hTR RNA Component as a Marker of Cellular Proliferation in Oral Lichen Planus

Previously, we have shown that the telomerase RNA component hTR is highly expressed in the epithelium of non-dysplastic Oral Lichen Planus (OLP) lesions. We concluded that it is possible that this high expression might be related to the increased cellular proliferation seen in OLP rather than being an indicator of potential malignant transformation. In the present study, and in order to confirm our finding in the previous study that hTR might be a marker for cellular proliferation in OLP, we analysed OLP biopsies known to be positive for RNA component of telomerase (hTR) for the expression of Ki-67 as a marker for cellular proliferation. Fourteen OLP tissue biopsies known to be positive for telomerase RNA component hTR, were investigated using an immunohistochemical approach to determine the rate of cellular proliferation in OLP, looking at the expression of Ki-67 protein as a marker for cellular proliferation. A statistically significant increase was found between Ki-67 expression in OLP in comparison to normal control buccal mucosa samples. The expression of hTR component in OLP might thus be a marker for cellular proliferation.     

端粒酶基因在脑胶质瘤中的原位表达

目的 :研究端粒酶基因 (hTR)在脑胶质瘤中的原位表达状况及其与分型、分级的关系 ,评估其对胶质瘤诊断的价值。方法 :用原位杂交技术检测了 79例甲醛固定、石蜡包埋的胶质瘤蜡块标本中端粒酶基因 (hTR)的表达状况并分析与组织学分级、WHO分型之间的关系。用ABC法检测PCNA的表达。 6例正常脑组织作对照。结果 :端粒酶基因 (hTR)在脑胶质瘤中的检出率为 5 9 2 % (4 7/ 79)。端粒酶基因 (hTR)在胶质瘤组织学分级中的分布为Ⅱ级 11/ 32 ,Ⅲ级 12 / 2 0 ,Ⅳ级 2 4 / 2 7,各级胶质瘤组间比较 ,差异有显著意义 ,P <0 0 5。端粒酶基因 (hTR)的表达强度与胶质瘤的组织学分级、WHO分型之间有相关性 ,P <0 0 5 ,Ⅲ级、Ⅳ级的检出率明显高于Ⅱ级。正常脑组织中未检出端粒酶基因。端粒酶阳性组与阴性组PCNA阳性细胞密度差异有显著意义 ,P <0 0 5。结论 :胶质瘤中端粒酶基因 (hTR)表达状况可能与胶质瘤的分化程度、组织学分级相关 ,提示端粒酶基因的过表达可能对胶质瘤的演化和进展具有一定重要作用 ,并可能作为胶质瘤的一个新的诊断标志物 ,结合PCNA评价胶质瘤的生物学行为更为可靠。     

胶质瘤细胞端粒酶基因表达对其增殖及凋亡影响的研究

背景与目的：端粒酶逆转录酶（hTERT）和端粒酶相关蛋白1（hTP1）是人端粒酶的重要组成部分。已知胶质瘤细胞有端粒酶异常再活化，而hTERT和hTP1基因表达是否也相应增加，尚不清楚。本研究是为了探讨胶质瘤细胞hTERT和hTP1基因表达、增殖活性及凋亡程度变化的相互关系及其在胶质瘤发生、发展中的作用。方法：采用mRNA原位杂交、免疫组织化学染色及原位细胞凋亡检测（TUNEL）等方法观察了70例不同级别的人胶质瘤组织标本。结果：本组70例胶质瘤hTERT mRNA和蛋白的阳性表达率分别为88．6％和82．9％．WHO Ⅰ～Ⅱ级组、Ⅲ级组及Ⅳ级组间比较二者的阳性表达率均无显著性差异（P〉0．05），hTP1 mRNA和蛋白及增殖细胞核抗原（PCNA）蛋白的阳性表达率均为100％。以上五种阳性肿瘤细胞的密度彼此间均呈显著性正相关（r=0．589～0．882，P〈0．0005），并均随肿瘤级别升高而相应增加，不同级别组间比较差异均有显著性（P〈0．05～0．01）。凋亡肿瘤细胞的检出率也为100％，但凋亡肿瘤细胞密度随肿瘤级别升高而相应减少，不同级别组间比较差异均有显著性（P〈0．01），且与前五种阳性肿瘤细胞密度间均呈显著性负相关（r=-0．551 ～-0．775，P〈0．0005）。结论：以上指标对评价胶质瘤的生物学行为均有重要参考价值，胶质瘤细胞hTERT和hTP1基因异常表达是其端粒酶再活化的先头条件，后者通过抑制肿瘤细胞衰老凋亡和促进其增殖在胶质瘤的发生、发展过程中均起重要作用。     

