猪孤雌胚胎来源单倍体细胞的研究

【作者】 刘强；

【导师】 李荣凤；

【作者基本信息】 南京医科大学 ， 人体解剖与组织胚胎学， 2014， 硕士

【摘要】 哺乳动物单倍体细胞系可以为基因功能研究和转基因动物生产提供有力的工具。为了得到猪单倍体细胞,本研究主要进行了三组实验。首先我们利用染色体核型分析技术检测了猪早期(8细胞到16细胞)孤雌胚胎的基因组完整性。所分析的总共81个卵裂球染色体中期相中48.15%为遗传完整的单倍体细胞。基于以上实验结果的支持,两种主流的获得大量单倍体细胞的技术被应用到了本实验中,包括利用孤雌激活的卵母细胞得到类胚胎干细胞和建立孤雌胎儿成纤维细胞系。总共建立了六个孤雌胚胎类胚胎干细胞系。通过染色体分析,在这六个细胞系中没有观察到包含有19条染色体的猪单倍体细胞。其中的四个细胞系主要由多于38条染色体的多整倍体细胞组成。总共建立了28个猪孤雌胎儿细胞系,染色体分析显示其主要细胞类型为19到38条染色体的非整倍体(59.48-60.91%),二倍体细胞也占有较高的比率(16.17%-22.73%),明显不同于猪孤雌类胚胎干细胞系。在27%的猪孤雌胎儿成纤维细胞系中观察到了低比例的单倍体细胞(3.4%-8.3%)。但是这些细胞都不是真正的单倍体细胞,虽然都含有19条染色体,但都存在染色体的随机丢失与补充。通过流式细胞筛选获得很低的单倍体细胞富集效率(5.63%,5.97%)和较高的二倍体细胞富集效率(86.36%,89.06%)。富集之后的二倍体细胞呈正常的二倍体核型,经基因芯片检测父源印记基因表达水平极低。综合上述结果可见：从猪早期孤雌胚胎可以得到少量的真正的单倍体细胞。体外培养猪孤雌胚胎得到的类胚胎干细胞和通过分离体内发育的孤雌胎儿所得到的成纤维细胞中的单倍体细胞呈现异常核型。猪二倍体孤雌胎儿成纤维细胞可能是研究父系印迹基因的良好的材料。更多还原

【Abstract】 Mammalian haploid cell lines could provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from8-cell to16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by48.15%of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day30PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spreads with exactly19chromosomes (the normal haploid complement) were found. Four cell lines showed a tendency to develop to polyploidy (more than38chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with19-38chromosomes were the predominant cell type (59.48-60.91%).The diploid cells were the second cell type (16.17%-22.73%). Although a low percentage (3.4-8.3%) of cells with19chromosomes were detected in27%of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels.We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes or diploidization. The porcine PA embryo derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.更多还原