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.     

γ射线诱导细胞死亡过程中端粒酶活性的研究

目的 观察γ射线照射人肿瘤细胞和正常细胞后端粒酶的变化及其与细胞死亡的关系。方法 检测照射后不同时间细胞端粒酶活性，细胞内端粒酶原位表达以及死亡细胞总数和形态观察。结果 在1－5Gy吸收剂量范围内A549和L02细胞辐射诱导死亡以凋亡为主要途径；在凋亡发生的过程中，端粒酶原位检测显示60％－90％的细胞中存在强烈的端粒酶信号，TRAP检测示端粒酶活性表达增强，提示放射损伤可诱导端粒酶活性增高，高端粒酶活性可持续至细胞发生凋亡。结论 γ射线诱导人肿瘤和正常细胞凋亡可能不是通过直接抑制端粒酶活性的机理，辐射诱导端粒酶活性增高可能是细胞修复辐射损伤的机理之一。     

60Coγ射线对hOGG1低表达细胞株放射敏感性的影响

目的研究^60 Co γ射线对DNA碱基切除修复基因hOGG1低表达细胞株放射敏感性的影响.方法以A549细胞和通过稳定转染hOGG1核酶而获得的hOGG1低表达的A549-R细胞为研究对象,用MTT法测定不同剂量γ射线照射后两种细胞的存活率;单细胞凝胶电泳检测^60 Coγ射线处理后两种细胞DNA损伤与修复的差异;流式细胞术检测照射后两种细胞的周期分布、凋亡率和细胞增殖指数.结果照射后两种细胞的存活率均随照射剂量的增加而下降,但A549-R细胞组的存活率显著低于相同剂量组的A549细胞（P＜0.05）;所设剂量均可诱导细胞DNA损伤,但DNA迁移长度和彗星细胞率在两种细胞间差异无统计学意义（P＞0.05）;损伤后的修复在两种细胞间差异有统计学意义（P＜0.05）,A549-R细胞的修复能力远远低于A549细胞.流式细胞术检测结果表明：照射后两种细胞都表现为G0/G1期细胞阻滞,细胞凋亡随照射剂量的增加而增加,细胞增殖指数随照射剂量的增加而降低,这些变化均以A549-R细胞更为明显.结论hOGG1低表达使得细胞DNA修复能力降低,细胞周期阻滞于G0/G1期、细胞增殖减慢、凋亡增加、存活率下降,从而使细胞对^60Coγ射线的放射敏感性增强.     

端粒、端粒酶与再生

端粒位于真核细胞染色体末端，起保护染色体末端的作用。端粒酶位于端粒末端，作用是合成端粒DNA序列，以抵消或延缓端粒随细胞分裂的不断缩短。端粒酶活性的表达在一定程度上意味着细胞分裂、组织增生，甚至可以看作是损伤修复、再生的迹象。     

鳞癌细胞株放射敏感性与端粒酶活性及端粒长度变化的关系

目的探索放射抗拒鳞癌细胞株放射敏感性的改变与端粒酶活性、端粒长度变化的关系。方法以人喉鳞癌细胞株Hep2为实验对象,用γ射线反复照射获得具有放射抗拒性细胞株Hep2R,拟合细胞存活曲线比较两种细胞株的放射生物学参数及端粒酶抑制剂AZT(azidothymidine,叠氮胸苷)作用后的变化,用TRAP-ELISA法测定端粒酶活性(OD值),Southernblotting法分析端粒平均长度(TRF),比较端粒酶活性、端粒平均长度的变化。结果辐射诱导出一个具有放射抗拒性的Hep-2R细胞株,并已稳定传代培养30代以上且放射抗拒性能稳定,测得其SF2=0.6798、D0=3.24,Hep-2R细胞的端粒酶活性较Hep-2细胞升高(0.982±0.005vs0.604±0.015),端粒长度延长了2倍(11.12kbvs3.76kb),AZT作用后Hep2R细胞株SF2=0.4892、D0=2.51,端粒酶活性下降为0.708±0.011、端粒长度缩短为10.18kb。Hep2细胞对应的参数SF2=0.4148、D0=2.06,AZT作用后Hep2细胞SF2=0.3843、D0=1.81,端粒酶活性下降为0.364±0.003、端粒长度缩短为2.76kb。结论辐射诱导细胞获放射抗拒性后其端粒酶活性升高、端粒平均长度增长,端粒酶抑制剂能通过降低端粒酶活性、缩短端粒长度而对其有增敏效应。     

