Direct Correlation among Telomere Length, Cellular Aging, and Rejuvenation Effects of Honey Child Powder

Purpose: Telomere length (TL) is an indicator of age;however, hormonal influences complicate individual aging. It remains unclear whether TL shortening is a direct factor in both individual and cellular aging. Therefore, we examined the direct relationship between TL and cellular senescence at the cellular level. Methods: Telomerase activity, TL, and gene expression were measured in cultured human lung-, fetal-, and skin-derived fibroblasts, human skin keratinocytes, and telomerase reverse transcriptase (TERT) gene-immortalized cells using detection kits, Cawthon’s method, and reverse transcription-quantitative polymerase chain reaction, respectively. Novel substances that elongate telomeres were screened to confirm cell rejuvenation effects. Results: Long-term cell culture of TIG-1-20 normal human fibroblasts resulted in TL shortening, decreased division rate, and senescence progression, whereas in OUMS-36T-2 cells, TL elongation via TERT gene transfer increased the division rate, reduced endoplasmic reticulum stress, and upregulated genes associated with young individuals, indicating that cellular rejuvenation occurs via TL elongation. In addition, a honey child powder (HCP) extract was found through screening, and the HCP extract strongly suppressed the menin gene, resulting in increased telomerase activity and extended cell lifespan. Upon addition of the HCP extract to skin fibroblasts, gene expression of moisturizing components, including collagen, hyaluronic acid, and elastin, increased, and exhibited a rejuvenating effect with an increase in elastin amount. Conclusions: TL elongation or shortening is involved in cell proliferation rate and cellular aging, and TL elongation rejuvenates cells. In addition, HCP extract has a rejuvenating effect on cells and is expected to be a rejuvenating compound.

Influence of the h TERT rs2736100 polymorphism on telomere length in gastric cancer

AIM: To investigate the functional consequences of rs2736100 polymorphism in telomere length and examine its link to gastric cancer risk.METHODS: Telomere length and human telomerase reverse transcriptase(h TERT) m RNA expression were measured in 35 gastric cancer tissues and 5 cell lines and correlated to rs2736100 polymorphism. The relationship between rs2736100 polymorphism and the risk of gastric cancer were examined in 243 gastric cancer patients and 246 healthy individuals.RESULTS: The rs2736100 A allele carrier is closely associated with reduced h TERT m RNA expression and shortened telomere length in gastric cancer tissue and cell lines. When gastric cancers were stratified by histological subtype,telomere length and h TERT m RNA levels were significantly increased in those with the C/C genotype in intestinal-type gastric cancer,but not in diffuse-type gastric cancer. Interestingly,there was no significant difference in the genotype and allele frequencies of the rs2736100 polymorphism between the patients with gastric cancer and healthy controls.CONCLUSION: The rs2736100 polymorphism of the h TERT gene is involved in the regulation of h TERT expression and telomere length,but not in the risk of gastric cancer.

Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus

AIM: To observe the interaction between the expression of telomerase activity (TA) and its associate genes in regulation of the terminal restriction fragment length(TRFL) in esophageal squamous cell carcinoma (SCC).METHODS: Seventy-four specimens of esophageal SCC were examined. The TA was measured by telomeric repeat amplification protocol (TRAP) assay, and the associated genes [human telomerase-specific reverse transcriptase (hTERT), hTERC, TP1, c-Myc, TRF1,and TRF2] were detected using RT-PCR method. The TRFL was measured by Telomere Length Assay Kit and Southern blotting. The correlations between the expression of telomerase and its associated genes with the TRFL and survivals were examined.RESULTS: Expressions of the TA, hTERT, hTERC, TP1,c-Myc, TRF1, and TRF2 genes were observed in 85.1%,64.9%, 79.7%, 100.0%, 94.6%, 82.4%, and 91.9% of the tumor tissues, respectively. The TRFL of the tumor and normal esophageal tissues were 2.70±1.42 and 4.93±1.74 kb, respectively (P＜0.0001). The TRFL of the telomerase positive and telomerase negative tumor tissues were 2.72±1.44 and 2.58±1.32 kb, respectively (P = 0.767).The TRFL ratios (TRFLR) of the telomerase positive and telomerase negative tumor tissues were 0.55±0.22 and 0.59±0.41, respectively (P = 0.742). The expression rates of h-TERT (P = 0.0002), hTERC (P＜0.0001), and TRF1(P = 0.002) in the tumor tissues are higher than those of the normal paired tissues. Though TA is markedly activated in tumor tissues (P＜0.0001), its expression is not related to clinicopathological parameters including gender, tumor differentiation, and TNM stages. The cumulative 4-year survival rates of telomerase positive and telomerase negative cases were 35.86% and 31.2%,respectively (P = 0.8442). The cumulative 4-year survival rates of patients with their TRFLR ≤85% and ＞85%were 38.7% and 15.7%, respectively (P = 0.1307).CONCLUSION: Though telomerase expression is not related to tumor stages and prognosis, our data support that the TA increased as the TRFL decreased,probably under the control of hTERT, hTERC, and TRF1.When telomerase expression was activated, only TRF2overexpression persisted to stabilize T-loop formation.Furthermore, as the TRFLR decreased to 85%, a trend of better prognosis was observed. Cox model analysis indicates a higher t/n TRFLR and distant metastasis are independent poorer prognostic factors (P = 0.035 and P = 0.042, respectively).

Telomeres,telomerase and colorectal cancer

Colorectal cancer(CRC)is the third most common cancer worldwide and,despite improved treatments,is still an important cause of cancer-related deaths.CRC encompasses a complex of diseases arising from a multistep process of genetic and epigenetic events.Besides heterogeneity in the molecular and biological features of CRC,chromosomal instability is a hallmark of cancer and cancer cells may also circumvent replicative senescence and acquire the ability to sustain unlimited proliferation.Telomere/telomerase interplay is an important mechanism involved in both genomic stability and cellular replicative potential,and its dysfunction plays a key role in the oncogenetic process.The erosion of telomeres,mainly because of cell proliferation,may be accelerated by specific alterations in the genes involved in CRC,such as APC and MSH2.Although there is general agreement that the shortening of telomeres plays a role in the early steps of CRC carcinogenesis by promoting chromosomal instability,the prognostic role of telomere length in CRC is still under debate.The activation of telomerase reverse transcriptase(TERT),the catalytic component of the telomerase complex,allows cancer cells to grow indefinitely by maintaining the length of the telomeres,thus favouring tumour formation/progression.Several studies indicate that TERT increases with disease progression,and most studies suggest that telomerase is a useful prognostic factor.Plasma TERT mRNA may also be a promising marker for the minimally invasive monitoring of disease progression and response to therapy.

The Cloning and Fluorescence In situ HybridizationAnalysis of Cotton Telomere Sequence

Telomeres form the ends of eukaryotic chromosomes and serve as protective caps that keep chromosomes structure independency and completeness. The first plant telomere DNA was isolated from Arabidopsis thaliana and was shown to have tandemly repeated sequence 5-TTTAGGG-3： The Arabidopsis-type telomere has been found in many plants, but several reports indicate that this sequence is absent in some plants. Up to now, no research has been conducted on the telomere of cotton. In this paper, the Arabidopsis-type telomere sequence was amplified and cloned using the primers designed based on the fragment containing telomere sequence in an Arabidopsis bacterial artificial chromosome （BAC）. Fluorescence in situ hybridization （FISH） with cotton metaphase chromosomes using the Arabidopsis-type telomere sequence as probes indicated that the signals were located at all chromosome ends of seven diploid and two tetraploid cotton species with different signal intensities among chromosome complements of different cotton species, even between long and short arms of the same chromosome. To identify the signals of FISH, the genome DNA of Xinhai 7, a cultivar of Gossypium barbadense, digested by BAL-31 nuclease was introduced in this study. The result of BAL-31 digestion indicated that the hybridization signals of FISH represent the outermost DNA sequence of each cotton chromosomes. So we first proved that the telomeric repeats of cotton cross-hybridize with that of Arabidopsis. The results of terminal restriction fragment （TRF） showed significant variation in telomere length among cotton species. The telomere length of cultivated cotton was close to 20 kb and was larger than those of wild cotton species whose telomere length rahged from 6 to 20 kb.

