Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.     

小鼠卵母细胞和早期胚胎端粒酶活性的测定

为了解小鼠早期发育过程中端粒酶的活性变化,本研究利用端粒重复序列扩增法(TRAP)进行了小鼠卵母细胞和附植前胚胎端粒酶活性的测定。结果表明,小鼠卵母细胞、桑椹胚和囊胚为端粒酶阳性,而2-细胞和8-细胞为阴性。依据电泳条带在成像系统下的光密度,计算相对总产品产量(TPG)的定量比较发现,囊胚端粒酶活性最高,为269.331;桑椹胚次之,为96.231;卵母细胞最低,为85.782。小鼠桑椹胚和囊胚胚胎记数及单细胞端粒酶活性比较结果显示,囊胚单细胞TPG最低,为3.45;桑椹胚单细胞略高于囊胚,为4.00;而卵母细胞最高,为85.782,大约是囊胚细胞的25倍。     

端粒和端粒酶的结构与功能及其应用

端粒是构成真核生物线状染色体末端重要的DNA-蛋白质复合结构,DNA由简单的串联重复序列组成．它的合成由一个特殊的具有反转录活性的核糖核蛋白端粒酶完成．端粒对染色体、整个生物基因组,甚至对细胞的稳定都具有重要意义．端粒酶是由RNA模板和蛋白亚基组成的核蛋白颗粒．它解决染色体的末端问题,归属于逆转录酶家族又和逆转录酶有一定的差别．端粒酶的过度表达和细胞的永生化和癌变直接相关．端粒酶的结构和功能决定了它在肿瘤与癌症治疗等方面具有广泛的应用前景．     

Inhibition of telomerase in canine cancer cells following telomestatin treatment

Telomere shortening in normal somatic cells has been proposed as a major barrier to unlimited cellular proliferation. Telomerase is an enzyme capable of maintaining telomere length, and thus bypassing this barrier. In human beings, telomerase activity is restricted to cancer cells and cells of stem or germ cell lineages. Dogs represent a potentially useful clinical model for the development of telomerase‐based therapies because telomerase activity is also restricted to cancer cells and stem cells in this species. We examined the ability of telomestatin to inhibit telomerase activity in telomerase‐positive D17 and CMT7 canine cancer cell lines. At a concentration of 2 μM, telomestatin treatment resulted in a decrease in telomerase activity, telomere shortening, growth inhibition and apoptosis in telomerase‐positive cancer cells. These effects were not seen in telomerase‐negative skin fibroblasts or negative controls. These results confirm that telomestatin specifically inhibits telomerase activity in canine cancer cells and strengthens the usefulness of dogs as a model for testing telomerase‐based therapies.     

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT‐MECs)

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post‐parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT‐MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF‐7 cells. hT‐MECs on passage 42 grew vigorously and ‘S’ sigmoid curves of growth were observed. Moreover, hT‐MECs maintained a normal chromosome modal number of 2n = 60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta‐casein protein was synthesized and secreted. Beta‐casein expression was enhanced by prolactin (P < 0.05). Lipid droplets were found in hT‐MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT‐MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT‐MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time.     

酵母端粒酶RNA的制备及其构型影响因素分析

【目的】体外转录并纯化酵母端粒酶RNA,明确缓冲液中组分对其构型的影响,初步研究四膜虫端粒酶相关蛋白p65与酵母端粒酶RNA的结合情况,为酵母端粒酶活性鉴定及构型功能研究奠定基础。【方法】利用核酶的自剪切功能,通过在3′端引入HDV核酶序列获得完整的酵母端粒酶RNA;并用分子筛SuperDeX-200/16/60凝胶过滤纯化酵母端粒酶RNA;琼脂糖凝胶电泳检测RNA Buffer组分中NaCl和MgCl2浓度对酵母端粒酶RNA构型的影响;非变性聚丙烯酰胺凝胶电泳(PAGE)检测四膜虫端粒酶相关蛋白p65与酵母端粒酶RNA的结合情况。【结果】通过基因序列合成,结合PCR扩增方法获得了酵母端粒酶RNA基因,并将其构建到体外转录载体上;体外转录获得大量的酵母端粒酶RNA(Yeast-RNA-239);使用SuperDeX-200/16/60成功分离得到了纯净的Yeast-RNA-239。NaCl、MgCl2浓度对酵母端粒酶RNA构型有一定的影响,表现为没有NaCl或MgCl2时,RNA的构型均一且致密;随NaCl、MgCl2浓度的增大,RNA构型逐渐变得松散;而四膜虫端粒酶相关蛋白p65能与酵母端粒酶RNA有效结合。【结论】在体外成功制备了高纯度的酵母端粒酶RNA,NaCl、MgCl2浓度对其构型有影响,四膜虫端粒酶相关蛋白p65能够帮助端粒酶RNA正确折叠。     

端粒和端粒酶与肿瘤关系的研究进展

端粒是末端染色体 ,维持染色体的稳定。端粒酶是染色体末端转移酶 ,是合成端粒DNA的酶 ,对端粒的结构起着稳定的作用。端粒、端粒酶的研究始于 2 0世纪初期 ,但直到近几年科研人员才发现他们与肿瘤有着密切的关系。在人类端粒酶的研究过程中发现端粒酶在约 85 %以上的恶性肿瘤中呈阳性 ,而动物肿瘤与端粒酶的研究还是空白。文章主要介绍端粒、端粒酶的结构功能及它们与肿瘤关系的研究进展 ,并讨论世界上首例蛋鸡 J亚群禽白血病的自然发病的病例的端粒酶 TERT表达。作者曾用辣根过氧化物酶标记的链酶卵白素进行免疫组化检测 ,结果在病鸡的心、肝、脾、肺、肾、气管、十二指肠、睾丸、骨髓、脑、胸腺、法氏囊等组织中发现 TERT表达呈阳性 ,说明在蛋鸡 J亚群禽白血病发生时端粒酶活性升高 ,提示端粒酶参与了该病的发生发展机制 ,并且该结果与人类肿瘤端粒酶的活性的研究结果相一致     

人参皂苷-Rh2抗肿瘤机理的研究进展

论述了从人参中提取的有效成分人参皂苷-Rh2（G-Rh2）的抗肿瘤机理的研究进展，并探讨了G-Rh2在调节肿瘤细胞信号通路系统、逆转肿瘤细胞的异常分化与耐药性、降低肿瘤细胞端粒酶活性、增强机体免疫力、提高抗肿瘤能力以及阻断肿瘤细胞某些重要成分的合成与代谢等方面的抗肿瘤活性及其作用机理。