用于保种的蒙山牛耳成纤维细胞的培养研究

本研究首先利用组织块培养法成功分离培养了蒙山牛耳皮肤成纤维细胞,接着对培养的细胞进行了传代培养和冷冻保存及复苏检测,然后对细胞进行了形态观察和生长曲线测定,最后分别对不同汇合程度和不同保存时问细胞的核型正常率和细胞周期进行分析.结果表明,培养的细胞具有典型的成纤维细胞形态,具有正常的分裂增殖特性,没有转化倾向.在处于S期细胞数目上,60%～70%和80%～90%汇合程度组差异不显著,但显著高于90%～100%汇合程度组(P<0.05).而在处于G_0+G_1期细胞数目上,60%～70%和80%～90%汇合程度组显著低于90%～100%汇合程度组(P<0.05).冷冻保存1个月和3个月组细胞复苏率显著高于12个月组(P<0.05),但保存时间对核型正常率、处于S期细胞数目和处于G_0+G_1.期细胞数目上差异不显著(P>0.05).说明在本实验室条件下,可以利用体细胞培养进行蒙山牛种质资源保存,这也为稀有或濒危物种种质资源保存提供一种有效手段.

Culture of Mengshan Cattle Ear Fibroblasts Used for Conservation of Species Resources

Fibroblasts derived from Mengshan cattle ear were firstly successfully cultured with a normal tissue culture method, and then subcuhured for passages, stored in liquid nitrogen and survival rate evaluated after thawing. Finally, cultured cells were analyzed by their morphology, growing curve, chromosomes and cell cycle. Results demonstrated that cultured cells were typical fibroblasts with normal morphology and proliferation. There was no significant difference in the S and G_0/G_1 stage rate of 60% -70% and 80% -90% confluence, but were both higher/ lower than 90% -100% confluence. The survival rates after storage for 1 month and 3 month were higher than storage for 12 month, but storage time had no significant effect on normal chromosomes, S and G_0/G_1 stage rates of cultured cells. In conclusion,the culture of Mengshan cattle ear fibroblasts provided an effective method for conservation of rare and endangered species.