Genetic Variation of <i>hTERT</i>, Leukocyte Telomere Length and the Risk of Breast Cancer: A Case-Control Study in Egyptian Females

Background: hTERT is a key player in telomere biology and its activity is directly related to cell senescence and development of many health-related problems including cancer. Although previous studies investigated this association, the results greatly vary among populations. This study aimed to investigate the association of hTERT gene SNPs and the risk of breast cancer (BC) in Egyptian females and their impact on telomere length (TL). Methods: 218 BC patients and 178 age-matched healthy females were genotyped for hTERT variants rs2736098G > A, rs2735940C > T using PCR-RFLP and for MNS16A tandem repeat using PCR to determine their association with breast cancer risk. Telomere length was measured using qPCR. Results: hTERT rs2736098G > A results indicated that both AG and GG genotypes and G allele were associated with an increased risk of BC. The rs2735940 TT genotype was significantly associated with BC risk, however, the MNS16A tandem repeat region polymorphism didn’t show any correlation with the risk of developing BC. TL showed a significant reduction in BC patients with age 40 years compared with controls. However, it didn’t show a significant difference above the age of 40 years. Conclusions: hTERT rs2736098 and rs27365940, not MNS16A may be associated with an increased risk of developing BC in Egyptian females. Also, telomere length can be a promising screening marker of BC especially in young population.

Estrogen deficiency reversibly induces telomere shortening in mouse granulosa cells and ovarian aging in vivo

Estrogen is implicated as playing an important role in aging and tumorigenesis of estrogen responsive tissues;however the mechanisms underlying the mitogenic actions of estrogen are not fully understood.Here we report that estrogen deficiency in mice caused by targeted disruption of the aromatase gene results in a signi-ficant inhibition of telomerase maintenance of telomeres in mouse ovaries in a tissue-specific manner.The inhibition entails a significant shortening of telomeres and compromised proliferation in the follicular granulosa cell compartment of ovary.Gene expression analysis showed decreased levels of proto-oncogene c-Myc and the telomerase catalytic subunit,telomerase reverse transcriptase(TERT),in response to estrogen deficiency.Estrogen replacement therapy led to increases in TERT gene expression,telomerase activity,telomere length and ovarian tissue growth,thereby reinstating ovary development to normal in four weeks.Our data demonstrate for the first time that telomere maintenance is the primary mechanism mediating the mitogenic effect of estrogen on ovarian granulosa cell proliferation by upregulating the genes of c-Myc and TERT in vivo.Estrogen deficiency or over-activity may cause ovarian tissue aging or tumorigenesis,respectively,through estrogen regulation of telomere remodeling.

链霉菌中一类同源非典型端粒的克隆及分析

采用自连接-PCR的方法克隆和测序了Streptomyces cattleya DSM46488、Streptomyces mobaraensisDSM40847及Streptomyces davawensisJCM4913的端粒,序列比对发现,这3种端粒具有较高的序列相似性,前60个核苷酸的序列相似性达到了80%以上;在二级结构水平,这些端粒含有多个回文序列,其中,包含相同的回文序列I和II,但并不能通过折返形成超级发夹结构;Streptomyces cattleya DSM46488、StreptomycesmobaraensisDSM40847及Streptomyces davawensis JCM4913的端粒与线性质粒pSHJG1端粒及Streptomyces albus J1074染色体端粒均具有较高的序列相似性;系统发育分析显示它们与典型端粒及其他非典型端粒处于不同的进化分支,S.cattleyaDSM46488、S.mobaraensis DSM40847、S.davawensisJCM4913、S.albus J1074及S.hydroscopicussubsp.Jinggangensis 5008都编码同源的潜在末端蛋白-端粒相关蛋白,暗示了这类同源的非典型端粒系统广泛存在于链霉菌中。

