金华猪胎儿成纤维细胞系的建立与生物学特性分析

建立并保存家畜的成纤维细胞在保护畜禽种质资源研究中发挥着重要作用。本研究以40日龄金华猪胚胎为实验材料,采用组织贴壁法建立了金华猪胎儿成纤维细胞系,并对所建细胞系进行生物学特性研究。结果表明,原代细胞生长迅速,贴壁后2~3d可长满培养瓶,获得的成纤维细胞生长态势良好;细胞生长曲线为典型的S形,细胞群体倍增时间(population doubling time,PDT)约为36h;细胞冻存前、复苏后的活率分别为95.2%和92.8%;细胞中期染色体二倍体(2n=38)占主体约为91%,达到了建立成纤维细胞系的要求;苹果酸脱氢同工酶(malic dehydrogenase,MDH)和乳酸脱氢同工酶(lactic dehydrogenase,LDH)电泳结果表明,本细胞系没有被其他细胞污染,细胞纯度较高;细菌、真菌和支原体三类微生物检测结果均为阴性,细胞没有受到微生物的污染;15代细胞的凋亡率为2.0%,细胞没有大规模的凋亡现象发生;当阳离子脂质体(Lipofectamine2000)剂量为0.3μL、荧光蛋白报告质粒(pEGFP-N3)为0.5μg时转染成纤维细胞的效率最高,可达32.4%。细胞系各项指标均达到美国典型培养中心(American Type CultureCollection,ATCC)标准。金华猪胎儿成纤维细胞系的成功建立使金华猪种质资源在细胞水平得到保存,并可为胚胎克隆以及转基因等研究提供必要的实验材料;也可以为以后其他种质资源在细胞水平上的保存提供理论与技术支持。

Establishment and Biological Characteristic Analysis of Fetal Fibroblast Cell Line for Jinhua Pig

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 μL 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.