Expression of Antisense Human Telomerase RNA Gene in SMMC-7721Cells

背景与目的 :探讨反义端粒酶RNA(hTR)对人肝癌细胞端粒酶活性的影响。材料与方法 :构建反义hTR基因逆转录病毒载体 ,脂质体介导转染人肝癌SMMC_7721细胞 ,通过Southernblot、TRAP_PCR、流式细胞术检测hTR表达及端粒酶活性变化。结果 :经G418筛选 ,转染细胞形成稳定克隆 ,反义hTR表达增强 ,细胞生长受到抑制、倍增时间延长 ,出现凋亡。 结论 :反义hTR基因表达明显抑制肝癌细胞的生长 ,降低肿瘤细胞的恶性度和异型性。hTR基因是肝癌基因治疗良好的靶点     

苦参素改变端粒酶活性对人肝癌细胞株HepG2、QGY体外增殖影响的研究

[目的]观察苦参素对人肝癌细胞株HepG2、QGY体外增殖和端粒酶活性的影响.[方法]应用MTT法检测HepG2与QGY增殖抑制的程度;流式细胞仪分析细胞周期的分布及凋亡;PCR-ELISA法检测端粒酶活性的变化.[结果]苦参素对HepG2和QGY细胞增殖的抑制随着时间的延长而增加,具有时间依赖性,各浓度时间之间均有显著性差异(P＜0.001);从细胞周期分析,苦参素能阻滞HepG2和QGY细胞周期进程,同时诱导细胞凋亡,各细胞之间均有显著性差异(P＜0.001);苦参素对细胞端粒酶均有时间和剂量依赖性,各组间均有显著性差异(P＜0.001).[结论]对端粒酶活性的抑制可能是苦参素发挥抗肿瘤作用的机制之一.     

Expression of Human Telomerase RNA Is an Early Event of Stomach Carcinogenesis

Expression of human telomerase RNA (hTR) and telomerase activity in gastric cancer and corresponding non-cancerous mucosa were studied. Telomerase activity was detected in 23 (88%) of 26 carcinoma tissues. Although all tumor specimens and non-cancerous mucosa expressed various levels of hTR, 21 (81%) of 26 cases expressed hTR at a higher level in the tumor than that in the corresponding mucosa. All 8 gastric carcinoma cell lines also expressed hTR at high levels. Nine (35%) of 26 non-cancerous mucosa showed telomerase activity and all of them contained intestinal metaplasia. The incidence of telomerase-positive mucosa in grade 2 intestinal metaplasia was significantly higher than that in grade 0 or grade 1 intestinal metaplasia, whereas hTR overexpression was found in grade 0 or grade 1 intestinal metaplasia as well as grade 2 intestinal metaplasia. The degree of Heticobacter pylori infection increased in parallel with the level of hTR expression and telomerase positivity. These results overall suggest that Helicobacter pylori infection may he a strong trigger for hTR overexpression in intestinal metaplasia, and this may lead to telomerase reactivation.     

RNA干扰对肾癌细胞端粒酶活性及增殖、凋亡的影响

目的 探讨针对人端粒酶RNA（hTR）及其催化亚基（hTERT）的小干扰RNA（siRNA）对肾癌细胞端粒酶活性及其增殖、凋亡的影响。方法 将hTR-siRNA、hTERT-siRNA（100nmol／L）单独或联合转染人肾癌786—0细胞,采用RT-PCR法检测hTR、hTERT mRNA表达,TRAP-ELISA法检测端粒酶活性,MTT法检测细胞增殖,免疫组化TUNEL法检测细胞凋亡。结果 （1）hTR-siRNA可显著降低786—0细胞hTR mRNA表达（P〈0．01）,hTERT-siRNA可显著降低hTERT mRNA表达（P〈0．01）,但彼此互不影响。（2）二者均能显著抑制端粒酶活性（P〈0．01,P〈0．01）,并增加786—0细胞增殖抑制率及凋亡细胞阳性率（P〈0．01,P〈0．01）。二者联合应用与单独应用差异亦无显著性（P〉0．05）。结论 hTR、hTERT siRNA通过抑制各自基因表达,抑制人肾癌细胞端粒酶活性,进而抑制增殖、促进凋亡。     

Expression of G-CSF and GM-CSF in Human Meningiomas Correlates with Increased Tumor Proliferation and Vascularization

The hematopoietic growth factors granulocyte- and granulocyte-macrophage colony stimulating factor (G-CSF and GM-CSF) are nowadays widely used in routine cancer therapies as potent factors to control radiation and chemotherapy induced neutropenia, a side effect that frequently endangers the success of tumor therapies. However, there is little information about the role of G-CSF and GM-CSF for tumor growth or progression. We were interested in the expression and potential role of both factors in human meningiomas, tumors of arachnoidal origin that account for about 20% of all primary intracranial tumors. Therefore, we analyzed immunohistochemically the protein expression of G-CSF, GM-CSF and their respective receptors in 30 meningioma tissues of different malignancy and histopathological type. Both factors and receptors were not expressed in the corresponding normal tissue. In contrast, G-CSF, GM-CSF and their receptors were expressed to a varying degree in human meningiomas. Increasing expression of both factors and receptors correlated significantly with enhanced proliferation in the tumor and thus with higher malignancy. In addition, a strong perivascular expression of G-CSF was associated with a highly vascularized tumor type. Thus, expression of both G-CSF and GM-CSF is associated with the expression of proliferation vascularization, two markers of an increasingly malignant tumor phenotype, suggesting a contribution of both factors to tumor progression.     

