人皮肤微血管内皮细胞的分离、培养及鉴定  

Isolation, cultivation and identification of human skin microvascular endothelial cells

作　　者：王广宇[1] 王雨[1] 朱艳萍[1] 康煜冬[1] 王伏声[1] 丁毅[1] 董雨[1] 徐旭英[1] 

机构地区：[1]首都医科大学附属北京中医医院疮疡外科,北京市100010

出　　处：《中国组织工程研究》2016年第51期7678-7683,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目面上项目(81673975);首都中医药研究专项一般项目(15zy16);医院骨干课题(2014GG-01)~~

摘　　要：背景:目前,国内外研究多采用酶消化法结合免疫磁珠法分离微血管内皮细胞,然而步骤过于繁琐且对细胞损伤较大。因而,如何简化人皮肤微血管内皮细胞分离、培养,同时又能获得到高纯度的内皮细胞体外培养方法成为研究热点。目的:探索一种简便高效的来源于糖尿病患者皮肤微血管内皮细胞的体外培养方法,观察细胞生长状态并进行鉴定。方法:利用临床上糖尿病伴足部慢性创面截肢后,肢体近心端皮肤和局部创面周围皮肤真皮浅层组织。经剪碎后采用贴壁法和短时少量应用胰酶法得到人皮肤微血管内皮细胞,传代时利用胰酶消化法和反复贴壁法对细胞进行纯化。结果与结论:1实验成功获取人皮肤微血管内皮细胞,原代培养的人皮肤微血管内皮细胞在培养24 h后基本完全贴壁,第10天进入对数生长期,第12至13天细胞浓度达80%,传代细胞较原代细胞生长活跃,培养5-7 d可长满培养瓶底,呈"铺路石样"排布;2免疫组织化学染色结果显示,培养的细胞中FⅧ因子与CD31相关抗原呈阳性,细胞阳性率达100%;3MTT法测得培养第2、4、5代人皮肤微血管内皮细胞的生长曲线呈倒"S"形。4结果证实,应用贴壁法,并在培养过程中少量短时应用胰酶法,可获得大量高纯度的人皮肤微血管内皮细胞。BACKGROUND：Currently, the enzymatic digestion combined with magnetic activated cel sorting for isolating microvascular endothelial cel s are cumbersome and do harm to cel s. Therefore, how to simplify the isolation and culture of human dermal microvascular endothelial cel s to obtain highly purified endothelial cel s in vitro becomes a hotspot. OBJECTIVE：To explore a simple and effective cultivation method of microvascular endothelial cel s from diabetic patient skins in vitro, and to detect the cel growth. METHODS：Diabetic patients with chronic foot wounds after amputation were enrol ed to col ect the limb proximal skin and topical skin around the wound superficial dermal tissue. Human dermal microvascular endothelial cel s were obtained using adherent method and trypsin method, fol oewd by purified utilizing trypsin digestion and repeated attachment method when passage culture. RESULTS AND CONCLUSION：Human dermal microvascular endothelial cells were obtained successfully, Primary cultured endothelial cells completely adhered to the wall at 24 hours, entered the logarithmic phase at the 10^th day, and the cell concentration reached 80%at the 12^th-13^th day. While the passage cells grew more actively than primary cells, and fully covered the bottom in a“cobblestone”arrangement after 5-7 days of culture. Immunohistochemical staining showed that cultured cells were positive for FVIII and CD31-associated antigens with 100%positive rate. MTT assay showed that cell growth curves of 2, 4, and 5 generations of dermal microvascular endothelial presented the inverted＂S＂shape. These results suggest that abundant highly purified human dermal microvascular endothelial cells can be obtained through the adherent method and a small amount of short-term trypsin method.

关 键 词：皮肤 内皮细胞 血管 组织工程 组织构建 血管内皮细胞 微血管 人皮肤真皮浅层 原代培养 免疫组织化学染色 细胞鉴定 FⅧ因子相关抗原 CD31 MTT法 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]