原发性肺腺癌患者外周血端粒酶逆转录酶阳性白细胞检测方法的建立

目的:建立一种基于端粒酶逆转录酶(TERT)活性的外周血白细胞亚群检测方法,以评估恶性肿瘤患者外周血中白细胞的增殖潜能。方法:改造的重组I型单纯疱疹病毒(HSV1-hTERTp-eGFP)能够特异性地在TERT启动子高转录活性的白细胞中复制,并表达绿色荧光蛋白(GFP),通过观察绿色荧光识别具有高增殖潜力的TERT阳性白细胞,评估机体的免疫增殖潜能。分别将100、500、1 000个经HSV1-hTERTp-eGFP病毒感染24 h后的Jurkat阳性细胞,加入到未经病毒感染的2.5×106个外周血白细胞中,作为模拟样品进行检测,设置3个平行组,并对阳性细胞的回收率、重复性、线性回归系数等指标进行检验。同时,采集5例良性肺结节患者外周血,设置3个平行组,检测每组1 mL外周血中的TERT阳性白细胞比例,通过计算组内变异系数评估外周血样本检测技术的可靠性。此外,利用本检测技术,对80例良性肺结节患者及80例原发性肺腺癌患者外周血TERT阳性白细胞百分比进行检测和分析。结果:Jurkat细胞系能够被HSV1-hTERTp-eGFP病毒感染并启动GFP的表达,可以作为模拟样本进行技术验证。100、500、1 000个Jurkat细胞的模拟样品平均检出阳性细胞数分别为80.3、448.2、920.6个,回收率分别为80.3%、89.6%和92.1%;3次重复实验的批间变异系数分别为11.3%、7.2%、4.4%,而3个平行实验组内的批内变异系数均小于10%;5例良性肺结节患者外周血TERT阳性细胞百分比的组内变异系数也均在10%以内。说明本检测技术准确度高,且具有良好的稳定性。在80例良性肺结节患者中,随着年龄的增长,TERT阳性白细胞的百分比呈下降趋势(r=-0.61;P<0.01)。与良性肺结节患者对照组相比,早期肺腺癌患者的TERT阳性白细胞百分比降低(P<0.01);与非浸润性肺腺癌患者相比,浸润性肺腺癌患者的TERT阳性白细胞的比例显著降低(P<0.01)。结论:肺腺癌患者的外周血存在免疫细胞增殖能力下降的现象,且在浸润性肺腺癌患者中表现更明显。这一发现可能与年龄相关的免疫衰老有密切联系,为理解浸润性肺腺癌患者较差的预后提供了新视角,并为深入探索免疫衰老机制指明了方向。

端粒酶hTERT促进肿瘤细胞侵袭与转移及其机制研究

目的观察hTERT基因修饰对人骨肉瘤细胞系U-2OS生物学行为的影响。方法采用脂质体法将克隆有人全长cDNAhTERT的真核荧光质粒(pIRES2-EGFP-hTERT)转染端粒酶阴性的人骨肉瘤U-2OS细胞,经G418筛选,免疫组化和Westorn Blot鉴定后,检测其端粒酶活性的改变、生长周期以及生物力学的变化。结果成功转染人真核荧光表达载体的U-2OS细胞能有效抵抗G418,并可有效表达hTERT蛋白,细胞内端粒酶活性明显增强,G1期细胞比例下降,S期细胞比例升高,并且还明显增强了对细胞外基质的粘附力。进一步采用Transwell小孔迁移实验检测发现,hTERT/U2OS侵袭能力明显增强。结论转染hTERT基因后,U-2OS细胞内可同时通过端粒酶途径延长端粒。hTERT基因修饰可促进细胞周期进程,促使细胞从G1期→S期。通过替代途径延长端粒的肿瘤细胞在hTERT基因修饰后,增殖能力及侵袭能力明显增强。

重建端粒酶活性延长人成纤维细胞寿命的研究

正常人体细胞DNA的端粒随着细胞分裂而缩短,当缩短至一定长度时细胞将停止增殖并衰老死亡。细胞中的端粒酶对端粒起着补足长度的作用。但端粒酶在正常体细胞中不表达,只在生殖细胞、干细胞和肿瘤细胞中表达。最近已有将人端粒酶亚单位基因导入正常人体细胞而使细胞寿命延长的报道。本研究将人端粒酶催化亚基(hTERT)基因用电穿孔法转入正常人体成纤维细胞,筛选出阳性克隆后传代培养,确认外源性端粒酶基因表达和端粒酶活性的重建,证实细胞衰老延缓;同时,通过DNA整倍性和染色体核型分析,明确这些寿命延长的细胞并未发生恶性转化。目的在于通过在具有成骨潜能的成纤维细胞中重建端粒酶活性来延长它们作为骨修复种子细胞的寿命,并且对它们进一步用于临床的安全性进行考察。

