人2型糖尿病皮肤角质形成细胞的无血清培养及纯化  

Primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium

作　　者：张殿宝[1] 王竟[2] 庞希宁[1] 施萍[3] 苏航[2] 

机构地区：[1]中国医科大学干细胞与再生医学研究室,辽宁沈阳110001 [2]中国医科大学附属第一医院肛肠外科,辽宁沈阳110001 [3]中国医科大学附属第一医院全科医学教研室,辽宁沈阳110001

出　　处：《基础医学与临床》2013年第11期1418-1421,共4页Basic and Clinical Medicine

基　　金：国家重点基础研究发展计划(2012CB518103);辽宁省科学技术计划(2012225080);沈阳市科学技术计划(F11-262-9-01)

摘　　要：目的体外原代无血清培养人2型糖尿病皮肤角质形成细胞,并进行纯化。方法采用中性蛋白酶-胰蛋白酶两步消化法分离人2型糖尿病皮肤角质形成细胞,使用无血清培养基进行原代培养,通过两步消化传代纯化细胞,使用倒置相差显微镜观察细胞生长状态,WST-8法测定细胞增殖曲线,流式细胞术检测其表型和纯度。结果无血清培养并纯化的角质形成细胞呈典型的上皮形态,第3代角质形成细胞增殖曲线为S形,对数增长期的平均倍增时间为36.9 h,角蛋白阳性率为98.9%。结论建立高纯度的人2型糖尿病皮肤角质形成细胞的无血清培养方法。Objective To improve the methods for primary culture and purification of type 2 diabetic human kerati- nocytes in serum-free medium in vitro. Methods Type 2 diabetic human keratinocytes were isolated by 2 step di- gestion of neutral protease and trypsin, cultured in serum-free medium and purified by two-step subculture, the ke- ratinocytes were observed using inverted phase contrast microscope, the growth curve was obtained by the WST-8 assay and phenotype and purity were analyzed by flow cytometry. Results Type 2 diabetic human keratinocytes in serum-free medium showed typical epithelial morphology, the growth curve of the keratinocyte at passage 3 is S-shaped, the average doubling time is 36.9 hours during logarithmic growth phase and the keratin-positive rate was 98.9%. Conclusions The methods for culture of high-purity type 2 diabetic human keratinoeytes in serum-free media have been established.

关 键 词：角质形成细胞 2型糖尿病 原代培养 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]