Overexpression of Human Telomerase RNA in Helicobacter pylori-infected Human Gastric Mucosa

Telomerase, an enzyme associated with cellular immortality and malignancy, plays an important role in cellular immortalization and tumorigenesis. Furthermore, overexpression of the RNA component of the telomerase, called human telomerase RNA (hTR), has been demonstrated in various human cancers as an early event. The pattern of hTR expression following Helicobacter pylori ( H. pylori ) infection in human gastric mucosa was investigated by a radioactive in situ hybridization (ISH) assay. Paraffin-embedded sections of 50 biopsy specimens taken from the gastric antrum of individual patients infected to different extents with H. pylori , as well as normal gastric mucosa, were studied. In normal gastric mucosa, only weak hTR expression was noted and the expression was limited to basal cells of the gastric glands. However, the degree of hTR expression gradually increased in parallel with the degree of H. pylori infection. The mean scores of gastric mucosa with mild, moderate and severe degrees of H. pylori infection were 2.3, 2.8, and 3.7 times higher than that of normal gastric mucosa, respectively. The results of this study suggested that up-regulation of hTR expression is a frequent and early event associated with H. pylori infection in the gastric mucosa and may play some role in gastric carcinogenesis. Sufficient synthesis of hTR during this early stage may be a prerequisite for telomerase reactivation to occur in gastric cancer.     

苦参碱对肝癌细胞HepG2增殖的影响及端粒酶活性调控的体外研究

[目的]探讨苦参碱（Ma)对肝癌细胞株HepG2增殖的影响及其端粒酶活性，端粒酶逆转录酶（hTERT)表达的调控作用。[方法]将不同浓度的Ma加入肝癌细胞株HepG2细胞，MTT试验测定用药后细胞存活率和增殖情况。同时以TRAP-ELISA方法检测其端粒酶活性；采用Lipofect脂质体转染法将携带hTERT启动子和报告基因的质粒转染至肝癌细胞株HepG2细胞，2h后加入不同浓度的Ma，孵育48h后，分别检测其报告基因萤虫素酶活性。[结果]500μg/ml以上浓度可抑制HepG2的生长；750μg/ml浓度的Ma可抑制端粒酶活性，并下调hTERT启动子的表达。[结论]Ma可抑制肝癌细胞株HepG2细胞增殖，同时可调控hTERT的表达并影响端粒酶活性。     

端粒酶催化亚单位和c-myc在骨肉瘤中的表达

目的:探讨端粒酶催化亚单位和c-myc在骨肉瘤发生中的作用。方法:应用免疫组织化学方法检测60例骨肉瘤、12例骨软骨瘤中端粒酶催化亚单位和c-myc的表达,采用显微图像分析系统进行平均灰度值测定。结果:骨肉瘤中端粒酶催化亚单位和c-myc蛋白的平均灰度值均低于骨软骨瘤中两者的平均灰度值,且有统计学意义(P<0.01)。骨肉瘤中端粒酶催化亚单位与c-myc蛋白平均灰度值间无相关性(P>0.05)。结论:端粒酶催化亚单位、c-myc蛋白分别与骨肉瘤的发生有关。     

反义端粒酶(hTR)基因对人肝癌细胞系生物学行为的影响

背景与目的:探讨反义端粒酶RNA(hTR)对人肝癌细胞端粒酶活性的影响.材料与方法:构建反义hTR基因逆转录病毒载体,脂质体介导转染人肝癌SMMC-7721细胞,通过Southern blot、TRAP-PCR、流式细胞术检测hTR表达及端粒酶活性变化.结果:经G418筛选,转染细胞形成稳定克隆,反义hTR表达增强,细胞生长受到抑制、倍增时间延长,出现凋亡.结论:反义hTR基因表达明显抑制肝癌细胞的生长,降低肿瘤细胞的恶性度和异型性.hTR基因是肝癌基因治疗良好的靶点.     