端锚酶反义寡核苷酸联合反义端粒酶催化亚单位对人肺腺癌A549细胞端粒动力学的影响

背景与目的:端锚酶是真核生物体内的一种重要的功能蛋白,对调节细胞端粒长度及参与细胞衰老和永生化过程起着重要的作用。本研究探讨端锚酶及端粒酶反义寡核苷酸联合作用对人肺腺癌A549细胞端粒相关蛋白表达和翻译的影响,以及对端粒缩短效能和细胞周期的作用。方法:将培养的A549细胞分为空白对照组、端锚酶正义寡核苷酸对照组(tankyrase sense oligonucleotide,sTANKS)、端粒酶催化亚单位正义寡核苷酸对照组(human telomerase reverse transcriptase sense oligonucleotide,shTERT)、端锚酶反义寡核苷酸实验组(tankyrase antisense oligonucleotide,asTANKS)、端粒酶催化亚单位反义寡核苷酸实验组(human telomerase reverse transcriptase antisense oligonucleotide,ashTERT)、端锚酶及端粒酶催化亚单位反义寡核苷酸联合实验组(asTANKS+ashTERT)。分别与不同的正、反义寡核苷酸作用,采用RT-PCR(reverse transcription-polymerase chain reaction)法观察端粒酶催化亚单位的mRNA表达,ELISA-PCR法测定端粒酶活性,Western blot法观察端锚酶活性,Q-FISH法检测各组端粒的平均长度;并通过传代实验观察不同正、反义寡核苷酸对A549细胞传代寿命的影响。结果:ashTERT能明显抑制端粒酶催化亚单位的mRNA表达及蛋白质活性,不影响端锚酶活性;作用48h后细胞平均端粒长度明显缩短[(7.59±0.07)kb];细胞在经过56.92±0.46个倍增时间(population double,PD)连续培养终止。asTANKS不影响端粒酶催化亚单位mRNA表达及蛋白质活性,却能明显抑制端锚酶活性;作用48h后细胞平均端粒长度明显缩短[(7.33±0.09)kb];细胞在经过(53.33±0.57)PD连续培养终止。ashTERT+asTANKS联合作用同时抑制端粒酶和端锚酶,其细胞平均端粒长度缩短更为明显[(3.55±0.08)kb],流式直方图上FITC荧光"左偏"更显著,与ashTERT、asTANKS比较差异有显著性(t=37.33、32.50,P<0.001);ashTERT+asTANKS组细胞在经(24.53±0.40)PD后培养终止,与ashTERT、asTANKS比较差异也有显著性(t=53.38、43.39,P<0.001)。结论:端锚酶反义寡核苷酸对A549细胞端粒长度的抑制有别于端粒酶途径,它不仅能通过影响端锚酶活性,缩短A549细胞端粒的平均长度,而达到缩短肿瘤细胞生存寿命的目的;而且可与端粒酶抑制剂产生协同作用,明显缩短肿瘤细胞的生存周期,可能成为抗癌作用的新靶点。

