用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1．088)分离出小鼠血液中的淋巴细胞，直接用作核移植供体细胞，采用胞质内注射法成功构建的重构胚经常规培养2h后，SrCl2激活处理6h，然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上，添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化，然后接种培养。结果显示，小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育，核移植重构胚2-细胞率41．03％(128／312)，桑葚胚和囊胚发育率分别为9．29％(29／312)，1．92％(6／312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团，分离率为0．64％(2／312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的，可用于深入研究。

Developmental capacity of reconstructed embryos by transfer with peripheral blood lymphocytes in mouse]

Mouse peripheral blood lymphocytes were used as donor nuclei in nuclear transfer procedures to determine their feasibility. Lymphocytes were collected from peripheral blood by lymphocyte isolation liquid (density 1.088), and transferred into enucleated oocytes by intracytoplasmic injection. Two hours later, oocytes injected with donor cell nuclei were activated for 6 hours by treatment in modified M16 medium containing 10 mmol/L SrCl2, and then cocultured with mouse oviduct epithelial cell in mM16 medium. On early day 4 blastocysts were cultured on mouse embryonic fibroblasts in DMEM. After 4 days, ICMs ( inner cell mass) were trypsinized, seeded and cultured. Results showed that donor nuclei of lymphocyte could be reprogrammed to support the development of reconstructed embryos in vitro. The proportion of reconstructed oocytes (n = 312) that developed to 2-cell stage was 41.03%, while 9.29% and 1.92% of reconstructed oocytes developed to morula and blastocyst phase, respectively. In addition, 2 ICMs were isolated from reconstructed blastocysts, and the proportion of reconstructed oocytes developing to ICM was 0.64% (2/312). This study illustrates that it is feasible to construct embryos with peripheral blood lymphocytes in mouse, warranting further study in this area.