细胞外基质对表皮干细胞缝隙连接介导的细胞间通讯的影响

Gap junctions mediated modulation of epidermal stem cell phenotype and communication exerted by extracellular matrix

目的:探讨细胞外基质对表皮干细胞间通讯的调节机制,为理解皮肤创伤愈合的信号传递提供实验依据。方法:体外分离和培养人表皮干细胞(n=8),流式细胞仪检测抗原表达。细胞模型是2×106表皮干细胞接种在胶原包被的培养皿表面,实验组为表皮干细胞+Ⅳ型胶原基质组(n=8),对照组为表皮干细胞+I型胶原/层粘连蛋白基质组(n=8),和表皮干细胞培养于无胶原包被的60mm平皿组(n=8)。Epilife无血清培养基培养7天后,用激光共聚焦显微镜观察培养表皮干细胞缝隙连接蛋白(connexin)表达、单细胞显微注射荧光示踪剂检测在培养表皮干细胞之间缝隙连接细胞间通讯。结果:培养的人表皮干细胞抗C-Kit、CK14、CK19、β1-整合素、P63染色阳性,流式细胞学检查示C-Kit、CK14、CK19、β1-整合素、P63阳性率分别是(91.50±4.72)%、(88.54±6.28)%、(90.38±7.10%、(89.54±5.61)%和(87.38±4.64)%。培养7天后,在Ⅳ型胶原组,(90±4.11)%的细胞表达表皮干细胞表型;在无胶原基质组,细胞生长数量少,(50±6.20)%的细胞表达表皮干细胞表型;在I型胶原/层粘连蛋白基质组,只有少于(10±3.54)%的细胞表达表皮干细胞表型,(90±4.83)%的细胞表达表皮细胞表型;此外,保持特异性标志的表皮干细胞Connexin表达图不同于有表皮细胞特征的细胞。因为皮肤的创伤愈合过程伴随着细胞外基质和生长因子的改变,随之是广泛的表皮干细胞的细胞分裂,分化,和迁移,而相似的改变对于体内皮肤创伤的修复过程极为重要。结论:细胞外基质通过Connexin调节表皮干细胞间通讯,不同Connexin表达模式允许第二信使分子和细胞代谢产物的不同通透性,因此协调细胞和组织功能。

Objective To explore the effects of extracellular matrix exert on cellular signaling and communication by connexin in vitro. Methods In this study, we isolated and cultured human epidermal stem cell （hESCs） for 7days on different matrices, and the hESCs cultured on type IV collagen matrix were experimental group （n=8）, and on type I collagen/ laminin-5 matrix （n=8）, and on no collagen matrix were as control group （n=8）. After 7 days culture, the expression of connexin was detected by confocal microscope, and the intercellular communication was measured by dye coupling studies. Results The primary cultured hESCs were positive for anti-C-Kit,CK14,CK19,β1-integrine,P63 immunostaining,and FACs analysis revealed that the expression of C-Kit,CK14,CK19,β1- integrine.P63 on cultured hESCs were （91.50＋4.72） % %, （88.54＋6.28） %, （90.38＋7.10 %,（89.54＋5.61） % and （87.38＋4.64） %, respectively. After 7 days culture, （90＋4.11）% cells on type IV collagen matrix were positive for the phenotype of hESCs,the cultured cell numbers were fewer in no collagen matrix group, and （50＋6.20）% cells were positive for the phenotype of hESCs; but only less than （10＋3.54）% cells on type Ⅰ collagen/laminin-5 matrix （n=8）were positive for the phenotype of hESCs, and （90＋4.83）% cells were positive for the phenotype of epidermal cells in this culture system. Under these conditions, cultured hESCs cells displayed plot morphology, express the hESCs-specific marker β1-integrine, all were consistent with maintenance of hESCs phenotype. Dye coupling studies revealed functional gap junctions between and among every cell phenotype. Additionally, cultured cells that retained hESCs cell-specific markers expressed connexin profiles different from cultured cells with epidermal cells characteristics, hESCs had unique connexin profiles that allowed for functional coupling. Conclusions Because skin injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of epidermal progenitor cells, we suggest that similar changes may be vital to the skin recovery and repairing process in vivo.