CAJ | 학술논문

背景：昆明小鼠胚胎成纤维细胞是目前最常用的饲养层细胞，C57BL/6小鼠胚胎成纤维细胞作为饲养层的研究鲜有报道。 　　目的：体外分离和培养C57BL/6小鼠胚胎成纤维细胞，制备饲养层，力求扩大小鼠胚胎成纤维细胞的来源。方法：用不同浓度胰蛋白酶分步消化法体外分离和培养C57BL/6小鼠胚胎成纤维细胞，观察其生物学特性，研究其生长规律，并制备小鼠胚胎成纤维细胞饲养层，检测干细胞在所制备饲养层上的生长状态。 　　结果与结论：不同浓度胰蛋白酶分步消化法制备的C57BL/6小鼠胚胎成纤维细胞生长状态好，获得的成纤维细胞数量多，增殖活跃。在细胞冻存后1，2周、1，3，6个月内复苏的细胞存活率差异无显著性意义。C57BL/6小鼠胚胎成纤维细胞在第2-5代增殖旺盛，第6代以后细胞增殖出现明显下降。种植到培养皿上的C57BL/6小鼠饲养层细胞在种植后3 d内活力高，种植4 d以后细胞活力急剧下降。所以C57BL/6小鼠胚胎成纤维细胞来源的饲养层的最佳使用时间为灭活后3 d内，C57BL/6小鼠胚胎成纤维细胞饲养层和昆明小鼠胚胎成纤维细胞饲养层一样，能很好地支持胚胎干细胞及诱导多能干细胞生长。
배경：곤명소서배태성섬유세포시목전최상용적사양층세포，C57BL/6소서배태성섬유세포작위사양층적연구선유보도。 　　목적：체외분리화배양C57BL/6소서배태성섬유세포，제비사양층，력구확대소서배태성섬유세포적래원。방법：용불동농도이단백매분보소화법체외분리화배양C57BL/6소서배태성섬유세포，관찰기생물학특성，연구기생장규률，병제비소서배태성섬유세포사양층，검측간세포재소제비사양층상적생장상태。 　　결과여결론：불동농도이단백매분보소화법제비적C57BL/6소서배태성섬유세포생장상태호，획득적성섬유세포수량다，증식활약。재세포동존후1，2주、1，3，6개월내복소적세포존활솔차이무현저성의의。C57BL/6소서배태성섬유세포재제2-5대증식왕성，제6대이후세포증식출현명현하강。충식도배양명상적C57BL/6소서사양층세포재충식후3 d내활력고，충식4 d이후세포활력급극하강。소이C57BL/6소서배태성섬유세포래원적사양층적최가사용시간위멸활후3 d내，C57BL/6소서배태성섬유세포사양층화곤명소서배태성섬유세포사양층일양，능흔호지지지배태간세포급유도다능간세포생장。
BACKGROUND:Kunming mouse embryonic fibroblasts are the most common feeder layers at present, and there are rare reports addressing C57BL/6 mouse embryonic fibroblasts as feeder layers. OBJECTIVE:To separate and culture C57BL/6 mouse embryonic fibroblasts in vitro, and produce feeder layers to enlarge the resources of mouse embryonic fibroblasts. METHODS:C57BL/6 mouse embryonic fibroblasts were isolated and cultured by trypsin digestion method in vitro. The biological characteristics and growth rule of the fibroblasts were investigated, then the feeder layers for the cel culture were produced. The growth of cel colonies on the prepared feeder layer was tested. RESULTS AND CONCLUSION:C57BL/6 mouse embryonic fibroblasts grew wel with a large amount, by trypsin digestion method at different concentrations. There was no significance in the survival rate after cryopreservation for 1 week, 2 weeks, 1 month, 3 months and 6 months. The cel s were proliferative from the second to fifth passage and declined sharply after the sixth passage. The planted mouse embryonic fibroblasts feeder layers had a high activity within 3 days, but got a sharp decline after 4 days. So it is best to use C57BL/6 mouse embryonic fibroblast feeder layers within 3 days after they’re inactivated. C57BL/6 mouse embryonic fibroblast feeder layer can support embryonic stem cel s and induce pluripotent stem cel s to grow as Kunming mouse embryonic fibroblasts.