RNA干扰抑制端粒酶对受照后端粒长度的影响

目的 探索RNA干扰体内表达质粒抑制端粒酶表达的作用,以及端粒酶抑制后射线对端粒长度的影响.方法 构建RNA干扰体内表达质粒,脂质体介导转染A549细胞系,TRAPELISA法检测端粒酶活性,RT-PCR法检测hTERTmRNA表达,端粒限制性片段平均长度分析法检测端粒长度.结果 pPUR/hTERT质粒在A549转染株内表达可下调hTERT mRNA表达,抑制端粒酶活性;A549照射后端粒明显延长,而pPUR/hTERT表达株照射后未见端粒延长,抑制了射线诱导端粒延长作用.结论 RNA干扰体内表达质粒可抑制端粒酶表达及照射后端粒延长,表明射线诱导端粒延长可能是端粒酶在端粒断裂端合成端粒序列的结果.     

反义端粒酶RNA抑制胶质瘤细胞系BT—325细胞端粒酶活性表达

目的：观察反义端粒酶RNA对BT－325细胞的作用,探讨粒酶在恶性胶质瘤发生过程中的可能作用及反义端粒酶RNA在恶性胶质瘤基因治疗中的意义。方法：经脂质体介导的方法将pBBS 212-hTR(反义端粒酶RNA真核表达载体）导入BT－325细胞中,细胞克隆转移扩大培养后,采用TRAP－PAGE电泳,电镜和TUNEL技术检测BT－325／pBBS 212-hTR的细胞的端粒活性表达,细胞凋亡的情况。结果：与对照组比较,反义端粒酶RNA显著抑制BT－325细胞端粒酶活性,诱发细胞发生凋亡。结论：转染反义端粒酶RNA在封闭细胞粒酶活性表达的同时,可促进BT－325细胞凋亡,在研究恶性胶质瘤发病机制及治疗方面做了有益的探讨。     

电离辐射诱导端粒延长作用的研究

目的观察人细胞系A549照射后端粒酶活性的变化及其对端粒长度的影响。方法采用端粒重复序列扩增法（TRAP）检测照射前、后不同时间点细胞端粒酶活性，端粒限制性片段平均长度分析法（耵谭）检测端粒长度。结果在1—5Gy剂量范围内A549端粒酶活性表达呈剂量和时间依赖性增强；照射后TRF延长，照射剂量越高，TRF延长出现越早，随着时间的延长，较低剂量组耵谭也明显延长，表明这种诱导作用与端粒酶表达增强有相近似的剂量效应和时间效应。结论端粒放射损伤可通过端粒序列的合成（延长端粒）进行修复；端粒酶可能在端粒放射损伤修复中起到重要作用。     

转染P21WAF1基因对BT—325细胞端粒酶表达的影响

目的：观察外源性p231WAF1基因对BT－325细胞的作用,探讨p21WAF1表达水平的改变对细胞端粒酶活性的影响。方法：经脂质体介导的方法将PCEP－WAF1质粒导入BT－325细胞中,细胞克隆转移扩大培养后,采用TRAP－PCR－ELISA、TUNEL和电镜等技术检测外源性p21WAF1基因对BT－325细胞端粒活性和细胞凋亡的作用。结果：较对照组相比,转染外源性p21WAF1基因的BT－325细胞端粒酶表达水平显著下降,伴有显著细胞凋亡现象。结论：外源性p21WAF1基因的表达可促进BT－325细胞端粒酶活性降低,诱导细胞显著凋亡,提示端粒酶的活性表达可能与细胞周期调控存在密切联系。     

白藜芦醇的辐射防护及其分子机理的研究

目的 研究白藜芦醇（RES）对受照射小鼠的辐射防护作用及其分子作用机理。方法 采用^60Coγ射线照射小鼠模型，观察小鼠30d存活和残废动物存活时间。用原位末端标记法观察受照射小鼠脾细胞凋亡，流式细胞仪检测脾细胞凋亡率和凋亡相关蛋白的表达水平，同时检测脾细胞内凋亡相关酶活性大小。结果 照射前给予RES能明显提高受照射小鼠的存活率，延长死亡动物存活时间。RES能明显抑制受照射小鼠脾细胞凋亡，提高Bcl-2表达水平，对于Fas表达无明显影响，同时脾细胞内的Caspase-3和Caspase-8活性明显升高。结论 RES具有明显的辐射防护途径，其作用机理与抑制加速器射敏感细胞的细胞凋亡有关。     

全反式维甲酸诱导对胃癌细胞株端粒酶活性的影响

端粒酶（Ｔｅｌｏｍｅｒａｓｅ）是近年来发现的人体内唯一由ＲＮＡ和蛋白质组成的具有逆转录酶活性的核糖核蛋白酶（Ｒｉｂｏｎｕｃｌｅｏｐｒｏｔｅｉｎ）。在正常人体细胞中端粒酶一般不表达或活性极低,而在肿瘤组织中则明显增高,肿瘤组织中端粒酶的活化可以维持染色体端粒长度的动态平衡,因此,端粒酶活化作为肿瘤细胞无限制增殖的一个重要条件有可能是肿瘤发生过程中的一条必经通路［１］。维甲酸具有逆转肿瘤细胞的作用。本研究以维甲酸体外诱导的ＳＧＣ－７９０１细胞为模型,研究诱导后肿瘤细胞端粒酶活性的变化,并拟从端粒酶亚…     

