猪胎儿肾脏成纤维细胞体外培养体系的建立

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。

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

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 G0/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 G0/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 G0/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 G0/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 G0/G1 cycle stage.