猪胎儿肾脏成纤维细胞体外培养体系的建立  

Establishment of an in vitro culture system for porcine fetal kidney fibroblast cells

作　　者：卢晟盛[1,2] 刘红波[1,2] 吕培茹[1,2] 陆阳清[1,2] 杨小淦[1,2] 皮道元[1,2] 卢克焕[1,2] 

机构地区：[1]广西亚热带生物资源保护利用重点实验室,广西大学 [2]广西大学动物科技学院,南宁530004

出　　处：《动物学报》2007年第6期1054-1062,共9页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金项目(No.30660127);广西科学基金资助项目(桂科青0640002)~~

摘　　要：本研究旨在建立猪胎儿肾脏成纤维细胞体外培养体系,并探讨其作为猪体细胞克隆供体的可能性。使用组织块培养法从体长为10cm以上的猪胎儿分离得到猪胎儿肾脏成纤维细胞,绘制了生长曲线,鉴定了细胞类型并且进行了细胞周期同期化效果的研究。结果表明:该培养体系可以支持猪胎儿肾脏成纤维细胞的体外生长,单个细胞均为梭形细胞,抗波形蛋白免疫荧光染色显示为阳性,而抗角形蛋白免疫荧光染色为阴性,分离到的细胞为胎儿肾脏成纤维细胞。使用血清饥饿法和接触抑制法诱导细胞进入G0/G1期,并且分别比较两者同期化效率,结果显示:血清饥饿2d和4d的同期化效率差异不显著,但都比8d组的高(88.97%和87.69%比82.45%,P<0.05);接触抑制4d、6d组间同期化效率差异不显著,但都比0d组的高(85.56%和85.89%比81.82%,P<0.05)。本研究在国内首次分离得到猪胎儿肾脏成纤维细胞,已经在体外传代培养到32代,其同期化效果好,可以作为体细胞克隆供体。A method is described for isolation and culture of porcine kidney fetal fibroblast cells defined for porcine somatic nuclear transfer. Porcine fetal kidney fibroblast cells were first isolated and identified by morphology and Immunofluorescence. The synchronization efficiency of two protocols （serum starvation and contact inhibition） was determined using flow cytometry to measure cellular DNA, enabling the calculation of percentages of cells in GO/G1 phase of the cell cycle. The fibroblast cells expressed Vimentin, no expression of Cytoceratin. Flow cytometry measurements revealed that short periods of 2 d - 4 d of serum deprivation decreased the proportion of porcine fetal fibroblast cells in the GO/G1 phase （2C DNA content） from 88.97 to 87.69%. This effect was not significant statistically. Prolonged culture in serum-deprived medium for 8 d further decreased the proportion of the cells in the GO/G1 phase （82.45%）, and there was significant difference （ P 〈 0.05） for 8 d group when compared to 2 and 4 d groups （88.97 % and 87.69 %, respectively）. The contact inhibition protocol increased the proportion of porcine kidney fetal fibroblast cells at G0/G1 phase from 81.82% to 85.89% . There was no significant difference （ P 〉 0.05） in percentage of GO/G1 by contact inhibition between the 4 d （85.56%） and 6 d groups （85.89%）, but both differed significantly from the 0 d group （81.82% ： P 〈 0.05）. The method was successfully utilized to culture pure porcine fetal fibroblast cells.Serum starvation and contact inhibition were effective as protocols to synchronize the cells at the GO/G1 cycle stage [ Acta ZoologicaSinica 53 （6）： 1054-1062, 2007].

关 键 词：猪 胎儿肾脏成纤维细胞 组织块培养 间接免疫荧光 流式细胞仪 细胞周期同期化 

分 类 号：Q813[生物学—生物工程]