辛伐他汀对颅内动脉瘤大鼠端粒长度、端粒酶反转录酶及端粒重复序列结合因子2基因表达的影响

目的探讨辛伐他汀对颅内动脉瘤(IA)大鼠端粒长度、端粒酶反转录酶(TERT)和端粒重复序列结合因子2(TRF2)基因表达的影响。方法将36只SD大鼠分为对照组、模型组和辛伐他汀组,每组12只。对模型组及辛伐他丁组的大鼠切除双侧卵巢并结扎左侧颈总动脉及双侧肾动脉后支以构建IA模型,对照组仅暴露相应组织及血管后缝合。建模后模型组和辛伐他汀组分别给予生理盐水及辛伐他汀灌胃。于建模后4个月,测定各组大鼠外周血白细胞端粒相对长度、TERT和TRF2 mRNA的相对表达量。结果建模后4个月,模型组和辛伐他汀组各有8只大鼠形成IA,对照组无大鼠形成IA;对照组、辛伐他汀组和模型组大鼠的端粒相对长度依次缩短(P<0.05),而对照组、模型组和辛伐他汀组TRF2 mRNA相对表达量依次升高(P<0.05);模型组和辛伐他汀组的TERT mRNA相对表达量低于对照组(P<0.05),但两组间比较,差异无统计学意义(P>0.05)。结论 IA大鼠可能由于端粒酶活性降低而发生端粒长度缩短,辛伐他汀可通过促进TRF2的表达而发挥保护端粒作用。

端粒酶逆转录酶和端粒重复结合因子2在大鼠心肌细胞发育过程中的表达

目的:观察大鼠心肌细胞发育过程中端粒酶逆转录酶(TERT)和端粒重复结合因子2(TRF2)的表达,探讨TERT活性与心肌细胞分化和发育的关系。方法:应用免疫组化方法分别检测胎鼠孕16d、出生后2、5、10、20d的大鼠心脏标本,观察TERT和TRF2的表达和分布。结果:TERT分布于胎鼠心肌细胞的胞质和细胞核中,出生后2～20d,TERT在胞质的表达呈下降趋势。TRF2主要分布在胎鼠心肌细胞的胞核中,出生后5～20d,TRF2表达阳性的心肌细胞数目逐渐减少。结论:在大鼠心肌细胞发育过程中,TERT和TRF2的表达下调促使部分细胞永久地退出细胞周期而进入终末分化;TERT活性受到了心肌细胞发育过程的调节。

端粒酶逆转录酶基因在口腔癌前病变及鳞癌的表达

目的 观察端粒酶催化蛋白基因 (h TRT)在口腔癌前病变及鳞癌中的表达 ,探讨其与口腔鳞癌(OSCC)发生发展及临床病理特征之间的关系。 方法 用原位杂交技术检测 5 4例标本 ,其中正常口腔粘膜 6例、上皮非典型增生 15例、口腔粘膜鳞癌 33例。 结果 正常口腔粘膜组织中 h TRT的 m RNA表达较弱 ,阳性信号仅局限于上皮基底层及副基底层间 ,阳性率 16 .6 7% (1/ 6 ) ;上皮非典型增生中 h TRT的 m RNA阳性表达见于多层上皮细胞 ,并随细胞异形性增高而表达增强 ,阳性率 6 0 % (9/ 15 ) ;口腔粘膜鳞癌组织中 h TRT的 m RNA有较强阳性表达 ,阳性率 87.88% (2 9/ 33) ;OSCC组织中 h TRT m RNA的表达与肿瘤临床病理分化无关 ,与淋巴结转移密切相关。 结论 端粒酶 h TRT基因的表达在

精子发生中的端粒与端粒酶

端粒长度、端粒酶活性在生精细胞分化与增殖的各个阶段各不相同 ,从精原细胞到精子的成熟过程中 ,端粒酶活性的不断下降与端粒长度的依次增加呈负相关 ,在成熟精子细胞和精子中 ,端粒酶活性完全丧失。近年来研究表明 ,在少精子症和生精细胞成熟阻滞的无精子症病人之间 ,睾丸中端粒酶活性表达水平相近。人端粒酶活性、端粒酶逆转录酶为精子发生高度敏感和特异的标志 ,特别对原发性无精子症病人睾丸灶性精子发生的诊断具有重要的临床意义。