γ射线对小鼠主动脉性腺中肾区基质细胞氨基肽酶N/CD13活性的影响

目的探讨小鼠主动脉性腺中肾（AGM）区基质细胞氨基肽酶N／CD13（APN／CD13）的表达和放射损伤前后该酶活性的变化及意义。方法采用免疫组织化学染色、RT-PCR检测AGM区基质细胞APN／CD13的表达；^60Coγ射线8．0Gy照射细胞，在不同的时间点用分光光度计检测APN／CD13的酶活性。结果AGM区基质细胞APN／CD13呈强阳性表达；放射损伤后酶活性呈一过性降低，继而升高，4h达高峰，24—48h基本恢复至照射前水平。结论APN／CD13在AGM区基质细胞上呈强阳性表达；在放射损伤后酶活性增高，可能是促进造血功能恢复的一种代偿机制。     

高效表达的NO对肺腺癌细胞A549放射敏感性的影响

目的 研究同步、高效表达的一氧化氮（NO）对肺腺癌细胞A549放射敏感性的影响,探寻增强肿瘤放射敏感性的新途径.方法 利用前期构建的可调控目的 基因高效表达的载体pfos-iNOS/GFP转染肺腺癌细胞A549,将阳性转染细胞和未转染细胞给予2 Gy X线照射后,检测细胞照射前后不同时相点NO的产量;给予不同剂量的X线照射后,检测照射后各组细胞的存活率,根据细胞存活率分别绘制各组细胞的辐射剂量-存活曲线,并进一步通过计算Do值及放射增敏比（SER）分析各组细胞放疗敏感性的变化.结果 转染载体pfos-iNOS/GFP的细胞照射后,NO浓度较照射前明显增高,其中照射后16 h较照射前增强10倍.转染治疗载体组Do值（1.16 Gy）明显低于未转染组（3.93 Gy）,治疗载体组的放疗敏感性是对照组的3.5倍.结论 高效表达的NO使体外肺腺癌细胞的放射敏感性明显增强,为肿瘤放疗增敏提供了新的模式.     

CT导向肺活检标本端粒酶活性检测对肺癌的早期诊断价值

目的 研究肺活检标本端粒酶活性检测对肺癌的早期诊断价值。资料与方法 经X线平片与CT扫描疑诊为早期肺癌52例患者，分别进行纤维支气管镜活检与经皮针刺切割活检，并使用银染端粒酶重复序列扩增法（TRAP）检测活检标本端粒酶活性；而后将早期肺癌（T1NOM0）与非癌病变组对照研究。结果 经手术病理证实为早期肺癌患者22例，肺活检标本端粒酶活性表达率为86．4％（19／22）；经手术或随访2年以上证实为良性病变者24例（肺囊肿3例，结核6例，炎性假瘤5例，肺炎10例），端粒酶活性表达阳性率为4．2％（1／24）。两组有显著性差异（P＜．01）。结论 端粒酶可以作为肺癌定性的一个敏感的肿瘤标志物，结合病理学检查，有可能对肺癌作出早期诊断。     

A549肿瘤细胞中特异性hTERT-siRNA的干扰作用研究

目的 研究hTERT特异的siRNA对肿瘤细胞端粒酶活性及细胞生长的抑制作用。方法 设计和构建由U6启动子驱动的产生特异hTERT的siRNA表达质粒,转染至A549肺肿瘤细胞,以TRAP-ELISA方法观察细胞端粒酶活性,Western blot检测端粒酶蛋白的表达,MTT法检测对细胞生长的影响。结果 构建的针对hTERT基因745靶位点的U6表达质粒具有明显的干扰效果,受到特异hTERT-siRNA干扰的A549肿瘤细胞端粒酶表达下调,细胞生长受抑。结论 应用RNA干扰技术可阻止hTERT基因的表达,有望成为肿瘤基因治疗新的研究方向。     

