Triple-mutated oncolytic herpes virus for treating both fast- and slow-growing tumors

Oncolytic virus therapy has emerged as a promising treatment option against cancer. To date, oncolytic viruses have been developed for malignant tumors, but the need for this new therapeutic modality also exists for benign and slow-growing tumors. G47∆ is an oncolytic herpes simplex virus type 1 (HSV-1) with an enhanced replication capability highly selective to tumor cells due to genetically engineered, triple mutations in the γ34.5, ICP6 and α47 genes. To create a powerful, but safe oncolytic HSV-1 that replicates efficiently in tumors regardless of growth speed, we used a bacterial artificial chromosome system that allows a desired promoter to regulate the expression of the ICP6 gene in the G47∆ backbone. Restoration of the ICP6 function in a tumor-specific manner using the hTERT promoter led to a highly capable oncolytic HSV-1. T-hTERT was more efficacious in the slow-growing OS-RC-2 and DU145 tumors than the control viruses, while retaining a high efficacy in the fast-growing U87MG tumors. The safety features are also retained, as T-hTERT proved safe when inoculated into the brain of HSV-1 sensitive A/J mice. This new technology should facilitate the use of oncolytic HSV-1 for all tumors irrespective of growth speed.     

端粒酶催化亚单位基因转染对人胚胎成纤维细胞表型及寿命的影响

目的探讨外源性人端粒酶蛋白催化亚单位(hTERT)基因转染对人胚胎成纤维细胞(hEFs)寿命的影响以及基因转染后细胞是否存在恶性表型。方法应用脂质体法将pIRES2-EGFP-hTERT正义重组质粒及pIRES2-EGFP空载质粒分别导入原代培养hEFs,Westernblot检测hTERT蛋白及I、III型胶原表达。染色体核型分析、裸鼠皮下致瘤性实验、DNA倍体实验以及形态学观察分析转染细胞是否存在恶性表型。结果外源性hTERT基因转染的胚胎成纤维细胞hTERT蛋白和I、III型胶原表达增加,体外传代次数增加,传至75代仍保持以2d的倍增周期生长,并且染色体仍为23对,均为正常二倍体细胞;裸鼠皮下不具有成瘤的能力;而hEFs和空载转染细胞传至60代左右平均五六天传代1次,出现生长停滞现象。结论外源性hTERT基因转染使胎儿成纤维细胞增殖旺盛,寿命延长,且不具有恶性表型。     

survivin和hTERT在大肠黏膜癌变中表达及其与c-myc关系

目的研究survivin和端粒酶催化亚单位(hTERT)在大肠黏膜癌变中表达及其与c—myc关系。方法采用原位杂交和免疫组化(SP法)检测hTERTmRNA、survivin、c—myc蛋白在45例大肠腺癌、20例腺瘤、10例正常组织的表达，并对各指标阳性率和相关性分析。结果肠腺癌中stirvivin阳性率66．7％，hTERTmRNA85％，c—myc53％，三者在腺瘤中分别为30％、45％、20％，正常黏膜为0％、10％、0％，stirvivIn和hTERTmRNA表达与大肠癌的分化程度及临床参数无明显相关性，与c—myc明显相关，Survivin与hTERTmRNA呈正相关性(x^2=10．235，P=0．001)。结论Survivin和hTERT在大肠癌中高表达，非癌组织中低表达，可成为诊断治疗的新靶点，但不能作为肿瘤进展的标志物，survivin、hTERT、myc可能在大肠癌变中起协同作用。     

端粒酶hTERT mRNA在乳腺癌中的表达及与c-myc相关性分析

目的 探讨人乳腺癌中原癌基因c-myc与hTERT mRNA表达的关系.方法 收集癌旁正常乳腺组织、导管原位癌、浸润性导管癌各12、18和25例,用原位杂交法检测hTERT mRNA表达,并用免疫组织化学法检测乳腺导管癌c-myc表达.结果 ①hTERT mRNA在癌旁正常乳腺组织中未见表达,在导管原位癌、浸润癌中的阳性率分别为83.33%和88.00%,后两组hTERT mRNA表达明显高于正常组;②hTERT mRNA表达与浸润性导管癌肿块大小及淋巴结转移与否无相关性（P＞0.05）;③43例乳腺导管癌中hTERT mRNA与c-myc表达呈正相关（γ=0.388 4,P＜0.05）.结论 端粒酶hTERT的表达可能在乳腺导管癌的组织发生中起关键作用,c-myc的过度表达可能与乳腺导管癌hTERT基因转录激活有关.     

