农业生物技术学报2012，Vol.20Issue(5)：536-542,7.DOI:10.3969/j.issn.1674-7968.2012.05.010
金华猪胎儿成纤维细胞系的建立与生物学特性分析
Establishment and Biological Characteristic Analysis of Fetal Fibroblast Cell Line for Jinhua Pig
摘要
Abstract
Establishment and preservation of livestock fibroblasts play a important role in protecting of animal genetic resources. The fetal fibroblast cell line of Jinhua pig was successfully established from 40 d's embryos by direct explant technique, and the cell morphology, growth dynamic, vitality, chromosome, isoenzymes, microbial inspection, cell apoptosis, as well as the transfection of fluorescent protein report plasmid (pEGFP-N3) had been studied in the line. The results showed that the primary cells proliferated rapidly, and overgrew on the bottom of culture flast in 2-3 days after explanted with a good situation, the growth curve had an obviously typical S shape and the population doubling time (PDT) was 36 h. The viabilities before freezing and after recovery were 95.2% and 92.8%, respectively. The frequency of chromosome number (2n=38) was 91% in metaphase, and it had met the establishment standard of fibroblast cell line. The analysis of lactic dehydrogenase(LDH) and malic dehydrogenase(MDH) isoenzymes showed no cross-contamination among the cells. No bacteria, fungi and mycoplasm were found, and there were without the microbial pollution in fibroblasts. The cell apoptosis rate of fibroblasts in the 15th generation was 2.0%, and without large-scale cell apoptosis. The plasmid transfection efficiency peaked at 32.4% , when the dose of cationic liposome (lipofectamin 2000) was 0.3 |iL and fluorescent protein report plasmid(pEGFP-N3) was 0.5 μg. The individual item of the cell line had met the standard quality controls of American Type Culture Collection (ATCC). Establishment of Jinhua fetal fibroblast cell line not only preserves genetic resources of Jinhua pig but also provides a necessary experimental material for bioresearch, embryonic cloning and transgenosis animal. Moreover, the establishment of Jinhua fetal fibroblast cell line can provides both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.关键词
金华猪/胎儿成纤维细胞系/生物学特性Key words
Jinhua pig/ Embryo fibroblast cell line/ Biological characteristics引用本文复制引用
郝柱,王颖,彭静,沈一飞,郭晓令,徐宁迎..金华猪胎儿成纤维细胞系的建立与生物学特性分析[J].农业生物技术学报,2012,20(5):536-542,7.基金项目
国家转基因生物新品种培育重大专项(No.2009ZX08006-009B) （No.2009ZX08006-009B）
中国博士后科学基金(No.2011M501018) （No.2011M501018）
浙江省重点科技创新团队项目(No.2010R50027) （No.2010R50027）
