胃癌组织中端粒酶活性表达的初步研究

目的 :端粒酶的激活与胃癌发生密切相关 ,通过检测胃癌组织中端粒酶活性表达水平 ,为胃癌及其发生的早期诊断提供一种有效的方法。方法 :应用PCR ELISA技术 ,把端粒酶重复序列扩增技术与微孔液相杂交酶联呈色技术有机结合 ,检测胃肠癌组织和腹水标本中的端粒酶活性表达。结果 :1 7例晚期胃癌组织标本中检出 1 3例端粒酶活性表达 ,阳性率为 76 47% ;2 3例中期胃肠癌组织标本中检出 1 9例端粒酶活性表达 ,阳性率为 78 2 6% ;66例早期胃癌组织标本中检出5 7例端粒酶活性表达 ,阳性率为 86 36% ;36例癌旁近端组织标本中检出 1 1例端粒酶活性表达 ,阳性率为 30 5 6% ;36例癌旁远端组织标本中检出 4例端粒酶活性表达 ,阳性率为 1 1 1 1 % ;2 6例胃肠癌腹水标本检出 1 9例端粒酶活性表达 ,阳性率为 73 0 8% ;5 4例正常人胸腹水标本中没有检出端粒酶活性表达。结论 :胃癌组织标本中有较高的端粒酶活性表达 ,端粒酶活性表达可以作为胃癌发生的有效指标 ,而PCR ELISA技术可以定量评价作为端粒酶活性表达从而作为诊断胃癌发生的一种快速简便有效的方法。     

人类癌组织中端粒酶活性与p16基因表达的相关性

端粒酶是一种由RNA和蛋白质组成的RNA酶,依其自身序列逆转录合成端粒序列并添加到染色体末端,以保证细胞分裂的稳定性。近年来的研究已证实端粒酶的激活是许多恶性肿瘤发生的先决条件。p16基因作为一种重要的抑癌基因,其编码产物P16蛋白可通过p16-cyclinD/CDK-pRB途径调控细胞周期。在多种肿瘤中均出现p16基因表达异常,其表达缺失与人类肿瘤的发生发展关系密切。就端粒酶活性与p16基因表达在人类癌组织中的相关性及其可能作用机制进行总结分析。     

胚胎发育过程中端粒酶活性水平的变化

本文采用液体闪烁计数法观察了不同胎龄的６个胚胎，共３６个组织样品的端粒酶活性水平。结果表明端粒酶活性水平在胚胎发育过程中表现为阶段性变化。端粒酶活性水平不仅随胎龄而异，而且同一胎儿不同组织其差异也极为显著。     

端粒酶活性检测的几种方法

端粒酶活性检测方法的不断发展改进为癌症的诊断治疗以及人们对衰老的进一步研究提供了新途径和新思路。近年来端粒酶活性的检测方法有：(1)基本方法。(2)TRAP法。(3)改良的TRAP法。(4)间接检测法等。     

多形胶质母细胞瘤细胞系端粒酶活性的测定

端粒缩短见于星形细胞瘤发展过程中,但其长度在胶质母细胞瘤／细胞系相对稳定,提示胶质瘤细胞内存在端粒修复机制的可能性．为证实此点,利用端粒重复片段扩增技术（ＴＲＡＰ）,对８株人／大鼠多形胶质母细胞系的蛋白提取液中端粒酶活性加以测定．结果显示：８例胶质瘤样本的反应液均可见端粒ＰＣＲ扩增片段;用无ＤＮａｓｅ的ＲＮａｓｅ事先处理蛋白提取液,可明显降低或消除ＰＣＲ产物的出现,说明ＴＲＡＰ反应中的ＰＣＲ扩增是在端粒酶的介导下进行而非ＤＮＡ污染或其它端粒修复因子所致．从而不但建立起检测人癌细胞内端粒酶活性的可靠方法,也为针对端粒酶的胶质母细胞瘤生物／药物治疗提供了实验依据．     

全组织提取物检测A-to-I RNA编辑酶活性

目的:建立一种简便、快速、可靠的检测A-to-IRNA编辑酶活性的方法。方法与结果:一步法制备的C57BL/6小鼠十种组织的全组织提取物,在各提取物中检测到A-to-IRNA编辑酶的非特异性编辑活性,不同组织中A-to-IRNA编辑酶活性的强度依次为脑>肺>胸腺>脾>淋巴结>肝>肾>睾丸>心脏>骨骼肌,编辑活性与加入反应体系中的蛋白量成正相关。结论:一步法制备的全组织提取物可用于检测A-to-IRNA编辑酶活性,该方法操作简便、可控、省时。     

检测人结肠癌组织端粒酶活性的TRAP方法研究

对检测人结肠癌组织端粒酶活性的端粒酶重复序列扩增法(TRAP)进行了研究.当Mg^2＋浓度为1.8 mmol/L、退火温度为55℃时,能得到可靠的检测结果.影响检测结果可靠性的另两个因素是抽提物的反应用量和阳性片段的污染.     

