鲤鱼体细胞系的构建及鱼类核移植技术的初步研究-生物化学与分子生物学专业论文.docxVIP

摘要摘 摘要 摘 要 本文以鲤科鱼类鲤鱼和金鱼为实验材料．取鲤鱼吻端上皮及鲳条组织进行体外培养． 并对其进行传代建成了体细胞系，分析了建系过程中培养细胞的生物学特性，其中包括细 胞形态学分析、细胞生长曲线的绘制、克隆生成率的检测、细胞对温度适应性的观察、细 胞染色体数目的统计及其细胞冻存、复苏程序的摸索等．同时利用继代培养细胞做供体移 入鲤鱼和金鱼的其核未受精卵中。实验结果如下： 1．建立了鲤鱼吻端上皮细胞和尾鲳细胞系，现已传至第65代。 2．分析了原代培养细胞及继代培养细胞的生物学特性。 ①形态学观察，细胞在培养过程中呈梭形细胞增多的趋势。 ②从绘制的生长曲线来看，继代培养细胞在48-72h期间生长最旺盛，分裂速度较快。 ③克隆形成率的计算结果显示，吻端上皮和尾鳕第31代细胞克隆生成率都在70％以上。 ④对细胞对温度适应性观察可见，鲤鱼细胞对温度的适应性很强。并且对低温的耐受力大 大超过对高温的耐受力。 ⑤细胞染色体数目统计表明，原代细胞染色体数目和继代培养细胞染色体数目均为 2n=100，为正常的二倍体，未出现变异。 ⑥在冻存、复苏过程中。我们采取了逐步降温及其悬至液氮罐口两种程序，同时使用甘油 和DMSO(二甲基亚砜)两种冻存液，对冻存前、复苏后细胞进行了计数统计。结果为逐步 降温和使用D惦O冻存的细胞复活率稍高于吊至液氮罐口和使用甘油的方法，复苏后细胞 形态学没有变化。 3．采用继代培养到第4一10代的鲤鱼体细胞为供体，鲤鱼和金鱼未受精具核卵为受体，进行 移核试验，最终获得发育至原肠期的胚胎。这是鲤鱼体细胞核移植的首次报道。 关键词 鲤鱼；金鱼；体细胞；核移植 Ill Preliminary Preliminary studies on somatic cell lines construction for common carp(Cyprinus carpio) and nuclei—transfer techniques for fish Abstract Common carp and golden carp，both called cyprinidae were chosen for experimental materials in this research．Epithelia of rostral side and fins were taken to culture in vitro．As a result,cells of tail fins were successfully passed from generation to generation,and consmacted cell lines．During the culture procedure，the passage cells were observed and their biological characteristics were analyzed，which consist of morphologic analysis，drawing of growth curve， detection of producing rate for clones，insight of adaptation幻temperatures，calculation of chromosomes and investigation for cells frozen to thaw．Meanwhile，subcullural cells regarded as donors were transferred into unfertilized eggs with nuclear ofcommon carp and golden carp， the results arc as follows： 1． Cell lines from rostral side and caudal fin of cornHlon carp were respectively established successfully,and passed to 65 generations at present． 2． Biological characteristics were analyzed for primary and secondary cultural cells (1)Morphologic observation．There is an inclinable increase for spindle-shaped cells during cell cuimm． (2)As the growth curve described，subcultural cell