低剂量辐射对细胞凋亡相关基因蛋白表达的影响

目的研究低剂量辐射对细胞凋亡相关基因蛋白ｐ５３、ｂｃｌ－２和ｂａｘ表达的影响，以揭示低剂量辐射免疫兴奋效应以及辐射诱导细胞凋亡“Ｊ”型曲线的分子机理。方法采用间接免疫荧光流式细胞术。结果７５ｍＧｙＸ射线全身照射后２４小时，ｐ５３基因蛋白产物的表达显著降低，ｂｃｌ－２基因蛋白表达呈增高趋势，ｂａｘ基因蛋白表达呈降低趋势，但均无统计学意义。然而，ｂｃｌ－２／ｂａｘ比值则明显增加。结论低剂量辐射全身后，ｐ５３和ｂｃｌ－２／ｂａｘ比值的变化表明：ｐ５３、ｂｃｌ－２／ｂａｘ在辐射诱导细胞凋亡的调控中起着重要作用。该结果为揭示低剂量辐射免疫兴奋效应和辐射诱导细胞凋亡“Ｊ”型曲线的分子机理提供了有意义的实验数据     

骨关节病滑膜细胞凋亡和癌基因表达

为了探索骨关节病在细胞和分子水平上的发病机理，观察了骨关节病的关节滑膜细胞的凋亡和癌基因表达。采用ＤＮＡ末端转移酶介导的凋亡细胞原位检测技术，检测正常滑膜和病理滑膜细胞程序化死亡，同时，采用免疫组化法观察ｐ５３，ｅｒｂＢ－２和ＰＣＮＡ在滑膜细胞中的表达。结果发现骨关节病中有，７７．８％出现滑膜细胞凋亡，与正常对照有明显差别。病理组中癌基因表达昨３阳性占７１．４％，ｅｒｂＢ－２阳性占７１．４％，ＰＣＮＡ在正常和病理滑膜细胞中均呈阴性表达。说明细胞凋亡和多种癌基因的产物有关，ｐ５３和ｅｒｂＢ－２对细胞凋亡有调节作用。     

胃癌、结肠癌中端粒酶活性的检测及临床意义

目的:检测胃癌、结肠癌及其相应正常组织中端粒酶活性,探讨其在胃癌、结肠癌发生中的作川以及与临床病理特征之间的关系。方法:采用PCR-TRAP法检测92例胃癌、结肠癌和61例正常组织的端粒酶活性,并州端粒酶活性与临床病理特征关系进行分析。结果:胃癌和结肠癌中端粒酶活性阳性率为84.9％,而正常组织为9.83％,胃癌和结肠癌组织端粒酶活性显著高于正常组织,P<0.01;胃癌、结肠癌组织中端粒酶活性在不同分化程度及淋巴转移之间未发现显著差异,P>0.05。结论:端粒酶在胃癌、结肠癌的发生中起重要作用;端粒酶在胃癌、结肠癌组织中高表达,而在正常胃肠组织低表达的特性,使其有望成为一种较为理想的胃肠道肿瘤诊断的标志物。     

大蒜素对人胃腺癌SGC-7901细胞株端粒酶活性和细胞凋亡的影响

为观察大蒜素对胃腺癌SGC 790 1细胞端粒酶活性和细胞凋亡的影响。采用不同浓度的大蒜素处理SGC 790 1细胞后 ,用流式细胞仪检测细胞周期和凋亡的改变 ;用TRAP PCR ELISA法检测端粒酶活性的变化。结果显示 ,大蒜素能够改变细胞周期的分布 ,增加G2 /M期细胞比例 ;呈剂量依赖性诱导其凋亡 ;大蒜素对端粒酶活性的抑制作用也有时间和剂量依赖性。提示大蒜素能够抑制端粒酶的活性 ,促进细胞凋亡     

Radiation-induced effects on telomerase in gynecological cancer cell lines with different radiosensitivity and repair capacity

