体细胞克隆绵羊胚的构建及其发育的研究.docVIP

分类号 S821；Q813 10129 I C S 11.220 学 号 体细胞克隆绵羊胚的构建及其发育的研究 Cloned Embryo Reconstruction in Sheep somatic and the research of the embryo development 申 请 人：陈洋 学科门类：农学 学科专业：动物遗传育种与繁殖 研究方向：发育生物学与胚胎工程 指导教师：周欢敏 教授 论文提交日期：二〇〇六年五月 摘 要 本研究分别以成年绵羊皮肤成纤维细胞和胎儿皮肤成纤维细胞为核供体，绵羊卵母细胞为核受体构建重构胚，并比较了两种重构胚的融合率、卵裂率和桑椹胚率。 在37℃、5%CO2、DMEM-F12+10%FBS培养液适宜于绵羊皮肤成纤维细胞体外生长。组织块培养法对于原代细胞的培养要优于酶消化法。传代培养时，酶消化法和反复贴壁法可分离纯化绵羊皮肤成纤维细胞，以D-Hanks为缓冲液配制的胰蛋白酶适合绵羊皮肤成纤维细胞的消化。成年绵羊皮肤成纤维细胞较胎儿皮肤成纤维细胞生长缓慢，群体倍增时间分别为48h和24h；并发现至少在传至第10代时有80.0%的染色体还保持正常的倍数（2n=4）1500v/cm，脉冲时间为40μsec较适合重构胚的融合。采用盲吸法去核，保证卵母细胞成熟率的条件下，体外成熟19～21h(P0.05)。 关键词：绵羊；成纤维细胞；体外培养；核移植 Cloned Embryo Reconstruction in Sheep somatic and the research of the embryo development Abstract This study reconstructed the embryos with adult sheep skin fibroblasts - sheep oocytes and fetal sheep skin Fibroblasts -sheep oocytes, and compared the rate of fusion, cleavage, morula of the two kinds of reconstructed embryos. The condition of 37℃ 、100% humidity and 5% CO2, DMEM-F12 supplemented with 10% FBS was used to support the sheep skin fibroblasts growth in vitro. The method of explanted tissue culture is better than the method of enzyme digestion for the primary cells. The sheep skin fibroblasts could be separated and purified by enzyme digestion and repeatitial attachments for two or three times of subcultures during the course of subcultures. The trypsin dissolved in D-Hanks was feasible for digestion of sheep skin fibroblasts; The adult sheep fibroblasts grew more slowly than fetal cells, the time of group multiplication of the fibroblasts were 48 hours and 24 hours, respectively; Sheep skin fibroblast had 80.0% normal chromosome content after at least ten passages (2n=54). Matured sheep oocytes were efficiently activated by Ionomycin together with 6-DMAP and 30.6 rate of blastocyst were achieved. It was higher than the rate of blastocyst achieved by the method of cytochalasin B. The condition of 1500