一种分离人表皮角质形成细胞的新方法  

A new method to isolate human epidermal keratinocytes

作　　者：邹德波[1] 潘婧[2] 张平 吴训伟[3,4] 

机构地区：[1]山东省千佛山医院脊柱外科,山东济南250014 [2]济南市妇幼保健院妇科,山东济南250001 [3]济南磐升生物技术有限公司,山东济南250101 [4]山东大学口腔医学院组织工程与再生研究室,山东济南250012

出　　处：《临床皮肤科杂志》2016年第6期424-429,共6页Journal of Clinical Dermatology

基　　金：山东省泰山学者海外特聘教授基金(tshw201502065)资助项目

摘　　要：目的:探索一种简单快速无需分开表皮和真皮即可分离得到人表皮角质形成细胞(KC)并进行体外培养的方法。方法:取皮肤组织切碎后,先用含分散酶和Ⅰ型胶原酶的消化液37℃浸泡60 min,然后加入胰酶消化30 min,再加入DNA酶继续消化5 min以获得单个细胞;加入含10%胎牛血清(FBS)的DMEM培养基中和消化,用100μm细胞过滤器过滤,离心后,用含10 mmol/L Rock蛋白激酶抑制剂的DMEM培养基(含10%FBS)培养,3 d后换成Cn T07培养基。每2 d换液1次,培养过程中,观察细胞生长状态。结果:与传统分散酶分离表皮KC方法相比,用该方法分离得到的表皮KC数量较多,第1代以克隆形式生长并且生长较快;传代后细胞呈角质细胞形态,主要表达未分化的角质蛋白K5,生长曲线和形成单细胞克隆能力和传统分离的表皮KC相近,并稍优于传统方法。采用新方法成功地从带毛发的头皮组织分离出大量的表皮KC并表达正常的角质蛋白。结论:该法得到的表皮KC形态、生长状态及形成单克隆的能力都接近或优于传统方法。该方法简单高效,具有较高的实用价值,尤其为以后临床上大量分离患者的表皮KC用于细胞治疗奠定基础。Objective： To explore a new simple and efficient method to isolate human epidermal keratinocytes from tissues without separating the epidermis from the dermis. Methods： The skin tissue, chopped into small pieces by scalpel, was transferred to a digestion solution containing dispase and type I collagenase, and incubated at 37℃ water bath for 1 h, followed by incubation with Trypsin solution for 30 minutes, finished by incubation with DNase I for another 5 minutes. The digested solution was neutralized by adding same volume of DMEM containing 10% FBS, then filtered and centrituged; The cell pellet was resuspended amt plated with 10% FBS DMEM medium containing 10 mmol/L Rock inhibitor. 3 days later, the cells were fed with CnT07 medium, and the medium was changed every other day. Results： Compared with the traditional dispase method, the new method isolated more keratinocytes; Cell growth and colony formation were faster; The passaged cells showed normal morphology and keratin 5 expression with a similar proliferative rate and the capability of colony formation to the cells isolated by traditional dispase method. Using the new method, epidermal kerafinocytes were also successfully isolated from hairy scalp tissues, and the passaged cells expressed keratin 5. Conclusions： We established a new method to quickly and efficiently isolate high quality of epidermal keratinocytes. The new method can be used to isolate epidermal keratinocytes from different types of skin tissues including hairy scalp. This is a promising method for large-scale isolation of epidermal cells, which provides a crucial opportunity for cell-base therapy for skin injury.

关 键 词：皮肤 角蛋白细胞 细胞分离 平板形成克隆实验 

分 类 号：R739.5[医药卫生—肿瘤]