人体外泌汗腺细胞的三维培养及形态学观察  

THREE-DIMENSIONAL CULTURE AND MORPHOLOGICAL OBSERVATION OF HUMAN ECCRINE SWEAT GLAND CELLS

作　　者：舒申友[1] 陈露[1] 李雪雪[1] 李海红[1] 

机构地区：[1]汕头大学医学院第二附属医院烧伤整形显微外科,广东汕头515041

出　　处：《中国修复重建外科杂志》2014年第2期162-166,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81071551);2010年广东省高等学校高层次人才项目~~

摘　　要：目的探讨人体外泌汗腺组织体外三维培养方法，并对构建的三维组织进行形态学分析。方法取整形手术患者自愿捐赠的正常腹部全层皮肤，经II型胶原酶消化分离人体外泌汗腺组织，以含5ng／mL重组人EGF、25mg／mL牛垂体提取物、100U／mL青霉素和100μg／mL链霉素的无血清角质细胞培养基进行培养。倒置相差显微镜下观察细胞生长情况。待原代细胞达80％融合后，调整细胞浓度至l×10^5个／mL；取0-3mL细胞悬液与0．3mLMatrigel基底膜基质混匀培养，倒置相差显微镜下观察细胞生长情况。培养14d后，取标本行冰冻切片，HE染色观察细胞形态结构，免疫组织化学染色检测细胞角蛋白7（cytokeratin7，CK7）和CKl9抗原表达。结果倒置相差显微镜观察示，经II型胶原酶消化后大量汗腺组织游离；贴壁后3～5d长出汗腺细胞，细胞持续分裂达2～3周，形成围绕汗腺组织块的环状单层；3～4周后细胞衰老。汗腺细胞接种于Matrigel基底膜基质中2～3d，细胞开始分裂，随后形成小细胞团、管状结构以及类球状结构；约2周时混合培养物开始液化，培养终止。HE染色示部分细胞形成中空管状结构，管壁由1～2层细胞构成，类似于在体汗腺的分泌部和导管部；免疫组织化学染色见培养的汗腺细胞CK7和CKl9表达均呈阳性。结论人汗腺细胞接种于Matrigel基底膜基质三维培养，形成的组织结构模拟了在体汗腺的组织形态结构。Objective To investigate the three-dimensional （3D） culture and morphology of human eccrine sweat gland cells. Methods The human eccrine sweat gland cells were isolated from normal abdominal full thickness skin by digestion of type II collagenase, and cultured in defined-keratinocyte serum free medium supplemented with 5 ng/mL recombinant human epidermal growth factor, 25 mg/mL bovine pituitary extract, 100 U/mL penicillin, and 100μg/mL streptomycin at 37℃ in a humidified atmosphere of 5%CO2/95% air incubator. When the cell fusion reached above 80%, the cells were harvested and the concentration was adjusted to 1 × 10^5 cells/mL. The mixture of 0.3 mL cell suspension and 0.3 mL Matrigel basement-membrane matrix was cultured in 12-well plate. The cell growth was observed under an inverted phase contrast microscope. At 14 days after culture, frozen sections were prepared and were stained with HE to observe the cells morphology, and immunohistochemical analysis was used to detect the antigen expressions of cytokeratin 7 （CK7） and CK19. Results Inverted phase contrast microscope observation showed that many free eccrine sweat gland tissues were seen after digestion of type II collagenase; eccrine sweat gland cells grew adhering to the wall at 3-5 days and continued division for 2-3 weeks to form single ring around the block sweat glands; cellular senescence were observed after 3-4 weeks. During the process of 3D culture, the single eccrine sweat gland cell divided into 2-4 cells after 2-3 days, and these cells subsequently formed small cell dusters, tubular-like structures and finally spheric-like shapes. After cultured for about 2 weeks, there was crack in part of the gelled mixture or liquefaction occurred. HE staining of frozen sections of the 3D cultures showed some of the tubular-like structures composed of 1-2 layers of epithelial cells, which were similar to the secretion part and the duct part of the eccrine sweat gland. Immunohistochemical analysis showed that CK7 and CK19 antigens expressed posi

关 键 词：人汗腺细胞 Matrigel基底膜 三维培养 类汗腺结构 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]