人胎儿皮肤皮脂腺细胞和外泌汗腺细胞的分离培养及鉴定

目的建立人胎儿皮肤皮脂腺、外泌汗腺细胞的体外分离培养与鉴定方法。方法通过分离人胎儿皮肤皮脂腺腺体和外泌汗腺腺管,以DMEM/F12(1∶1)为基础培养基,分别添加不同浓度的胎牛血清、表皮生长因子、L-谷氨酰胺、氢化可的松、霍乱毒素、青霉素、链霉素、重组人表皮生长因子、三碘甲状腺氨酸、胰岛素、转铁蛋白、亚硒酸钠作为皮脂腺细胞培养基及外泌汗腺细胞培养基,置入37℃、体积分数5%CO2孵箱中进行原代及传代培养。倒置相差显微镜下观察人胎儿皮肤皮脂腺、外泌汗腺细胞的形态及变化,并进行细胞克隆形成率测定。采用油红染色和细胞角蛋白(CK)4.62、上皮膜抗原(EMA)免疫组织化学染色对传代培养的皮脂腺、外泌汗腺细胞进行鉴定。结果分离的人胎儿皮脂腺腺体和外泌汗腺腺管可以在体外贴壁生长繁殖;其皮脂腺细胞的克隆形成率为2.7%,明显低于人胎儿角质形成细胞(8.0%,P<0.01).人外泌汗腺细胞的克隆形成率为7.3%,与人胎儿角质形成细胞(7.7%)比较差异无统计学意义(P>0·05).油红染色显示,皮脂腺细胞含有少量脂质小滴,CK4.62、EMA免疫组织化学染色均为阳性;外泌汗腺细胞CK7、CK19免疫组织化学染色均为阳性。结论用酶消化法和显微分离法可体外分离人胎儿皮肤皮脂腺、外泌汗腺细胞,两者均具备上皮细胞的标志和生物学特点,但皮脂腺细胞增殖速度较为缓慢。

In vitro isolation, cultivation and identification of sebocytes and eccrine sweat gland cells from human fetal skin

OBJECTIVE: To explore the preliminary methods of in vitro isolation, culture and identification of sebocytes and eccrine sweat gland cells from human fetal skin. METHODS: Human fetal skin was digested with dispase or type II collagenase, and then by micro - sieving to isolate human sebaceous gland and eccrine sweat gland cells. DMEM/F12 (1: 1) was used as the basic culture medium, supplemented with fetal bovine serum, recombinant human epidermal growth factor, L-glutamine, Hydrocortisone, choleratoxin, penicillin and streptomycin as the medium for sebocytes, or fetal bovine serum, recombinant human epidermal growth factor, triiodothyronine, hydrocortisone, insulin, transferrin, sodium selenite to the medium for eccrine sweat gland duct cells. Primary cultures and subcultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2/95% oxygen. Cell morphology was observed by inverted phase contrast microscopy, and the cultured cells were identified with cell clone efficiency determination. The cultured sebocytes were identified with oil red staining and CK4.62, Epithelia Membrane Antigen (EMA) immunohistochemistry staining. The cultured eccrine sweat gland duct cells were identified with CK7, CK19 immunohistochemistry staining. RESULTS: The isolated sebocytes and eccrine sweat gland cells from human fetal skin could grow by adhering to the wall and proliferate in vitro. The cell clone efficiency of human fetal sebocytes was 2.7%, which was obviously lower than that of human fetal keratinocytes (8.0%, P < 0.01). There was no obvious difference in the cell clone efficiency between human fetal eccrine sweat gland cells (7.3%) and human fetal keratinocytes (7.7%, P > 0.05) . The results of oil red staining indicated that a small quantity of lipid droplets in sebocytes, and immunohistochemistry staining of CK4.62, EMA were positive in subculture sebocytes. The immunohistochemistry staining of CK7, CK19 was positive in subculture eccrine sweat gland duct cells. CONCLUSION: In vitro cultured human fetal sebocytes and eccrine sweat gland duct cells displayed the markers and biological characteristics of epithelial lineage, but human fetal sebocytes proliferated more