反义端粒酶RNA基因对肝癌细胞的抑制作用

目的：研究逆转录病毒载体介导的反义端粒酶RNA基因对肝癌细胞的抑制作用，探讨通过抑制端粒酶活性治疗肝细胞癌的有效途径。方法：采用电穿孔法将携带正反义端粒酶RNA基因的逆转录病毒载体导入PT67包装细胞，G418筛选获得稳定产病毒细胞株，收集逆转录病毒上清并感染人肝癌HepG2细胞，G418筛选获得稳定转染细胞克隆并扩增培养，经PCR鉴定后，通过MTT法分别检测转染正反义端粒酶RNA基因组肝癌细胞（HepG2-hTR—EcoRⅠ和HepG2-hTR—BamHⅠ）以及未转染端粒酶RNA基因组细胞（HepG2）的生长，免疫荧光化学检测肝癌细胞增殖，TRAP—PCR—ELⅠSA法检测细胞的端粒酶活性，流式细胞术检测各组细胞所处的细胞周期及凋亡率。结果：基因转染后经PCR扩增可于约500bp处检测到目的基因的表达。转染反义端粒酶RNA基因的HepG2一hTR—BamHⅠ组细胞生长受到明显抑制；抗中性粒细胞核增殖抗原（PCNA）表达减少；实验组端粒酶活性为（2．31±0．16），较HepG2-hTR—EcoRⅠ组（3．24±0．20）及HepG2组细胞（3．22±0．17）明显下降（P〈0．01）；流式细胞术检测显示实验组细胞的凋亡率为（9．58±1．38）％，与对照组相比差别具有显著性（P〈0．01）；实验组细胞出现G2／M期阻滞。结论：端粒酶RNA是端粒酶活性表达的一个重要组成部分，通过反义技术下调其表达能够抑制肝癌细胞生长增殖，并诱导其凋亡。     

食管鳞癌端粒酶活性、端粒酶逆转录酶及bcl-X/L表达的研究

目的：探讨食管鳞癌组织中端粒酶活性、端粒酶逆转录酶（hTERT)及bcl-X/L的表达及其它们之间的关系。方法：应用TRAP－银染法检测45例食管鳞癌组织的端粒酶活性，采用原位杂交及免疫组织化学方法对癌组织进行了hTERT及bcl-X/L表达的检测。结果：癌组织端粒酶活性阳性率为82．2％，hTERT及bcl-X/L的表达阳性率分别为64．4％及71．1％，bcl-X/L的蛋白表达及hTERT的mRNA表达与端粒酶活性均有相关性。结论：食管鳞癌组织中端粒酶活性、hTERT及bcl-X/L的表达阳性率均较高。hTERT及bcl-X/L的表达对端粒酶活性有一定调节作用。     

人端粒酶催化亚单位及其相关蛋白在骨肿瘤中的表达

目的：研究骨肿瘤中端粒酶相关基因中,人端粒酶催化亚单位（human elomerase catalytic subunit,hTERT)和人端粒酶相关蛋白（human telomerase associated protein,TP1)两个重要成分的表达及对评估肿瘤良、恶性的意义。方法：提取50例手术切除骨肿瘤组织、2种人成骨内瘤细胞系Saos－2、OS732和原代培养人成骨细胞的总RNA。用一步RT－PCR方法检测hTERT和TP1mRNA的表达。结果：所有骨肿瘤标本及细胞系均检测到TP1的表达。hTERT在17例良性骨肿瘤、33例骨肉瘤、2种人成骨肉瘤细胞系和人成骨细胞的表达率分别为64．7％、57．6％、0％和0％。良、恶性肌肿瘤间hTERT表达的差异不具有显著性（P＞0．05）,不同分化程度骨肉瘤间hTERT表达的差异也不具有显著性（P＞0．05）。结论：一步RT－PCR是检测端粒酶亚单位的有效方法。hTERT和TP1表达水平的上调在骨肿瘤恶性进展中可能不起主要作用,在骨肿瘤发展和维持中可能有替代性（ALT）机制导致细胞永生化。     

三氧化二砷抑制恶性黑色素瘤B_(16)细胞生长及对端粒酶活性的影响

背景与目的:研究三氧化二砷(As2O3)对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的影响,探讨砷及其化合物治疗恶性黑色素瘤的作用机制,为临床治疗恶性黑色素瘤提供新的理论和实验依据。材料与方法:四甲基噻唑氮蓝(Methylthiazolyltetrazolium,MTT)比色法检测As2O3对B16细胞的生长抑制作用:端粒重序列扩增酶联免吸附实验(Telomericrepeatamplificationprotocolenzyme_linkedimmunosorbantassay,TRAR_ELISA)和聚内烯酰胺凝胶电泳银染(Telomericrepeatamplificationprotocol,polyacrylamidegelelectrophoresissilver_staining,TRAP_PAGE)法检测B16细胞的端粒酶活性。结果:As2O3可显著抑制B16细胞的生长并可下调端粒酶活性,其抑制作用有显著的时间和剂量依赖关系。结论:As2O3对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的抑制作用随药物浓度的增加和作用时间的延长而增强。