hTERT、HPV与宫颈癌的研究进展

端粒酶活性与恶性肿瘤密切相关,调节端粒酶活性的最主要成分是端粒酶逆转录酶(hTERT).宫颈癌中端粒酶活性高表达,而高危型人乳头瘤病毒(HPV)感染是导致宫颈癌的主要原因,HPVE6、E7通过整合到宿主细胞,可调节hTERT的转录,激活端粒酶,研究hTERT、HPV的相关性,有助于宫颈癌发病机理的阐明.     

端粒酶与宫颈癌的相关研究进展

端粒酶是一种能以自身RNA为模板逆转录合成端粒的核糖核酸酶,其激活可能是体细胞向肿瘤转变的关键步骤,是细胞癌变的早期事件。人端粒酶包括2个重要部分,即人端粒酶催化亚单位（hTERT）和人端粒酶RNA（hTERC）。研究揭示,hTERT和hTERC对维持端粒酶活性至关重要。发现端粒酶的活化、hTERT/hTERC的表达变化与宫颈癌的发生、发展有密切关系。开展三者在宫颈癌中的相关研究,将为宫颈癌早期筛查提供新思路和手段。     

端粒酶及其与宫颈癌关系研究进展

端粒酶是一种核糖核酸蛋白，可以自身RNA为模板逆转录合成端粒末端重复序列，维持端粒的长度。端粒酶的激活与细胞增殖和癌变关系密切，其活性受到多种因素调控，端粒酶催化亚单位hTERT可能是其激活的关键性限速步骤。在宫颈癌及其癌前病变中，端粒酶活性不同程度地升高，hTERT mRNA表达上调，并与高危型HPV感染、抑癌基因p53失活密切相关。端粒酶激活是高危型HPV感染后导致宫颈上皮内瘤变和宫颈癌的关键性步骤，端粒酶及其催化亚单位有可能成为宫颈癌筛查的一种重要肿瘤标志物。     

端粒酶与宫颈癌的相关研究进展

端粒酶是一种能以自身RNA为模板逆转录合成端粒的核糖核酸酶,其激活可能是体细胞向肿瘤转变的关键步骤．是细胞癌变的早期事件。人端粒酶包括2个重要部分,即人端粒酶催化亚单位（hTERT）和人端粒酶RNA（hTERC）。研究揭示,hTERT和hTERC对维持端粒酶活性至关重要。发现端粒酶的活化、hTERT／hTERC的表达变化与宫颈癌的发生、发展有密切关系。开展三者在宫颈癌中的相关研究,将为宫颈癌早期筛查提供新思路和手段。     

人端粒酶逆转录酶在子宫颈癌组织中的表达变化及其意义

目的研究端粒酶在宫颈癌及其癌前病变组织中的表达,探讨人端粒酶逆转录酶(hTERT)作为宫颈癌早期诊断标志物的可能性。方法采用RTPCR方法测定端粒酶mRNA在3株宫颈癌细胞、2株正常的人二倍体细胞、38例宫颈癌、16例宫颈上皮内瘤变(CIN)和20例正常宫颈组织中的表达水平;端粒重复序列扩增酶联免疫吸附法测定端粒酶活性;免疫组化链酶菌抗生物素蛋白过氧化物酶(SP)连接法检测3株宫颈癌细胞、2株正常的人二倍体细胞、21例正常宫颈组织、2例CIN和55例宫颈癌切片中hTERT蛋白的表达水平。结果hTERTmRNA在3株宫颈癌细胞中均表达,而在2株正常的人二倍体细胞中无表达,宫颈癌组织中的阳性表达率为81.6%,高于CIN的37.5%和正常宫颈组织的5.0%,差异有统计学意义(P<0.05);hTERTmRNA的阳性表达率与端粒酶活性有相关性(P<0.01);hTERT蛋白在3株宫颈癌细胞中有表达,2株正常的人二倍体细胞中无表达,正常宫颈组织中阳性表达率为4.8%、CIN为28.0%,均低于宫颈癌组织的65.5%,差异有统计学意义(P<0.05)。结论宫颈癌组织和细胞中hTERTmRNA和蛋白阳性表达率均高于正常宫颈组织,其有可能作为宫颈癌早期诊断的标志物。     

人宫颈癌脱落细胞端粒酶活性检测的意义

目的 研究宫颈上皮内瘤样病变(CIN )和宫颈癌脱落细胞端粒酶激活的意义。方法 采用端粒酶PCR-ELISA法检测13例CIN和17例宫颈癌脱落细胞端粒酶活性,并与相应的组织标本中端粒酶活性相比较,11例正常宫颈脱落细胞作为对照。结果8例(61.54％)CIN、14例(82.35％)宫颈癌脱落细胞表达端粒酶活性,正常宫颈无端粒酶活性,CIN、宫颈癌及正常宫颈脱落细胞端粒酶活性平均值分别为0.328±0.192、1.329±0.259和0.039±0.084,三组间端粒酶表达率和量存在显著差异。CINⅠ、Ⅱ、Ⅲ级之间端粒酶活性无明显差异。CIN及宫颈癌组织中端粒酶活性和相应脱落细胞一致。结论 端粒酶的激活是宫颈癌的早期改变,在宫颈癌发生发展过程中起重要作用;宫颈脱落细胞和组织中端粒酶活性可能成为宫颈癌早期诊断、预后判断和肿瘤浸润的标记物。     

