大鼠毛囊干细胞体外培养及基因转染方法

A method of hair follicle stem cells culture in vitro and gene transfection in rats

目的：探讨大鼠毛囊干细胞体外培养和基因转染的方法.方法：采用组织块法分离、培养大鼠触须隆突部毛囊干细胞,经Ⅳ型胶原差速贴壁法纯化后,采用碱性磷酸酶染色和细胞免疫荧光染色进行细胞鉴定,测定细胞生长曲线,并分别采用脂质体和慢病毒质粒进行毛囊干细胞基因转染.结果：经两次Ⅳ型胶原贴壁筛选后,毛囊干细胞呈克隆性生长,具有典型的干细胞生物学特征,碱性磷酸酶染色阳性,角蛋白-15、整合素-α6和整合素-β1表达均为阳性.细胞生长曲线显示毛囊干细胞在接种后第3天时出现干细胞克隆;第5天和第6天时细胞处于对数生长期.脂质体介导的毛囊干细胞基因转染率为1.55％～17.21％,中位数9.63％;慢病毒介导的毛囊干细胞基因转染率为62.14％ ～79.42％,中位数71.52％.结论：采用组织块法分离、培养大鼠触须隆突部毛囊干细胞,经Ⅳ型胶原差速贴壁法纯化后,毛囊干细胞可保持较好的生长状态和较强的克隆形成能力,且慢病毒介导毛囊干细胞基因转染比脂质体转染更高效、稳定.

Objective：To explore the method of hair follicle stem cells （HFSCs） culture in vitro and gene transfection in rats.Methods：The HFSCs in palpus eminence of rats were isolated and cultured by using tissue explant method,and were purified by using type-Ⅳ collagen differential adherence method.Then,the cells were identified through alkaline phosphatase staining and cell immunofluorescence staining,and the cells growth curve were also determined,meanwhile,the gene transfection of HFSCs were conducted by using liposome and lentivirus plasmid respectively.Results：The HFSCs grew in clone after screening 2 times by type-Ⅳ collagen differential adherence,and they showed typical biological characteristics of stem cells.The alkaline phosphatase staining and the expression of keratin-15,integrin-α6 and integrin-β31 were all positive.The cells growth curve showed that HFSCs presented in clone at the 3rd day after inoculation,and the cells entered on the logarithmic growth phase at the 5th and 6th day after inoculation.The rate of HFSCs gene transfection mediated by liposome were 1.55% to 17.21% with a median of 9.63 %,while the rate of HFSCs gene transfection mediated by lentivirus were 62.14% to 79.42% with a median of 71.52%.Conclusion：After isolated and cultured through tissue explant and purified through type-Ⅳ collagen differential adherence,the HFSCs in palpus eminence of rats can keep good growth state and strong ability clonality.Moreover,the HFSCs gene transfection conducted by lentivirus plasmid was more efficient and stable than that conducted by liposome.