人端粒酶逆转录酶在肿瘤免疫治疗中的应用

寻找有效的抗原表位是近期肿瘤免疫治疗的热点．人端粒酶逆转录酶(hTERT)的免疫学特性使其成为肿瘤免疫治疗中一个吸引人的目标。从人和鼠系统获得的数据证实，细胞毒淋巴细胞(CTL)能识别hTERT特异的肽并杀死多种组织类型hTERT表达的肿瘤细胞。由于hTERT在人肿瘤组织中的广泛表达，在极少正常组织的低水平表达，临床试验已开始检验将hTERT作为肿瘤免疫治疗目标的可行程度。近期树突状细胞(DC)转运系统及其相关技术的发展和成熟提供了一个快速有效筛选抗原肽的方法。     

构建MYC反应元件修饰hTERT核心启动子引导Fcy1表达的腺病毒载体

目的：构建串联的MYC反应元件修饰人端粒酶逆转录酶（hTERT)核心启动子引导酵母胞嘧啶脱氨酶基因Fcy1表达的复制缺陷型腺病毒载体。方法：将串联的MYC反应元件修饰的hTERT核心启动子及下游酵母胞嘧啶脱氨酶基因Fcy1克隆到穿梭质粒pDC316上，上辅助质粒pBHGlox(delta)E1,3Cre共转染HEK293细胞获得重组复制缺陷型腺病毒并在HEK293细胞中扩增重组病毒。PCR法及斑形成实验鉴定重组腺病毒和病毒滴度。结果：成功获得由6倍和18倍MYC反应元件修饰hTERT核心启动子引导Fcy1基因表达的复制缺陷型腺病毒载体。结论：MYC反应元件修饰hTERT核心启动子引导Fcy1基因表达的腺病毒载体为进一步研究骨肉瘤转录靶向基因治疗奠定了基础。     

肝癌组织端粒酶逆转录酶hTERT基因及GGT-mRNA-H亚型表达与肝癌早期诊断的研究

目的：研究肝癌组织中端粒酶逆转录酶hTERT基因及谷氨酰转移酶GGTmRNA-H亚型表达，探讨其在肝癌早期诊断中的意义。方法：应用RT—PCR法，检测65例原发性肝癌患者癌组织及癌周组织、108例良性肝病肝组织、6例健康对照肝组织的hTERT与GGTmRNA—H亚型，观察这两种基因的表达情况。结果：在65例原发性肝癌中，癌组织hTERT阳性56例（86．2％），GGTmRNA-H亚型阳性6l例（93．8％），AFP阳性40例（61．5％）（P〈0．05）；hTERT在20例小肝癌癌组织阳性表达16例（80．0％），GGTmRNA—H亚型阳性表达18例（90．0％），与健康对照组差异有统计学意义（P〈0．05）。急慢性肝炎组、肝硬化组与健康对照组比较，端粒酶逆转录酶、GGTmRNA-H亚型阳性表达率虽明显升高，但其差异无统计学意义（P〉0．05）。结论：肝癌组织中hTERT、GGTmRNA-H亚型联合分析可弥补AFP检测的不足，对提高肝癌、癌前病变的早期诊断具有重要意义。小肝癌中端粒酶活性增高在癌形成中扮演重要的角色。hTERT基因检测比AFP更敏感、特异性更高，有望成为肝癌早期诊断的理想指标。     

Human telomerase catalytic subunit gene re-expression is an early event in oral carcinogenesis