携带hTERT I540-PTD表达框重组复制缺陷型腺病毒的构建与鉴定

目的：构建携带人端粒酶逆转录酶亚基（hTERT）I540-PTD,I540-PTD4融合基因表达框的重组复制缺陷型腺病毒.方法：将pDC315-I540-PTD,pDC315-I540-PTD4与腺病毒基因组质粒pBHGloxdeltaE13Cre共转染HEK293细胞,出现典型的细胞病变反应后收获细胞,反复冻融法制备病毒粗提液,经扩增和纯化获得重组复制缺陷型腺病毒Ad-I540-PTD和Ad-I540-PTD4.使用终点稀释实验和空斑形成实验测定纯化后病毒滴度.采用聚合酶链式反应（PCR）对Ad-I540-PTD,Ad-I540-PTD4进行鉴定.使用病毒感染人骨肉瘤U2OS细胞后,进行免疫组化定性并观察I540-PTD,I540-PTD4的表达情况.结果：共转染10d后,HEK293细胞出现典型的细胞病变反应.经扩增、纯化后,终点稀释法和孔板形成法测定Ad-I540-PTD滴度分别为（8.9×10^12）,（6.3×10^12）pfu/L;Ad-I540-PTD4滴度分别为（5.8×10^12）,（5.4×10^12）pfu/L.以纯化的病毒为模板,使用自行设计的鉴定引物进行PCR均获得167bp目标片段.抗组胺酸标签抗体对病毒感染后的U2OS细胞免疫组化分析发现Ad-I540-PTD,Ad-I540-PTD4转染的U2OS细胞均呈强染,表明Ad-I540-PTD,Ad-I540-PTD4中携带的I540-PTD,I540-PTD4真核表达读框可在人类细胞内高效表达.结论：成功构建可高效表达I540-PTD,hTERTI540-PTD4的重组复制缺陷型重组腺病毒Ad-I540-PTD,Ad-I540-PTD4,为其作为基因疫苗诱发针对hTERT I540表位的抗瘤免疫奠定了基础.     

吲哚美辛和hTERT-siRNA对胆囊癌细胞的作用

目的研究吲哚美辛和hTERT-siRNA对胆囊癌细胞株GBC-SD的端粒酶活性、细胞增殖、运动的抑制作用;并试从β-catenin／hTERT信号转导途径揭示其分子机制。方法采用端粒酶TRAP、半定量RT-PCR和Western Blot印迹方法检测GBC-SD端粒酶活性、hTERT基因和β-catenin基因mRNA、蛋白质表达水平。四甲基偶氮唑盐（MTT）光吸收法检测琥珀酸脱氢酶（SDH）活性并做细胞增殖计数、运用Transwell小室和划线了解癌细胞侵袭与运动情况。结果质粒pGCsi-H1／GFP-hTERT转染GBC-SD细胞6～24h再加入吲哚美辛后对GBC-SD细胞的端粒酶、SDH活性、细胞增殖、运动、侵袭力均具有很明显的抑制作用,分别同阴性对照pGC-si-H1／NEGative联合吲哚美辛组、单用吲哚美辛组进行比较,差异有显著性（P〈0．01）。pGCsi-H1／GFP-hTERT作用于GBC-SD细胞24h再加入吲哚美辛,检测hTERT mRNA和hTERT蛋白表达,分别与pGCsi-H1／NEGative联合吲哚美辛组、单用吲哚美辛组进行比较,差异有显著性（P〈0．01）。pGCsi-H1／GFP-hTERT作用于GBC-SD细胞24h再加入吲哚美辛,检测β-catenin mRNA和p-catenin蛋白表达．分别与pGCsi-H1／NEGative联合吲哚美辛组、单用吲哚美辛组进行比较,无明显差异（P〉0．05）。结论吲哚美辛可增强hTERT-siRNA抑制GBC-SD细胞端粒酶、SDH的活性,及其增殖、运动、侵袭力,降低hTERTmRNA和hTERT蛋白表达,其机制与β-catenin／hTERT信号转导途径受抑存在一定联系。     

Expression of a mutant hTERT in human bladder carcinoma cell line T24 and its clinical significance

Objective: To construct a mutant pEGFP- hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods:PCR amplification was performed by using primers based on the known gene sequence of hTERT. PCR production was cloned into plasmid pGEMT-T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sa/I sites of the pEGFP-C1 vector. After transfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and senescence correlated [3-galactosidase staining. Results: Identification of pEGFP-hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated ~-galactosidase in transfected cells gradually increased with extended cultured time and cell growth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.