PURPOSE: Telomerase activation in response to irradiation might enhance the radioresistance of cells. Thus, we have investigated radiation-induced effects on telomerase in six gynecological cancer cell lines, with different intrinsic radiosensitivity and capacity for sublethal damage repair (SLDR). MATERIALS AND METHODS: Three endometrial adenocarcinoma (UM-EC-1, UT-EC-2B and UT-EC-3) and three vulvar squamous cell carcinoma (A431, UM-SCV-2 and UM-SCV-7) cell lines were irradiated with doses of 5, 10 and 25 Gy and the effects on telomerase were evaluated at 0.5, 6, 24 and 48 h post-irradiation. Telomerase activity was quantitatively measured by SYBR Green real-time telomeric repeat amplification protocol. RESULTS: The most radioresistant cell line A431 had the strongest stimulatory effects (approximately 2.0 - 2.5-fold) on telomerase activity 24 and 48 h post-irradiation with the highest radiation doses. In contrast to that, telomerase activities in the highly radiosensitive cell line UT-EC-2B remained below the basal level throughout the 48-h period of post-irradiation with the highest doses, and even a decline to approximately 50% of the basal level was found 24 h after exposure. In other cell lines being either moderately or highly radiation resistant, telomerase activity levels in response to irradiation remained mainly at the basal level or gradually increased. CONCLUSIONS: The present findings indicate that there might be a connection between the radiation-induced telomerase response and radiosensitivity. However, no correlation was found between the radiation-induced effects on telomerase and the sublethal damage repair capacity of the cells.     

大肠癌组织中的端粒酶活性变化

目的：研究端粒酶活性与结直肠癌恶性进展的关系。方法：应用端粒重复扩增（ＴＲＡＰ）方法检测２２例大肠腺瘤、４３例结直肠癌及邻近组织中的端粒酶活性,并分析其与临床病理的关系。结果：端粒酶活性阳性率在大肠癌中为９５．４％,腺瘤中为９．１％,而邻近组织中仅为２．３％,在低、未分化癌及粘液腺癌中端粒酶活性呈中、强表达者占多,而在中、高分化癌包括乳头状腺癌及管状腺癌中存在相当低、弱表达,但其总的表达在大肠癌Ｄｕｋｅｓ各期中无甚差异。结论：大肠癌的恶性转化与端粒酶的活化有关,检测端粒酶活性对临床大肠癌尤其是早期大肠癌具有特别重要的诊断意义。     

Telomerase--new diagnostic and therapeutic options in oncology

One of the new directions in study of malignant tumor pathogenesis is telomerase theory. Investigations in the culture of human cells have shown that telomerase activation can lead to tumor development. Prospective investigations of telomerase could be able to supplement the data about tumor pathomorphology and will permit to conduct monitoring on patients with malignant tumors by studying telomerase in fractionated cells from different body tissues and biological media. Telomerase inhibitors are possible drugs for antitumoral therapy. It is supposed that the abovementioned agents will decrease telomerase activity of proliferating tumoral cells that will cause destabilization of their genome, stopping of proliferation and death of tumoral clone.     

癌细胞株及胃癌组织端粒酶催化亚基基因表达研究

目的：建立人端普酶催化亚基蛋白质ＲＮＡ逆转录聚合酶链反应（ＲＴ－ＰＣＲ）,进行恶性肿瘤细胞和胃癌组织检测,探讨其在癌变中的作用,提供一种癌症检测的新方法。方法：制备癌细胞株和胃癌组织端粒酶提取液和总ＲＮＡ,运用端粒重复扩增法（ＴＲＡＰ）和ｈＴＲＴ ＲＮＡ ＲＴ－ＰＣＲ扩增法检测癌细胞株和胃癌组织端粒酶活性和ｈＴＲＴ ＴＮＡ表达。结果：８株癌细胞和６例胃癌组织均有端粒酶活性和ｈＴＲＴ ＲＮＡ表达;９     

Radiation-induced effects on telomerase in gynecological cancer cell lines with different radiosensitivity and repair capacity

Purpose: Telomerase activation in response to irradiation might enhance the radioresistance of cells. Thus, we have investigated radiation-induced effects on telomerase in six gynecological cancer cell lines, with different intrinsic radiosensitivity and capacity for sublethal damage repair (SLDR).

Materials and methods: Three endometrial adenocarcinoma (UM-EC-1, UT-EC-2B and UT-EC-3) and three vulvar squamous cell carcinoma (A431, UM-SCV-2 and UM-SCV-7) cell lines were irradiated with doses of 5, 10 and 25 Gy and the effects on telomerase were evaluated at 0.5, 6, 24 and 48 h post-irradiation. Telomerase activity was quantitatively measured by SYBR Green real-time telomeric repeat amplification protocol.

Results: The most radioresistant cell line A431 had the strongest stimulatory effects (～2.0 – 2.5-fold) on telomerase activity 24 and 48 h post-irradiation with the highest radiation doses. In contrast to that, telomerase activities in the highly radiosensitive cell line UT-EC-2B remained below the basal level throughout the 48-h period of post-irradiation with the highest doses, and even a decline to ～50% of the basal level was found 24 h after exposure. In other cell lines being either moderately or highly radiation resistant, telomerase activity levels in response to irradiation remained mainly at the basal level or gradually increased.

Conclusions: The present findings indicate that there might be a connection between the radiation-induced telomerase response and radiosensitivity. However, no correlation was found between the radiation-induced effects on telomerase and the sublethal damage repair capacity of the cells.