免疫外科法制备牛供体细胞核胞体及其核移植研究

Preparation of Donor Nuclei by Immunosurgery and Application in Bovine Somatic Cell Nuclear Transfer

本研究利用自制的兔抗牛脾细胞免疫血清裂解供体细胞膜而获得核胞体,结果表明,自制的免疫血清可以成功的裂解供体细胞膜而获得核胞体。将该法制备的核胞体应用于牛体细胞核移植的胞质内注射中,从而为核移植中供体细胞核胞体的获得提供一种新的思路。在比较不同供体细胞对重构胚发育的影响时,结果表明,颗粒细胞与耳成纤维细胞构建的重构胚卵裂率没有显著差异(62.62%versus67.89%,P>0.05),囊胚率也没有明显的差异(23.88%versus18.92%,P>0.05);在4℃低温冷藏一定时间的供体细胞与未经冷藏的供体细胞,其卵裂率和囊胚率都没有显著性差异(63.34%versus62.03%;26.67%versus24.45%,P>0.05);同样,耳成纤维细胞也得到相同的结果(70.76%versus66.67%;21.74%versus19.56%,P>0.05)。颗粒细胞构建的重构胚在注核后3～5h进行激活较1～3h有更高的卵裂率,表现出显著性差异(73.91%versus56.52%,P<0.05),囊胚率无显著性差异(23.53%,versus15.38%,P>0.05)。

This study was designed to assess the effects of the donor nuclei islation which was performed by the rabbit anti-bovine-spleen serum lysising the membrane of the donor cells. The nuclei gained by this method were applicated to the bovine oocyte intraplasmic nuclear injection, which provided a new method of acquiring donor nuclei in nuclear transfer accordingly. The results indicated that the serum could successfully lyse the membrane of the donor cells. In this study, the effects of different donor cells in the development of reconstructed embryos were compared, the result showed that the cleavage rates of granular cells and ear fibroblasts were 62.62% and 67.89%, the blastocyst rates were 23.88% and 18.92%, respectively, these data have no significant difference（P〉0.05）between each other. The study also researched whether it can effect the nuclei transplant or not if the donor cell treated by low temperature（4℃）preservation. The result showed that the there was no obvious difference（P〉0.05）in the cleavage rates and the blastocyst rates between the two groups which are treated with 4℃ preservation or not, no matter what the donor cell is granular cells or ear fibroblasts. The cleavage rates of granular cells with or without 4℃ preservation is 63.34% vs 62.03%, the data is 70.76% vs 66,67% on the ear fibroblasts. The blastocyst rates were 26,67% vs 24.45% on the granular cells, 21.74% vs 19.56% on the ear fibroblasts. In the research of the influence of delaying activation with different time after nuclear injection on the cleavage rate and the development of reconstructed embryos, the reconstructed embryos with the delaying time of 3- 5h has the higher cleavage rates than that of the embryos with 1 -3h delaying time in the granular cells, and there had significant difference （73.91% versus 56.52%, P〈0.05）between them. However, there has no significant difference between the blastocyst rates （23.53%, versus 15.38%, P〉0.05）. In a word, these results demonstrated that it was feasible to prepare the donor nuclei by immunosurgery which was applicated to bovine somatic cell nuclear transfer（SCNT）.