端粒酶RNA反义核酸联合其催化亚单位反义核酸对子宫颈癌Hela细胞生长的抑制作用

目的　探讨端粒酶RNA反义核酸 (反义hTR)联合端粒酶RNA催化亚单位反义核酸(反义hTERT) ,对宫颈癌Hela细胞生长的抑制作用及作用机理。方法　实验分为空白对照、脂质体对照、正义hTR、正义hTERT、反义hTR、反义hTERT及反义hTR +反义hTERT组。分别采用四甲基偶氮唑蓝 (MTT)法、PCR 端粒重复序列扩增 (TRAP) 酶联免疫吸附实验 (ELISA)、吖啶橙染色法、流式细胞术检测宫颈癌Hela细胞转染反义hTR和反义hTERT后细胞的增殖、端粒酶活性及细胞形态学、细胞凋亡和细胞周期阻滞的变化。结果　宫颈癌Hela细胞转染端粒酶反义核酸后 ,出现明显的细胞增殖抑制、端粒酶活性下降、细胞凋亡形态学改变、细胞凋亡率升高及细胞周期阻滞。反义hTR、反义hTERT组与空白对照、脂质体对照及正义hTR、正义hTERT组比较 ,差异有统计学意义 (P <0 0 1)。Hela细胞转染反义核酸 (0 2 μmol/L) 3d后 ,反义hTR +反义hTERT组 ,细胞增殖抑制率、相对端粒酶活性 (相对于空白对照组 )、细胞凋亡率分别为 6 9 3%、19 6 %、2 8 6 % ,与反义hTR组和反义hTERT组比较 ,差异均有统计学意义 (P <0 0 1) ,Q值分别为 0 86 7、0 919、1 0 75。反义hTR +反义hTERT组G0 /G1期细胞比例增加。结论　端粒酶反义核酸能通过特异性抑制端粒酶活性、诱导细     

端粒酶逆转录酶与子宫内膜癌的研究进展

人端粒酶逆转录酶(hTERT)是端粒酶的核心组分,与包括子宫内膜癌在内的多种肿瘤的发生、发展密切相关。hTERT表达和端粒酶活性增高可以促进肿瘤的发生。检测hTERT表达和端粒酶活性有助于子宫内膜癌的诊断。调控hTERT转录,抑制端粒酶活性有望成为治疗子宫内膜癌的新方法。现将hTERT与子宫内膜癌的研究进展作一综述。     

子宫内膜样腺癌与正常子宫内膜中端粒酶hTERT mRNA和端粒酶活性的定量分析

端粒酶表达是癌症细胞逃逸正常细胞衰老的主要机制,对其研究已经成为肿瘤判断预后和诊断性标志物的重要课题。功能件端粒酶全酶由端粒酶RNA(hTR)和催化蛋白亚基(hTERT)组成,前者作为端粒DNA合成的模板引物。hTERTmRNA的表达与端粒酶酶活性的水平呈平行关系,提示诱导hTERT基因表达可调节端粒酶活性。为验证定量测定hTERT     

宫颈癌盆腔淋巴结微转移分子检测及其意义

目的:研究宫颈癌患者盆腔淋巴结人乳头状瘤病毒(HPV)和端粒酶活性的表达及其临床意义。方法:应用FQ-PCR和PCR-ELISA方法检测61例Ia~IIb期宫颈癌组织和盆腔淋巴结HPV载量和端粒酶活性表达,并与常规病理结果比较。同时取15例良性疾病患者的淋巴结作为阴性对照组;分析宫颈癌淋巴结转移与各种病理因素的关系。结果:宫颈癌淋巴结HPV16/18和端粒酶阳性率显著高于常规病理阳性率(P<0.01),对照组淋巴结HPV16/18和端粒酶均为阴性。常规病理阳性的淋巴结HPV16/18拷贝数和端粒酶活性高于病理阴性的淋巴结,但低于原发灶,差异有统计学意义。淋巴结HPV16/18和端粒酶表达呈显著正相关(r=0.60,P<0.01)。临床分期晚和癌灶浸润深的患者易发生淋巴结转移。结论:与常规病理相比,采用FQ-PCR和PCR-ELISA技术检测HPV和端粒酶活性表达能显著提高宫颈癌淋巴结微转移的检出率。     

