【作者】 李吉霞；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2010， 博士

【摘要】 乳腺上皮细胞具有合成和分泌乳汁的功能,在体外培养条件下仍可保持其特性。乳腺细胞的这一特性对于通过核移植生产转基因动物,尤其是制备乳腺生物反应器具有其它细胞无法比拟的优越性。然而,乳腺细胞在体外很难长期培养,因此,通过转染外源基因使乳腺细胞永生化是非常有意义的。细胞永生化的方法很多,比如转染SV40大T抗原、HPV病毒、过表达一些原癌基因等等。但是这些方法都有使细胞特性发生改变的潜在危险。hTERT能够使细胞发生永生化,又不会使细胞的生物学特征发生改变,因此受到了越来越多的关注。本研究以荷斯坦奶牛的乳腺组织为材料,通过转染hTERT,以期获得了永生化的牛乳腺细胞系。在分离培养乳腺细胞的同时,发现了乳腺细胞中有乳腺干细胞(Mammary stem cells, MSCs)的存在。参照小鼠和人乳腺干细胞分离和培养的方法,分离了牛的乳腺干细胞(Bovine Mammary stem cells, BMSCs),并对其表面抗原标记特征和分化特征进行了研究;另外,本试验还以乳腺上皮细胞(Bovine mammary epithelial cells, BMECs)和转染了端粒酶的乳腺上皮细胞(hTERT-BMECs)作为供核细胞进行核移植,研究了端粒酶对核移植重构胚发育率的影响。本研究主要获得以下结论:(1)试验分离得到了牛乳腺上皮细胞,对细胞进行鉴定,结果表明乳腺上皮细胞表达细胞角蛋白;通过体外诱导,牛乳腺上皮细胞可以分泌β-酪蛋白。(2)通过比较不同培养环境对乳腺上皮细胞生长的影响,发现添加生长因子EGF或HGF的培养液对牛乳腺上皮细胞的增殖具有重要作用,17β-E2不能单独促进牛乳腺上皮细胞的增殖,但17β-E2可与EGF协同促进牛乳腺上皮细胞的增殖,而与HGF无协同作用。(3)通过转染外源基因hTERT诱导细胞发生永生化,转染后的细胞形态正常、生长活力旺盛,核型正常,无致瘤性,并且有端粒酶活性。目前牛乳腺上皮细胞已传至68代。在激素刺激下,转染后的细胞仍能表达β-酪蛋白,表明细胞功能正常。(4)通过Real Time PCR检测,发现hTERT-BMECs中抑癌基因p53、p16、p14和p21的表达下调,表明hTERT可能引发牛乳腺细胞的某些抑癌基因的下调,从而促进牛乳腺细胞永生化的发生。(5)通过悬浮培养,从乳腺细胞中分离得到了乳腺干细胞;通过免疫组化、RT-PCR以及western blotting等方法对牛乳腺干细胞进行鉴定,发现细胞表达β1-整合素、α6-整合素,以及Oct4、Nanog、Sox2等干性基因。体外诱导分化试验结果表明,乳腺干细胞可以分化为腺上皮样细胞、肌上皮样细胞和导管样结构。(6)以hTERT-BMECs作为供核细胞,观察核移植重构胚的发育情况,结果表明端粒酶活性的高低对核移植胚胎的发育率无显著影响,说明hTERT-BMECs可以作为体细胞核移植的供体细胞。更多还原

【Abstract】 Bovine mammary epithelial cells (BMECs) are perfect cells for bioreactor due to their secretion funtion. However it is difficult to culture BMECs for long time in vitro and the cell life span is limited. So it is very meaningful to get immortalized BMECs. There are many immortalization methods, such as transfection of SV40 large antigen, HPV E6 and E7, silence of anti-ongene, overexpression of ongene and so on. Recently, the hTERT got more and more attention because of its security, the cell transfected by hTERT could keep normality. The aim of the present study was to immortalize BMECs by hTERT. During immortalization, we found there were some bovine mammary stem cells (BMSCs) in bovine mammary epithelial cells. At the same time, we compared the BMECs with hTERT-BMECs for nuclear transfer embryos developmetal potential. We got the results as followed:(1) Bovine mammary epithelial cells had been successfully separated in the test, and the cells could be stained by Keratin. In vitro, the cells could secretβ-casein under induction condition.(2) The media added EGF or HGF can promote the bovine mammary epithelial cells proliferation,but when only 17β-E2 was added, there was no effect on the cells proliferation. When 17β-E2 and EGF were both added in the medium, 17β-E2 could enhance the effect of EGF on bovine MECs, but 17β-E2 has no synergistic effect with HGF.(3) After transfection with hTERT, the bovine mammary cell grew rapidly, but the cell still kept diploid karyotype, and no tumor formed by injection of the cells in nude mouse and the cells have telomerase activity. All of those domenstrated that the cells is immortalized cells.The cells could expressβ-casein under hormones induction, which showed that the cells had normal mammary cell function.(4) By Real Time PCR, we found the expression of anti-oncogene p53, p16, p14 and p21 decreased, which suggested that expression of anti-oncogene could be induced to be decreased by hTERT, and made the bovine mammary cell immortalization.(5) The bovine mammary stem cells were isolated by suspension culture. The bovine mammary stem cells expressedβ1-integrin,α6-integrin, Oct4, Nanog and Sox2 by immunochemistry, RT-PCR and western blot. In vitro the mammary stem cells could differentiate into mammary epithelial-like cells, myoepithelium-like cells and ductal-alveolar structure.(6) The BMECs and the BMECs transfected hTERT (hTERT-BMECs) were used as donor cells for comparing the development of nuclear transferred (NT) embryoes, we founded that telomerase could not improve the NT-embryoes development. And which domenstrated that the hTERT-BMECs we got could be used for NT.更多还原