蚜肠霉素处理及细胞传代数对小型猪体细胞核移植胚胎发育的影响

Effects of Different Donor Cells Treatment with Aphidicolin and Donor Cell Passages on the Preimplantation Development of Somatic Cell Nuclear Transfer Embryos in Minipig

目的 为优化小型猪体细胞核移植技术（SCNT）技术,探讨供体细胞不同类型、不同传代数以及蚜肠霉素（Aphidicolin,APC）处理各供体细胞对重构胚体外发育的影响.方法 小型猪输卵管上皮细胞（OEC）,耳成纤维细胞（EFC）和卵丘细胞（CC）分别经血清饥饿或血饥饿后再使用0.1μg/mlAPC处理.核移植重构胚体外培养7d,以检测其发育能力.结果 OEC经APC处理后,重构胚的卵裂率和囊胚形成均显著高于仅血清饥饿处理组（61.82％ vs 56.25％,24.55％ vs 17.86％; P＜0.05）.以EFC为供体细胞,APC组重构胚卵裂率显著高于血清饥饿组（63.36％ vs 57.01％; P＜0.05）.注入CC,APC处理能显著提高重构胚的囊胚形成率（29.63％; P＜0.05）.三种细胞经APC处理,CC作为核供体,重构胚的卵裂率和囊胚形成率最高.三种细胞中,无论哪一种作为核供体,其传代数对重构胚的发育没有影响.结论 以CC为供体细胞,血清饥饿后再使用APC处理,更适合小型猪的体细胞核移植.

Objective To optimize somatic cell nuclear transfer （SCNT） procedures in mini-pigs, the present study was designed to examine the effects of donor cell types, cell passage number and aphidicolin （APC） treatment on in vitro development of reconstructed embryos. Methods The Oviduct epithelial cells （OEC）, ear fibroblast cells （EFC） and cumulus cells （CC） derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 ktg/ml APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Results Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation （61.82% vs 56.25%, 24.55% vs 17.86%; P〈0.05）. The cleav- age rate from the EFC was significantly increased by APC treatment compared to serum starvation only （63.36% vs 57.01%; P〈0.05）. In the ooctyes with the CC, the reconstructed embryos could yield high blastocysts formation rate by APC treatment （29.63%; P〈0.05）. In the presence ofAPC, CC gave rise to highest cleavage and blastocyst formation rates among the three cell types. All of the three donors can be used as donor karyoplasts for nuclear transfer, and the efficiency is not influenced by culture passages. Conclusion The treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.