CAJ | 학술논문

背景：极小胚胎样干细胞是近年来发现的一种具有类似胚胎干细胞生物学特性的非造血干细胞，但对其体外培养扩增的方法报道极少。有研究推测，人胚胎成纤维细胞能为人骨髓极小胚胎样干细胞体外培养扩增提供良好的微环境。 　　目的：从人胚胎躯干中分离、培养人胚胎成纤维细胞，制备人胚胎成纤维细胞饲养层用于人骨髓极小胚胎样干细胞的培养。 　　方法：利用胰酶消化法从孕5-9周龄人胚胎躯干中分离培养人胚胎成纤维细胞。制作饲养层，使用不同浓度丝裂霉素C处理后，用于培养分选后的人骨髓极小胚胎样干细胞，以细胞形态、生长曲线作为胚胎成纤维细胞和饲养层的评价指标。 　　结果与结论：从人胚胎中成功分离培养出人胚胎成纤维细胞，该细胞可传代24代以上，且经过传代及冻存复苏后生物学特性无改变。丝裂酶素C低于12 mg/L时，人胚胎成纤维细胞增殖不能完全抑制；高于14 mg/L，人胚胎成纤维细胞可能死亡。12 mg/L丝裂霉素C作用3 h后能较好地抑制人胚胎成纤维细胞的增殖，并且保持其活力约2周，可以在很长一段时间内用作人骨髓极小胚胎样干细胞的饲养层。
배경：겁소배태양간세포시근년래발현적일충구유유사배태간세포생물학특성적비조혈간세포，단대기체외배양확증적방법보도겁소。유연구추측，인배태성섬유세포능위인골수겁소배태양간세포체외배양확증제공량호적미배경。 　　목적：종인배태구간중분리、배양인배태성섬유세포，제비인배태성섬유세포사양층용우인골수겁소배태양간세포적배양。 　　방법：이용이매소화법종잉5-9주령인배태구간중분리배양인배태성섬유세포。제작사양층，사용불동농도사렬매소C처리후，용우배양분선후적인골수겁소배태양간세포，이세포형태、생장곡선작위배태성섬유세포화사양층적평개지표。 　　결과여결론：종인배태중성공분리배양출인배태성섬유세포，해세포가전대24대이상，차경과전대급동존복소후생물학특성무개변。사렬매소C저우12 mg/L시，인배태성섬유세포증식불능완전억제；고우14 mg/L，인배태성섬유세포가능사망。12 mg/L사렬매소C작용3 h후능교호지억제인배태성섬유세포적증식，병차보지기활력약2주，가이재흔장일단시간내용작인골수겁소배태양간세포적사양층。
BACKGROUND:Very smal embryonic-like stem cells are a kind of non-hemopoietic stem cells, which have similar biological characteristics to embryonic stem cells. But the method of its culture and in vitro proliferation is rarely reported. Studies have speculated that human embryonic fibroblasts can provide a good microenvironment for in vitro culture and proliferation of very smal embryonic-like stem cells. OBJECTIVE:To isolate and cultivate human embryonic fibroblasts derived from human embryonic trunks and to establish a feeder layer culture system of human embryonic fibroblasts for culturing very smal embryonic-like stem cells derived from human bone marrow. METHODS:The human embryonic fibroblasts were isolated from the subcutaneous connective tissue of human embryos at pregnant 5-9 weeks using trypsin digestion method. Different concentrations of mitomycin C were used to pretreat feeder layers, which were used for cultivating very smal embryonic-like stem cells derived from human bone marrow. The effects of human embryonic fibroblasts and feeder layers were assessed by cel morphology and growth curves. RESULTS AND CONCLUSION:The human embryonic fibroblasts were successful y isolated and cultivated from human embryos, and they could be passaged beyond the 24th generation. The biologic characteristics of the cells had no changes after passage and cryopreservation. The optimal concentration of mytomcin C to inhibit proliferation of human embryonic fibroblasts was l2 mg/L for 3 hours. The human embryonic fibroblasts derived from human embryos are successful y isolated and cultivated and to produce feeder layers for very smal embryonic-like stem cells derived from human bone marrow.