人端粒酶逆转录酶启动子调控下胞嘧啶脱氨酶-胸苷激酶融合基因治疗卵巢癌的体外研究

目的　探讨人端粒酶逆转录酶 (hTERT)启动子调控下的融合双自杀基因———胞嘧啶脱氨酶 胸苷激酶 / 5 氟胞嘧啶 更昔洛韦 (CD TK/ 5 FC GCV)系统对人卵巢癌细胞及正常细胞的体外杀伤作用。方法　应用脂质体转染法将hTERT核心启动子报告质粒pBTdel 2 79转染人卵巢癌细胞系3AO、正常卵巢上皮细胞 (NOEC)和人胚肺成纤维细胞株HELF ,用荧光素酶分析检测hTERT启动子活性 ;应用RT PCR技术检测hTERTmRNA的表达水平。应用基因克隆技术分别构建hTERT启动子和巨细胞病毒 (CMV)启动子调控下的CD TK基因真核表达载体pBTdel 2 79 CD TK和pcDNA3 CD TK ,以及hTERT启动子调控下的TK基因表达载体pBTdel 2 79 TK。用四甲基偶氮唑蓝 (MTT)法和流式细胞术检测pBTdel 2 79 CD TK/ 5 FC GCV系统和pcDNA3 CD TK/ 5 FC GCV系统对上述 3种细胞不同的杀伤作用 ;应用RT PCR技术检测pBTdel 2 79 CD TK和pcDNA3 CD TK转染前后 3AO和NOEC细胞中CD和TK基因的表达情况。用扫描电子显微镜观察pBTdel 2 79 CD TK/ 5 FC GCV作用后 3AO细胞的形态变化。结果　卵巢癌细胞系 3AO中hTERT启动子活性增高 ,为 2 4 1% ,RT PCR检测hTERTmRNA为阳性 ,而在正常细胞NOEC和HELF中 ,hTERT启动子活性仅为 0 3%和 0 7% ,hTERTmRNA为阴性。与p     

VEGF- and LPA-induced telomerase in human ovarian cancer cells is Sp1-dependent

OBJECTIVE: Both vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) are secreted by ovarian cancer cells and are known to promote cancer cell growth though the exact mechanism(s) are not completely understood. Since telomerase, a ribonucleprotein expressed in 95% of ovarian cancers, plays an important role in cellular immortalization, growth, and tumor progression, we examined whether telomerase is a molecular target of LPA and VEGF in ovarian cancer. METHODS: Telomerase-positive ovarian carcinoma cell lines PA-1, SW 626, and one telomerase-negative, non-tumorigenic SV40 large-T antigen-transfected human ovarian surface epithelial (IOSE) cell line, FHIOSE 118, derived from normal ovarian surface epithelium were cultured with and without VEGF and LPA for 4 h and 24 h, respectively. Telomerase PCR-ELISA, RT-PCR, VEGF ELISA and luciferase assays were performed to determine the effect of VEGF and LPA on telomerase activity in ovarian cancer cells. Western blot analyses were used to examine the signaling pathway involved in telomerase regulation by VEGF and LPA. RESULTS: We report that: (1) both VEGF and LPA upregulate telomerase activity; (2) LPA induction of telomerase activity is VEGF-dependent; (3) VEGF and LPA induction of telomerase activity is ERK 1/2-dependent; and (4) Sp1 binding sites within the proximal 976- to 378-bp regions of the hTERT promoter are essential for VEGF- and LPA-induced hTERT promoter activity. CONCLUSION: Consequently, these data show the novel finding that VEGF can regulate telomerase activity in non-endothelial cells and that telomerase appears to be a novel molecular target of LPA.     

The telomerase activity and expression of hTERT gene can serve as indicators in the anti-cancer treatment of human ovarian cancer

OBJECTIVE: The aim of this study was to evaluate the clinicopathological significance of telomerase activity and expression of hTERT gene in human ovarian cancer. The potential value of them as indicators for chemotherapy in ovarian cancer cells was also studied. MATERIALS AND METHODS: A total of 73 samples and ovarian cancer cell lines HO-8910 and COC1 were studied. Telomerase activity was detected by PCR-TRAP-ELISA assay and the expression of the hTERT mRNA was analyzed by semi-quantitative RT-PCR. Alteration of the telomerase activity and hTERT mRNA were also analyzed in the ovarian cancer cells treated with different concentration and different time of cisplatin. Cytogenetic analysis was performed to compare the telomere status in the OH-8910 cells pre- and post-cisplatin treatment. The associations between these two markers and cisplatin induced-apoptosis were respectively analyzed in COC1 cells by the flow cytometry. RESULTS: Telomerase activity are highly increased in malignancy (0.795+/-0.168(A450-655 nm)) than borderline (0.389+/-0.174(A450-655 nm)), benign tumors (0.236+/-0.102(A450-655 nm)) and normal ovary (0.213+/-0.070(A450-655 nm)) (p < 0.05). Twenty samples showed detectable levels of hTERT. The hTERT gene positive lesion showed significantly higher telomerase activity than negative (p = 0.004). There is a significant correlation between the telomerase activity and expression of hTERT (r = 0.921). Both telomerase activity and expression of hTERT can reflect the chemotherapeutic effect of cisplatin in a time-dependent and dose-dependent manner. Treatment with 10 microM cisplatin, the hTERT mRNA decreased after 12h and completely disappeared after 48 h, whereas the telomerase activity did not decrease until 24h. Results from cytogenetic analysis and flow cytometry assay confirmed that the alterations of these two markers are associated with the anti-cancer treatment of cisplatin. CONCLUSION: Expression of hTERT gene is rate-limiting with the activation of telomerase. Both of they may be useful in the predicting of chemotherapeutic effect in ovarian cancer.