AIMS: Detection of telomerase catalytic subunit (hTERT) mRNA has been used as a surrogate marker for estimation of telomerase activity. The exact role and timing of telomerase re-activation, a key enzyme implicated in cellular immortalization and transformation, in the multistep process of oral carcinogenesis is still unknown. The aim was to test the hypothesis that (i) quantitative rather than qualitative differences exist in the level of hTERT mRNA expression between normal oral mucosa, different grades of oral epithelial abnormalities and squamous cell carcinomas of the oral cavity, and that (ii) hTERT gene re-expression is an important, probably early event in oral carcinogenesis. METHODS AND RESULTS: The relative quantity of hTERT mRNA was analysed in 45 frozen oral epithelia representing different morphological stages of oral carcinogenesis classified according to the Ljubljana classification and in 37 oral squamous cell carcinomas, using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. hTERT mRNA was not detected in normal or reactive hyperplastic oral epithelia, but was present in 43% of atypical hyperplasias (premalignant lesions), 60% of intraepithelial carcinomas and 68% of oral squamous cell carcinomas. Statistical analysis revealed two groups of oral epithelial changes, with significant differences in the levels of hTERT mRNA expression: 1, normal and reactive hyperplastic oral epithelium, and 2, atypical hyperplasia, intraepithelial carcinomas and squamous cell carcinomas. CONCLUSION: These data suggest that hTERT gene re-expression represents an early event in the multistep process of oral carcinogenesis, already detectable at the stage of precancerous oral epithelial changes. Nevertheless, other genetic aberrations appear to be necessary for progression of oral epithelial abnormalities towards invasive squamous cell carcinoma.     

肝细胞肝癌中hTERT基因表达及其与肿瘤临床病理特征的关系

目的:研究hTERT基因在肝细胞肝癌中的表达,探讨其在肝细胞肝癌发生发展中的作用及意义。方法:应用免疫组化(SP法)和半定量逆转录聚合酶链反应(RTPCR)同步检测62例肝细胞肝癌及对应癌旁组织、8例正常肝组织中hTERT蛋白及hTERTmRNA的表达情况,分析hTERT基因的表达与肝细胞肝癌临床病理参数的关系。结果:hTERT蛋白主要定位于肝癌细胞胞质内,偶见核内染色。hTERT蛋白和hTERTmRNA在肝细胞肝癌中的阳性率分别为88.71%(55/62)、82.26%(51/62),分别明显高于癌旁肝组织的阳性率9.68%(6/62)、6.45%(4/62),差异具有非常显著性(P<0.01)。hTERT蛋白及其mRNA在8例正常肝组织均未见表达,阳性率为0.00%(0/8)。统计学分析显示,在肝细胞肝癌中hTERT基因的表达与肿瘤分期分级、门静脉有无癌栓、血清中甲胎蛋白表达水平、是否合并HBV感染等有关(P<0.05),与患者年龄、性别、肿瘤大小等因素无关。结论:hTERT基因可能在肝细胞肝癌的发生发展中起着重要作用,其表达有望成为肝细胞肝癌的重要分子生物学指标。     

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。     

子宫内膜癌中的Bmi-1表达及其与hTERT的关系

目的:研究Bmi-1、hTERT在子宫内膜癌、子宫内膜不典型增生、正常子宫内膜组织中的表达。方法:应用免疫组化检测105例子宫内膜癌、40例子宫内膜不典型增生、20例正常子宫内膜组织中Bmi-1、hTERT的表达。结果:在子宫内膜癌、子宫内膜不典型增生和正常子宫内膜组织中Bmi-1、hTERT阳性率分别为86.7%、55%和25%，94.3%、72.5%和5%。Bmi-1在子宫内膜癌和子宫内膜不典型增生中的表达均高于正常子宫内膜组，差异有统计学意义（P<0.05）。子宫内膜癌中，Bmi-1与浸润深度、血管浸润相关，差异有统计学意义（P<0.05），hTERT与组织学分级、组织学类型、浸润深度和血管浸润相关（P<0.05）。Bmi-1和hTERT呈正相关（P<0.01)。结论:Bmi-1和hTERT共同参与子宫内膜病变的恶性转化，联合检测Bmi-1和hTERT对早期判断子宫内膜癌的发生、发展有一定价值。     

Telomerase activity,telomere length and hTERT DNA methylation in peripheral blood mononuclear cells from monozygotic twins with discordant smoking habits

Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter‐individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co‐twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co‐twins were contrasted, selecting as variables the intra‐twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke‐induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551–559, 2017. © 2017 Wiley Periodicals, Inc.