人贲门癌,食管癌细胞端粒酶RNA和端粒酶活性检测

目的：检测人贲门组织、贲门癌组织和食管组织、食管癌组织端粒酶ＲＮＡ组分表达及端粒酶活性，探讨其在贲门癌和食管癌中的作用。方法：采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和端粒重复扩增法（ＴＲＡＰ）检测了人贲门组织、贲门癌组织和食管组织、食管癌组织端粒酶ＲＮＡ组分及端粒酶活性。     

Long but dysfunctional telomeres correlate with chromosomal radiosensitivity in a mouse AML cell line.

PURPOSE: To compare the chromosomal radiosensitivity of C3H mouse acute myeloid leukaemia (AML) cell lines 7926 and 8709 and to investigate the mechanistic basis of the radiosensitivity observed in 7926. MATERIALS AND METHODS: Yields of chromosome aberrations following X-irradiation were determined in Giemsa-stained metaphases. Cell cycle phase distributions were determined by BrdU incorporation and microscopy, apoptosis was assessed by caspase assays. Telomerase activity (TRAP assay), telomere length (Q-FISH and Southern blotting) and telomere function (Robertsonian-like fusion formation) were also examined. The expression levels of telomerase components, telomerase regulators and DNA PKcs were determined on Northern blots. RESULTS: A total of 4.5-7.6-fold elevated chromosome aberration yields were found in 7926 by comparison with 8709 3-24h after 0.5 and 1 Gy X-ray exposure. This difference could not be accounted for by differences in chromatid break-rejoining rates, cell cycle phase distribution or the induction of apoptosis. Telomeres and telomerase were dysfunctional in 7926. However, average telomere length was approximately two-fold greater than in 8709. CONCLUSION: Defective telomere function in 7926 correlates with chromosomal radiosensitivity. This implicates telomere function in addition to telomere length as a determinant of chromosomal radiosensitivity.     

肝癌腹水脱落细胞端粒酶活性检测及临床意义

 目的研究肝癌腹水脱落细胞端粒酶活性.方法应用银染TRAP法检测肝癌腹水和非癌性腹水脱落细胞端粒酶活性,并将检测结果与细胞学诊断结果进行比较.结果50例肝癌腹水脱落细胞37例阳性(74.0%),29例非癌性腹水脱落细胞无1例阳性.结论结果提示端粒酶活性检测可能在癌性腹水诊断和鉴别诊断方面有重要辅助诊断价值.     

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.     

hTERT基因转染对人胚胎成纤维细胞端粒长度、端粒酶活性及其亚单位的影响

为了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)端粒长度、端粒酶活性及其亚单位的影响 ,采用基因重组技术构建hTERT全长cDNA正义荧光真核表达载体 ,并采用脂质体法将正义重组质粒pIRES2 EGFP hTERT及空载质粒pIRES2 EGFP分别转染原代培养hEFs,检测转染细胞端粒长度、端粒酶活性及端粒酶亚单位的变化。结果显示 ,外源性hTERT基因转染细胞 (hEF hTERT)端粒酶活性较未转染细胞 (hEFs)及空载体转染细胞 (hEF EGFP)显著增加 (P <0 0 1) ,hEF hTERT、hEFs及hEF EGFP端粒长度分别为 6 0kb、5 3kb及 5 4kb ,端粒酶亚单位中除hTERT在mRNA和蛋白水平表达均增加外 ,hTR和TP1mRNA无明显变化。以上结果提示 ,外源性hTERT基因转染能使原代培养的hEFs端粒酶活化 ,端粒长度不再继续缩短 ,hTERT在mR NA和蛋白水平表达增加     

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.     

Age estimation in dental pulp DNA based on human telomere shortening

Age estimation based on evidence found in teeth has received considerable attention within the field of forensic science. We determined the terminal restriction fragment (TRF) length, as telomere length, to estimate age. Using dental pulp DNA we found the average TRF length showed a tendency to shortening with aging. Our findings show that telomere shortening, based on dental pulp DNA is a new and useful approach to estimate age of the subject at the time of death.