定量分析诱导山羊体细胞重编程过程中端粒酶的表达变化

动物体细胞重编程为诱导多能干细胞(iPS)是目前干细胞生物学研究的热点。文中重点对山羊体细胞重编程过程中端粒酶(TERT)基因的相对表达量进行了检测,探讨了山羊重编程细胞的形成与端粒酶基因表达的关系。从关中奶山羊胎儿皮肤分离得到的胎儿成纤维细胞(GEF),其增殖能力较强,核型正常(60条XY),通过转录因子在体外诱导得到山羊重编程细胞。利用Real-timeRT-PCR方法首先对关中奶山羊胎儿各种组织的TERT表达进行了检测,结果表明睾丸组织中TERT的表达显著高于上皮组织(P0.01),在山羊胎儿的其他组织中TERT也有不同程度的表达。对原代重编程细胞和4株不完全重编程细胞株的TERT表达检测结果发现,碱性磷酸酶(AP)阳性的重编程细胞端粒酶表达量要显著高于AP阴性的重编程细胞(P0.01)。这一结果揭示,激活端粒酶活性并使其保持较高的表达水平对体细胞的重编程至关重要。     

Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.     

Immortalization of bovine germ line stem cells by c-myc and hTERT

The limited life span of bovine germ line stem cells in vitro is one of the obstacles to spermatogenesis analysis, genetic manipulation and generating transgenic animal. The aim of this study is to establish immortalized bovine germ line stem cells by c-myc or hTERT. We constructed pEMY and pETE expression vectors and transfected germ line cells from 5-month-old bovine. After G418 screening, four types of positive clones were obtained. The results showed that they expressed exogenous genes c-myc or hTERT at mRNA and protein level by RT-PCR and Western blotting detection. Presumable cell lines GM7, GT3, GMT5 all expressed germ-line-stem-cell-specific makers by immunocytochemical analysis, such as c-kit, Oct-4 and GFR-1. The putative cell lines also had higher capacity of proliferation than freshly isolated bovine spermatogonial stem cells. So we can conclude that exogenous genes c-myc or hTERT have integrated into the genome of bovine germ cells and upregulated the expression of telomerase.     

排卵前期卵泡颗粒细胞端粒酶的表达及其影响因素

用端粒酶重复扩增酶联免疫吸附分析法（telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA）观察体外培养的大鼠排卵前期卵巢颗粒细胞中端粒酶活性的表达及其影响因素,并用放射免疫分析法（radioimmunoassay,RIA）同步测定培养液中雌二醇（estradiol,E2）、孕西阿（progesterone,P0）含量的变化及MTT（四甲基偶氮唑盐）法测定颗粒细胞增殖指数,分析颗粒细胞中端粒酶活性的表达以及端粒酶活性表达的影响因素。本实验中大鼠排卵前期卵巢颗粒细胞中有端粒酶活性表达,且在人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）、卵泡刺激素（follicle-stimu1ating hormone,FSH）、二丁酰环磷腺苷（dbcAMP）及维拉帕米（verapamil）作用下活性明显升高,而在反义c-myb作用下活性明显降低。RIA测定培养液中雌激素及孕激素含量发现,在verapamil及FSH作用下E2与P0分泌量明显升高,在dbcAMP及HCG作用下分泌量无明显改变,而在反义c-myb作用下分泌量明显降低,在不同作用因素下的端粒酶活性与它相对应的E2及P0分泌量无相关性。MTT法测定显示,反义hTERT能明显抑制颗粒细胞的增殖。由此可以证实,排卵前期卵巢的颗粒细胞中表达有端粒酶活性,其活性受FSH、HCG、verapamil、dbcAMP及癌基因的影响,并且端粒酶活性与颗粒细胞增殖功能相关。     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Curcumin inhibits telomerase and induces telomere shortening and apoptosis in brain tumour cells

Curcumin, a polyphenolic compound isolated from Curcuma longa (Turmeric) is widely used in traditional Ayurvedic medicine. Its potential therapeutic effects on a variety of diseases have long been known. Though anti‐tumour effects of curcumin have been reported earlier, its mode of action and telomerase inhibitory effects are not clearly determined in brain tumour cells. In the present study, we demonstrate that curcumin binds to cell surface membrane and infiltrates into cytoplasm to initiate apoptotic events. Curcumin treatment has resulted in higher cytotoxicity in the cells that express telomerase enzyme, highlighting its potential as an anticancer agent. Curcumin induced growth inhibition and cell cycle arrest at G2/M phase in the glioblastoma and medulloblastoma cells used in the study. Gene and protein expression analyses revealed that curcumin down‐regulated CCNE1, E2F1 and CDK2 and up‐regulated the expression of PTEN genes resulting in growth arrest at G2/M phase. Curcumin‐induced apoptosis is found to be associated with increased caspase‐3/7 activity and overexpression of Bax. In addition, down‐regulation of Bcl2 and survivin was observed in curcumin‐treated cells. Besides these effects, we found curcumin to be inhibiting telomerase activity and down‐regulating hTERT mRNA expression leading to telomere shortening. We conclude that telomerase inhibitory effects of curcumin underscore its use in adjuvant cancer therapy. J. Cell. Biochem. 114: 1257–1270, 2013. © 2012 Wiley Periodicals, Inc.