端粒酶在干细胞研究中的主要进展

端粒酶的表达对于维持干细胞自我更新能力和复制潜能具有重要意义。影响干细胞中端粒酶表达的因素包括生长因子、基因调控及其它物理化学因素。利用人端粒酶催化亚单位 (hTERT)基因修饰干细胞 ,可有效地体外长期扩增并维持某些干 /祖细胞的多向分化潜能特性 ;通过异位表达hTERT使干细胞永生化 ,可为干细胞基础研究及临床应用提供药物筛选模型或细胞模型     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞(ES细胞)诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸(RA)和转化生长因子-β1(TGF-β1)诱导小鼠胚胎干细胞(ES细胞)的拟胚体(EB)分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶(hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT-PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95%具有血管内皮细胞的一些特有标志和管道化生长特性。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

hTERT启动子调控rVBMDMP基因表达载体的构建与功能研究

为了探讨端粒酶催化亚单位(hTERT)启动子调控重组血管基膜衍生多功能肽(rVBMDMP)基因表达抑制肺癌细胞生长的作用机制,采用PCR方法,克隆hTERT启动子核心区片段,并检测其功能活性.然后,构建pLNSX/hTERT/rVBMDMP逆病毒载体,获取逆病毒,感染肺癌A549细胞,检测hTERT启动子调控rVBMDMP基因表达对细胞形态、细胞生长、细胞凋亡以及Caspase-3表达的影响.另外,观察hTERT启动子调控rVBMDMP基因表达对裸鼠成瘤的抑制作用、瘤组织细胞的凋亡及Caspase-3表达情况.结果发现:a.hTERT启动子核心区能调控rVBMDMP基因的表达(n=3,P<0.05);b.hTERT启动子调控rVBMDMP基因的表达具有抑制肺癌A549细胞的生长和裸鼠成瘤作用(n=6,P<0.05);c.rVBMDMP基因表达能促进肺癌A549细胞凋亡和Caspase-3蛋白表达.以上结果说明,hTERT启动子调控rVBMDMP基因表达后,通过提高Caspase-3的表达水平,促进细胞凋亡而抑制肺癌A549细胞的生长.     

hTERT基因片段的克隆、表达和抗hTERT抗体用于端粒酶及hTERT检测的研究

端粒酶是一种重要的肿瘤生物学标志,其活性在生殖细胞、绝大多数肿瘤细胞和体外培养的永生细胞可以测知,但在大多数体细胞中不易测出。人端粒酶由两部分组成,包括hTERC和hTERT,hTERC在正常细胞和肿瘤细胞中均有表达,而hTERT的表达似乎受到严格的调控且和端粒酶活性一致。为了检测肿瘤细胞中端粒酶及hTERT的表达,我们制备了抗hTERT蛋白的特异性多克隆抗体。首先用RT-PCR方法克隆了hTERTcDNA的一个片段,将其连接到GST融合表达载体pGEX-5X-3后在大肠杆菌中融合表达。将纯化的融合蛋白抗原免疫动物,制备抗hTERT蛋白的多克隆抗体。不同的细胞抽提物用该抗体进行了Westernblot分析,结果表明该抗体可特异识别端粒酶阳性细胞株中的hTERT及端粒酶,为端粒酶及hTERT的检测初步提供了一个简单有效的检测手段。     

EBV LMPl通过诱导c-myc表达活化端粒酶hTERT

利用原代人胚鼻咽上皮细胞和Tet—on—LMP1 HNE2等良好的细胞体系,采用报道基因法和Western blot法等,分别检测Epstein—Barr病毒(EBV)潜伏膜蛋白1(LMPl)诱导的c—myc反式激活活性和蛋白表达水平。从LMPl诱导细胞内c—myc表达的角度,探讨LMPl诱导端粒酶表达的分子机制。结果表明,LMPl促使细胞内c-myc反式激活活性增强,c-Myc蛋白表达量升高;导入反义LMPl表达质粒阻断LMPl表达后。c—myc反式激活活性下降。将端粒酶hTERT启动子上c—myc：结合位点突变后,LMP1不能诱导端粒酶hTERT表达。因而认为,EB病毒LMPl通过诱导c—mvc表达而活化端粒酶hTERT。     

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.     

Critical and distinct roles of p16 and telomerase in regulating the proliferative life span of normal human prostate epithelial progenitor cells

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, alpha2beta1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.     

Comparison of early passage, senescent and hTERT immortalized endothelial cells

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.     

Transient expression of human telomerase extends the life span of normal human fibroblasts

We utilized the Cre/lox recombination system to transiently express the catalytic subunit of telomerase (hTERT) in normal diploid foreskin fibroblasts (BJ cells). A retroviral construct containing an hTERT cDNA, flanked by loxP-sites was introduced into near senescent BJ cells (population doubling 85). At population doubling (PD) 92, which exceeds the typical life span of these cells, we excised the gene via Cre-mediated recombination. All clones lost telomerase activity and showed telomere shortening over an additional 50 PDs. Interestingly, the average telomere length in these cells became shorter than in untreated BJ cells at senescence. This may be due to hTERT preferentially elongating the shortest telomeres, leading to greater length uniformity. In summary, transient telomerase expression and only a very small average telomere elongation by hTERT resulted in a 50% increase in life span of human fibroblasts. This suggests a potentially safe use of hTERT in tissue engineering.