梅山猪耳成纤维细胞系的建立及生物学特性分析  

Establishment and biological characteristic analysis of ear fibroblast cell lines of Meishan Pig

作　　者：张宏丽 葛雷 吴彩凤[2] 张树山[2] 张德福[2] 戴建军[2] 孙玲伟 ZHANG Hongli;GE Lei;WU Caifeng;ZHANG Shushan;ZHANG Defu;DAI Jianjun;SUN Lingwei(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Animal Husbandry and Veterinary Research Institute,Shanghai Academy of Agricultural Sciences,Division of Animal Genetic Engineering,Shanghai Key Laboratory of Agri-Genetics and Breeding,Shanghai 201106,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海市农业科学院畜牧兽医研究所,上海市农业遗传育种重点实验室动物遗传工程研究室,上海201106

出　　处：《上海农业学报》2020年第1期82-87,共6页Acta Agriculturae Shanghai

基　　金：国家自然科学基金(31372315);上海市农业科学院攀高计划(PG171);嘉定区农业和社会事业项目(2016004)。

摘　　要：以梅山猪耳缘组织为研究材料,采用组织块培养法建立了梅山猪耳成纤维细胞系,并对所建细胞系进行生物学特性研究。结果表明:所建立的梅山猪耳成纤维细胞系生长态势良好,冷冻前后的平均细胞存活率分别为95.21%、90.08%,解冻后细胞的生长曲线呈“S”型;细菌、真菌和支原体3类微生物检测结果均为阴性,细胞没有受到微生物的污染;细胞中期染色体二倍体(2n=38)占主体约为92.31%,达到了建立成纤维细胞系的要求;苹果酸脱氢同工酶和乳酸脱氢同工酶电泳结果表明,该细胞系没有被其他细胞污染,细胞纯度较高;绿色荧光蛋白(EGFP)基因转染耳成纤维细胞,5个细胞系的阳性率为45.23%—67.32%。经G418筛选后,各个细胞系均能有效形成单克隆集落。本研究中梅山猪成纤维细胞系的建立使梅山猪种质资源在细胞水平得到有效保存,并可为其他动物成纤维细胞系的建立以及种质资源保存提供参考。This study established ear fibroblast cells from Meishan ear tissues with direct explant cultural technique,and the biological characteristics of these cell lines were studied.The results showed that the ear fibroblast cells of Meishan pig had a good growth situation,and the survival rate of fibroblasts was 95.21%and 90.08%respectively before and after cryopreservation.The growth curves of cryopreserved fibroblasts were type.After bacteria,fungi and mycoplasm detection,all the cells were not contaminated by microorganisms.The percentage of metaphase diploid cells(2n=38)was 92.31%in metaphase,and it accorded with the requirement demands of the fibroblast cell line.The banding patterns of malic dehydrogenase(MDH)and lactic dehydrogenase(LDH)showed that there were no contaminations with other cells.After transfected with green fluorescent protein(EGFP)gene,the positive rates of 5 ear fibroblast cells were 45.23%to 67.32%,and all the 5 cells after G418 selection could form monoclonal colony.In conclusion,the method for establishment of ear fibroblast cells makes the preservation of genetic resource of Meishan pig more efficiency in cell levels,and it can be used as a reference for the preservation of other animal genetic resources.

关 键 词：梅山猪 耳成纤维细胞 生物学特性 

分 类 号：S828.81[农业科学—畜牧学]