组织工程新技术:毛乳头细胞高效培养方法探索(英文)

A new technique for tissue engineering:exploration of a more efficient method for culture of human scalp dermal papilla cells☆

背景:毛乳头细胞的分离培养技术是研究毛囊细胞生物学的基础,目前采用的显微解剖法分离毛乳头进行毛乳头细胞传代培养是一种原始的方法,存在着分离效率低、劳动强度大、毛乳头贴壁率低、细胞生长缓慢等缺点。目的:探索一种高效省时的分离毛乳头方法。设计:采用酶消化法与解剖法对照的实验研究。地点和对象:实验在第三军医大学大坪医院野战外科研究所完成。为正常人头皮毛囊。干预:采用胶原酶D直接将毛囊下部的真皮鞘组织消化成真皮鞘细胞和毛乳头,观察毛乳头的贴壁率和细胞生长曲线。主要观察指标:胶原酶消化法、立体解剖分离法及组织化学染色结果。结果:消化法可大量获得毛乳头,省去了在解剖显微镜下分离毛乳头的步骤,第1天贴壁率为96.5%,第3天贴壁率为99.7%。显微解剖分离法分离较困难,每小时仅能分离15个左右,悬浮培养前5d无一贴壁。消化法极大地促进了毛乳头的贴壁和细胞的生长,并保证了毛乳头的完整性,组织化学染色表明毛乳头细胞是一种高度特异的成纤维细胞。结论:胶原酶D消化法分离毛乳头是一种高效的培养毛乳头细胞的方法。

BACKGROUND:The technique of isolation and culture of human scalp dermal papilla(DP) cells is basic to study hair follicle biology.At present,microdissection to isolate the dermal papillae and culture DP cells is a primitive method,which has many drawbacks,such as laboriousness,low efficiency,uneasy adhesion and poor growth. OBJECTIVE:To establish a more efficient and time saving method so as to isolate the dermal papillae. DESIGN:An enzyme digestion and dissection controlled experimental study was conducted. SETTING and PARTICIPANTS:The experiment was completed in the Research Institute of Field Surgery,Daping Hospital,Third Military Medical University.The subjects were human normal scalp. INTERVENTIONS:Collagenase D was used to directly digest the dermal sheath below hair follicle,and the dermal sheath cells and dermal papillae were obtained.The adhesion rate and growth curve of DP cells were observed. MAIN OUTCOME MEASURES:Methods of collagenase digestion,microdissection and results of histochemical staining. RESULTS:The digestive method could obtain a large number of the DP and omit the step of microdissection under microscope that remarkably decreased working load and laboriousness,increased the efficiency,maintained their intactness and improved the adhesion and growth of DP cells.On the first day,its attached rate was 96.5%(277/287),on the third day was 99.7%(286/287).It was hard to isolate DP with microdissection method.Nearly 15 DPs could be isolated each hour,and on one was adhered during the first five days.Immunohistochemical staining showed that the DP cells were highly specific fibroblasts. CONCLUSION:The digestive method of collagenase D to isolate the dermal papillae is a more efficient method for culture of the DP cells.