｓｔｃｏｍｍｏｎｉｎｔｒａｃｒａｎｉａｌｔｕｍｏｒ，ｉｓｏｎｅｏｆｔｈｅｍｏｓｔｌｅｔｈａｌｃａｎｃｅｒｓｉｎｈｕｍａｎ．Ｔｈｅｍｅｄｉａｎｓｕｒｖｉｖａｌｔｉｍｅａｆｔｅｒｄｉａｇｎｏｓｉｓｉｓｌｅｓｔｈａ...     

单纯疱疹病毒-胸苷激酶基因转染骨髓基质干细胞治疗大鼠脑胶质瘤

目的研究转染单纯疱疹病毒-胸苷激酶(HSV-tk)基因的骨髓基质干细胞(BMSCs)对大鼠脑胶质瘤的治疗作用。方法原代培养BMSCs,AdCMV-tk转染BMSCs。逆转录-多聚酶链反应(RT-PCR)检测BMSCs/tk对tk基因的转录。以BrdU标记BMSCs/tk,观察其趋瘤性。四甲基偶氮唑盐(MTT)法检测其对C6细胞的旁观者效应。将BMSCs/tk注入脑胶质瘤大鼠肿瘤对侧脑组织,原位末端标记(TUNEL)法检测脑内肿瘤细胞凋亡,动态MRI监测肿瘤体积的变化,观察荷瘤鼠生存期。结果全骨髓细胞培养法可获得纯化的BMSCs。感染AdCMV-tk 21 d后,BMSCs仍有明显的tk基因表达。转染tk基因后的BMSCs仍具有明显的趋瘤性。BMSCs/tk不仅在体外可产生明显的旁观者效应,在荷瘤鼠脑内也显示了明显的诱导肿瘤细胞凋亡的旁观者效应,凋亡阳性率为20.38%±2.57%,与BMSCs组(2.56%±0.52%)和对照组(2.74%±0.38%)相比,差异均有统计学意义(P值分别为0.023和0.025)。BMSCs/tk移植3周时,BMSCs/tk组、BMSCs组和对照组肿瘤体积分别为(8.28±2.64)、(134.51±16.37)和(147.22±31.05)mm3,BMSCs/tk组远小于BMSCs组(P=0.001)和对照组(P<0.01),并且部分大鼠脑内肿瘤消失。BMSCs/tk组大鼠生存期为(52.60±13.11)d,与对照组相比明显延长(P=0.01)。结论HSV-tk转染的BMSCs可能成为脑胶质瘤治疗的有效手段。     

逆转录病毒介导的双自杀基因对K562细胞杀伤作用的实验研究

目的 :观察逆转录病毒介导的双自杀基因对K5 6 2细胞的杀伤作用 ,探讨慢性粒细胞白血病的基因治疗方法。方法 :通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G4 18筛选后大量培养产病毒的阳性克隆PA317 CD +tk细胞株 ,收集病毒上清 ,浓缩后转染K5 6 2细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的K5 6 2 CD +tk细胞株。用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocytosine ,5 FC)和 或无环鸟苷 (Ganciciovir,GCV)后MTT法测定转基因组及未转基因组K5 6 2细胞的存活率。结果 :双自杀基因在K5 6 2细胞中可稳定表达 ,联合使用 5 FC和GCV对细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 FC或GCV。结论 :逆转录病毒介导自杀基因可有效杀死K5 6 2细胞 ,双自杀基因较单一自杀基因具有更强的抗肿瘤作用     

逆转录病毒介导的双自杀基因对淋巴瘤细胞的杀伤作用研究

目的 观察逆转录病毒介导的双自杀基因对淋巴瘤细胞的杀伤作用，探讨淋巴瘤的基因治疗方法。方法 在脂质体的介导下将含有双自杀基因的逆转录病毒载体pWZLneoCDglytk导入包装细胞PA317，经G418筛选后大量培养产病毒的阳性克隆PA317／CD tk细胞株，收集病毒上清，浓缩后转染Raji淋巴瘤细胞，再次经G418筛选，获得稳定表达双自杀基因的Raji/CD tk细胞株。采用RT-PCR法检测双自杀基因的表达。MTT法测定转基因组及末转基因组Raji细胞的存活率。结果 双自杀基因在Raji细胞中可稳定表达，联合使用5-氟胞咳暖(5-FC)和无环鸟苷(QCV)对细胞增殖的杀伤作用及旁杀伤效应高于单独使用5-FC或GCV。结论 逆转录病毒介导双自杀基因较单一自杀基因具有更强的杀伤作用。     

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.     

