摘要
“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著(P<0.05);不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人-兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。
Therapeutic cloning is one of our major research objectives, and therapeutic cloning is based on human somatic cell nuclear transfer. Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos, the effects of type, passage and preparation method of donor cells on embryo development remain to be determined. In our experiment, cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell, skin fibroblast and cumulus cells. The cumulus cell embryos show significantly higher development rates than the other two （P〈0.05）. The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations show no significant difference. Also, FISH were conducted to detect nuclear derivation of the embryos. The result showed that the nuclei of the inter-species cloned embryo cells came from human. We conclude （1） that cloned embryos can be constructed through human-rabbit inter-species nuclear transfer; （2） different kinds of somatic cell result in different efficiency of nuclear transfer, while in vitro passage of the donor does not influence embryo development; （3） refrigeration is a convenient and efficient donor cell preparation method. Finally, it is feasible to detect DNA genotype through FISH.
关键词
治疗性克隆
核移植
体细胞核移植
异种克隆胚胎
Therapeutic cloning
Nuclear transfer
Somatic cell nuclear transfer
Inter-species cloned embryo