酶活性测定的误区和问题辨析

针对“能否使用斐林试剂检测淀粉酶活性”这一问题,中学教师普遍持否定观点,认为检测过程中的水浴加热会改变反应温度,影响原有实验结果。这一错误观点反映出许多教师对于酶活性测定实验的理解存在误区。本文从酶活性测定的原理和步骤出发,对此问题进行辨析,并设计、实施实验证明使用斐林试剂检测淀粉酶活性不仅是可行的,而且效果比使用碘液检测更好。最后,结合实验教学中的相关问题提出建议,以更好地开展实验教学。     

棉铃虫幼虫加单氧酶活性的组织分布

棉铃虫（Ｈｅｌｉｃｏｖｅｒｐａ ａｒｍｉｇｅｒａ）６龄幼虫不同组织的加单氧酶活性的测定结果显示,对－硝基苯甲醚Ｏ－脱甲基酶主要分由于外来物质的入口部位,以中肠和脂肪体的活性较高,在前肠、后肠和马氏管等组织中有相对较低的活性,而在体壁和精巢中未检测到Ｏ－ 甲基作用。体壁表现一定的艾氏剂环氧化作用,但其活性不及中肠的１０％,内源性制剂被证明并非体壁低加单氧酶活性的主要原因。不同组织生物量的差异及其动态     

Telomeres and telomerase activity in pig tissues

The current state of the art concerning telomeres and telomerase stems almost exclusively from the analysis of protozoa, yeast, and a small number of mammals. In the present study, we confirm that the pig telomeric sequence is indeed T(2)AG(3), as previously suggested. By making use of sequence analysis of pig telomeric DNA variant telomeric repeats in the medial region of the telomeres, interspersed with canonical T(2)AG(3) repeats, were identified. This telomere organization is similar to the one present in humans. Analysis of terminal restriction fragments showed that the majority of telomeres from different pig tissues are longer than in humans but shorter than in Mus musculus. Telomeres from spermatozoa were found to be longer, ranging in size between 13 and 44 kb. Most of the somatic pig tissues expressed significant levels of telomerase activity, a situation more similar to mouse and that contrasts with the one in humans and dog. Moreover, the analysis of sperm cells from different epididymal compartments of an adult animal showed that telomerase activity is absent in maturing spermatozoa, suggesting that sperm telomere elongation is restricted during spermatogenesis.     

Amadori-glycated phosphatidylethanolamine up-regulates telomerase activity in PANC-1 human pancreatic carcinoma cells

Several lines of experimental data have highlighted a key role of Amadori-glycated phosphatidylethanolamine (Amadori-PE) in the development of diabetic complications. Recent epidemiological studies suggest that diabetes mellitus could be a risk factor for some cancers. A characteristic of cancer cells is their immortal phenotype, and the enzyme telomerase contributes to the infinite replicative potential of cancer cells. The purpose of this study was to obtain new information about the effect of Amadori-PE on the regulation of telomerase in PANC-1 human pancreatic carcinoma cells. Amadori-PE enhanced cellular telomerase in a time- and dose-dependent manner by up-regulating hTERT expression through induction of c-myc. These results provide experimental evidence for a novel role of Amadori-PE in linking diabetes and cancer.     

荧光定量PCR 法检测肿瘤细胞端粒酶活性的变化

目的：利用荧光定量PCR法检测端粒酶抑制剂作用于人肝癌细胞SMMC-7721后端粒酶活性的变化,探讨其抑制端粒酶活性的可能机制,为端粒酶抑制剂的临床应用提供理论依据。方法：利用荧光染料SYBR—Green I建立一种新的端粒酶活性检测方法：FQ—TRAP法。利用FQ—TRAP法检测端粒酶抑制剂作用后肿瘤细胞端粒酶活性变化。结果：端粒酶抑制剂作用后,肝癌细胞端粒酶活性都有变化,其中以ASODN,EGCG,AZT抑制效果较明显。结论：端粒酶FQ—TRAP法是一种特异性、灵敏度、重复性都较好,可快速、简便及定量检测人端粒酶活性的方法,端粒酶抑制剂作用后癌细胞端粒酶活性的变化,为端粒酶抑制剂的临床应用提供理论依据。     

Single-molecule analysis of human telomerase monomer

Human telomerase is a ribonucleoprotein that is minimally comprised of protein (hTERT) and RNA (hTR) components. We have applied single-molecule fluorescence two-color coincidence detection to characterize complex formation between fluorophore-labeled components in solution. By systematic labeling and in vitro assembly of hTERT, hTR and telomerase's DNA substrate, we have established that catalytically functional human telomerase comprises a stable hTERT:hTR:substrate interaction in a 1:1:1 absolute stoichiometry.     

Regulation of catalytic activity and processivity of human telomerase

The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37 degrees C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G-quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.