缝隙连接蛋白43对双自杀基因系统旁观者效应的影响

Objective： To observe the influence of connexin 43 （Cx43） on the bystander effect induced by cytosine deaminase （CD） and herpes simplex virus thymidine kinase （HSV-tk） coexpression suicide genes system in human cholangiocarcinoma QBC939 cells and transplantation tumors in nude mice. Methods： In vitro, the CD＋tk＋ and CD＋tk＋Cx＋ cells were respectively treated with 5-fluorocytosine （5-Fc） and Ganciclovir （GCV）. The cytotoxic effect was evaluated by MTT method. In order to investigate the influence of Cx43 on the bystander effect, the size of transplantation tumors of the CD＋tk＋ and CD＋tk＋Cx＋ cells was measured before and after application of 5-Fc and GCV. Results： CD and tk genes were stably expressed in transfected QBC939 cells. The increased expression of Cx43 was determined by testing for the presence of Cx43 mRNA by RT-PCR and the presence of Cx43 protein by Western Blot in CD＋tk＋Cx＋ cells. The killing effect of 5-Fc and GCV on CD＋tk＋Cx＋ cells was more effective than that on CD＋tk＋ cells both in vitro and in vivo. Conclusion： Double suicide genes system CD/5-Fc＋tk/GCV could induce remarkable killing effect on cholangiocarcinoma cells in vitro and transplantation tumors in vivo. The cotransfection of Cx43 gene could enhance the bystander effect and hence the inhibition of carcinoma cells.     

Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1(-)E3(-) backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.     

Exogenous wt-p53 enhances the antitumor effect of HSV-TK/GCV on C6 glioma cells

Objective To study on the antitumor effect of combining wt-p53 gene with suicide gene therapy (HSV-tk+GCV) for malignant gliomas. Methods AdCMV-p53 was transfected into C6 glioma cells at MOI of (Multiplicity of infection) 0(G100), 10(TPG1), 100(TPG2), then AdCMV-tk was transducted to C6 glioma cells of G100, TPG1 and TPG2, respectively, at MOI of 100. The C6 glioma cells tranfected with both AdCMV-p53 and AdCMV-tk were exposed to various concentration of GCV. The cell survival rate was measured by MTT assay in vitro. Rat glioma model was established by injecting 5 × 10⁵ C6 glioma cells into right caudate nucleus of SD rats. AdCMV-p53 and AdCMV-tk were injected into glioma on day 5 and 6, respectively. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/kg/day for 14 days. The survival time of all rats was observed. The growth of intracerebral tumors was monitored dynamically by enhanced MRI. Cell apoptosis was evaluated by TUNEL method. Expression of HSV-tk gene was identified by in situ hybridization and expression of exogenous p53 gene was detected with Western blotting. Results In vitro, wt-p53 significantly enhanced antitumor effect of HSV-tk/GCV. The concentration of GCV for ID50 of TPG2 cells (0.001 μg/ml GCV) was 10 times lower than that for the cells of tk-GCV group (MOI = 100), while the concentration of GCV for ID100 of TPG2 (0.01 μg/ml GCV) and TPG1(0.1 μg/ml GCV) was 100 and 10 times lower than that for the cells of tk-GCV group (MOI = 100), respectively. Apoptosis of C6 glioma cells also could be induced by transfection with wt-p53 gene slightly. For in vivo study, the survival time of tumor-bearing rats treated with HSV-TK/GCV or wt-p53 combined with HSV-TK/GCV was significantly prolonged and the intracerebral tumors were regressed and disappeared earlier in the combined gene therapy group than those in the HSV-TK/GCV therapy group as shown in enhanced MRI. However, only half dose of GCV for the rats treated with both wt-p53 and HSV-TK/GCV was needed to obtain the same efficacy as those rats treated with HSV-TK/GCV alone. These results indicate that the transfection of wt-p53 potentiates the effect of HSV-TK/GCV therapy. Conclusions The combination of HSV-tk/GCV system with wt-p53 gene transduction is optimal for clinical therapeutic trials of suicide gene therapy for malignant gliomas.