端粒酶逆转录酶基因hTERT突变表达载体在膀胱癌细胞T24中的表达

目的 构建端粒酶逆转录酶基因 (h TERT)缺失突变的真核表达载体 p EGFP- h TERT,并转入膀胱癌细胞株 T2 4中 ,观察其稳定表达 .探讨其对端粒酶活性调控机制及成为膀胱肿瘤基因治疗新靶点的可能性 .方法 将 PCR扩增的h TERT缺失突变体片段用 Eco RI与 Sal I酶切 ,并连接到真核表达载体 p EGFP- C1上 ,构建成 h TERT缺失突变的真核表达载体 p EGFP- h TERT;利用 DNA-磷酸钙共沉淀法将p EGFP- h TERT导入膀胱癌细胞株 T2 4中 ,应用荧光显微镜、与衰老相关的β-半乳糖苷酶染色等方法观察转染细胞中h TERT- GFP融合蛋白定位表达及对膀胱癌细胞生长的影响 .结果 酶切鉴定证实 h TERT缺失突变体已克隆到p EGFP- C1的 Eco RI与 Sal I位点之间 ,在转染细胞中观察到与其融合的绿色荧光蛋白的稳定表达 ,定位于细胞核内 ;转染细胞 2 wk后与衰老相关 β-半乳糖苷酶表达增加 ,细胞生长受抑制 .结论 构建的端粒酶逆转录酶基因 h TERT缺失突变真核表达载体 p EGFP- h TERT可在膀胱癌细胞 T2 4中稳定表达 .突变型 h TERT蛋白可入细胞核中竞争性影响端粒酶的功能 。

hTERT永生化细胞的研究进展及应用前景

人端粒酶逆转录酶基因(hTERT)诱导的永生化细胞兼具原代细胞的生物学特性和传代细胞系连续传代的能力,是一种理想的细胞来源,在基础研究、临床应用和生物工程领域具有无可比拟的优势。本文主要就hTERT永生化细胞的研究进展及应用前景作一综述。

Expression of hTERT, p53 and PCNA in Cystitis Glandularis

To examine the expression of human telomere reverse transcriptase （hTERT）, p53 and proliferating cell nuclear antigen （PCNA） in cystitis glandulafis, 38 patients were divided into two grouips： group A （including 18 cases of papillary cystitis glandularis） and group B （including 20 subjects with normal bladder mucosa）. All the cases were immunohistochemically examined by using antibodies specifically against p53 and PCNA, and hTERT was determined by in situ hybridization. hTERT was found in 6 cases （33.3%） and p53 was detected in 4 cases （22.2%） in group A, while they were not detected in group B. There were significant differences in hTERT and p53 expression between groups A and B （P〈0.05 for both）. PCNA was detected in 7 cases （38.9%） in group A and 1 case （5.0%） in group B, and significant difference in PCNA expression was found between the two groups （P〈0.05）. The expressions of hTERT, p53 and PCNA were significantly higher in group A than in group B, suggesting that papillary cystitis glandularis is predisposed to cancerous change, and p53, PCNA, hTERT may be related to the malignant alteration.

转录hTERT基因shRNA的溶瘤腺病毒的构建及抗肿瘤效应

目的构建转录端粒酶逆转录酶（hTERT）基因小发夹RNA（hTERT—shRNA）的条件增殖腺病毒。方法设计能转录hTERT-shRNA的模板DNA序列，煺火后克隆至pCA13质粒，构建重组质粒pCA13-hTERT。用Bgl Ⅰ从pCA13-hTERT酶切出包含CMV启动子及hTERT—shRNA模板的表达框，将表达框克隆入条件增殖腺病毒质粒pZD55，构建重组质粒pZD55-hTERT。将pZD55-hTERT与腺病毒右臂质粒pBHGE3共转染293细胞，9～12天后出现病毒空斑。提取重组腺病毒的DNA，PCR鉴定正确者即为条件增殖腺病毒ZD55-hTERT。大量扩增，氯化铯梯度离心纯化，测滴度。感染人肝癌细胞株（BEL-7404），通过荧光显微镜、结晶紫染色法观察细胞病作用，MTT法检测细胞存活情况。结果酶切分析、测序鉴定表明pCA13-hTERT构建成功；PCR、酶切分析、测序鉴定表明pZD55-hTERT构建成功；PCR鉴定表明ZD55-hTERT包含目的基因且无野生型腺病毒的污染；ZD55-hTERT滴度为1×10^11 PFU／ml; ZD55-hTERT在肝癌细胞株BEL-7404中可导致明显细胞病变效应。结论成功构建的ZD55-hTERT为利用hTERT—shRNA靶向肿瘤治疗奠定了基础。