人诱导性多潜能干细胞向表皮样干细胞分化的实验研究  

Experimental study on the differentiation of human induced pluripotent stem cells into epidermal-like stem cells

作　　者：李勇铁[1,2] 刘德伍[1] 刘德明[4] 毛远桂[1] 彭燕[1] 宁璞[1] 胡翔[3] 邹萍[3] 邹永红[5] 余群洪[5] 

机构地区：[1]南昌大学第一附属医院烧伤科,330006 [2]江西省吉安市中心人民医院烧伤整形科,343000 [3]南昌大学第一附属医院药剂科,330006 [4]南昌大学医学院人体解剖学教研室 [5]江西省吉安市中心人民医院烧伤整形科

出　　处：《中华烧伤杂志》2012年第4期274-277,共4页Chinese Journal of Burns

摘　　要：目的探讨人诱导性多潜能干细胞（iPSC）向表皮样干细胞分化的可能性。方法（1）以经过灭活处理的小鼠胚胎Fb株作为滋养层，将人iPSC株接种于其上，加入胚胎干细胞完全培养液培养，Ⅳ型胶原酶消化法传代，倒置相差显微镜下观察人iPSC形态及生长状况并行碱性磷酸酶（AKP）染色。以胚胎干细胞不完全培养液悬浮培养iPSC，观察拟胚体形成能力。（2）将人iPSC接种于铺有人羊膜的6孔培养板培养，设为诱导组；另于未铺羊膜的6孔培养板上培养iPSC，作为对照组。观察2组iPSC形态，免疫细胞化学染色法检测整合素β1和细胞角蛋白19（CKl9）表达。结果（1）人iPSC在胚胎干细胞完全培养液中呈典型的干细胞克隆状生长，边界清楚，增殖旺盛；AKP染色阳性。iPSC在无滋养层条件下悬浮培养可形成拟胚体。（2）诱导组iPSC经培养4d后形成干细胞克隆，部分细胞整合素β1和CK19表达阳性。对照组细胞大量死亡，未见整合素β1和CK19表达。结论人iPSC在羊膜诱导下可定向分化为表皮样干细胞，有望成为皮肤组织工程新的种子细胞。Objective To investigate the feasibility of differentiation of human induced pluripotent stem cells （iPSCs） into epidermal-like stem ceils. Methods （1） Human strain of iPSCs were plated onto trophoblast of inactivated Fb strain of mouse embryos and cultured in complete medium of embryonic stem cells, iPSCs were subcultured by collagenase 1V digestion method. The morphology and growth of iPSCs were observed under inverted phase contrast microscope, and the cells were stained with alkaline phosphatase （AKP）. iPSCs were cultured in incomplete medium of embryonic stem cells to observe the ability of embryoid body formation. （2） Human iPSCs were inoculated onto 6-well plate covered with human amniotic membrane to culture as induction group. Other iPSCs were cultured on 6-well plate without human amniotic membrane as control group. Morphological changes in iPSCs in two groups were observed. Expressions of integrin β1 and CK19 of iPSCs in two groups were determined by immunocytochemical staining. Results Human iPSCs showed a typical stem cell clone-like growth with a clear boundary, and they proliferated vigorously in complete medium of embryonic stem cells. These cells were AKP-positve. iPSCs formed embryoid body in trophoblast-free and suspension culture conditions. After 4 days of co-culture, stem cell clones were formed on the surface of amniotic membrane in induction group, and part of the cells were integrin β1 and CK19 positive. Most of the cells died, and no integrin β1 and CK19 positive cells were found in control group. Conclusions Haman iPSCs can be differentiated into epidermal-like stem cells by amniotic membrane in- duction, and it lays an experimental basis for providing new source of seed ceils of skin tissue engineering.

关 键 词：诱导多功能干细胞 细胞分化 表皮样干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]