摘要
目的观察人骨髓间充质干细胞(MSC)与热休克的人汗腺细胞(SGC)间接共培养后,其表型转化情况及细胞外信号调节激酶(ERK)通路所起的作用。方法体外分离和培养人MSC、SGC,采用二步免疫细胞化学法鉴定其均为纯化的SGC、MSC。将原代培养的SGC于47℃下行热休克处理后收集上清液。将第3代MSC作为实验对象并分组。对照组:行常规培养;SGC上清液组:采用含体积分数30％SGC上清液、体积分数1％胎牛血清、1×105U／L青霉素和0．1 g／L硫酸链霉素的DMEM／F12培养基培养;SGC上清液+表皮生长因子(EGF)组、SGC上清液+PD98059组同SGC上清液组处理后,分别添加50μg／L EGF、10μmol／L PD98059继续培养。培养7 d时用流式细胞术检测各组细胞中细胞角蛋白(CK)7、癌胚抗原(CEA)的阳性表达率,以蛋白质印迹法检测ERK和磷酸化ERK(pERK)的表达水平。结果SGC上清液组CK7、CEA阳性表达率分别为(5.76±0．10)％、(2．01±0．09)％;SGC上清液+EGF组分别为(7．31±0.21)％、(7．27+0.12)％;SGC上清液+ PD98059组分别为(1．63±0．11)％、(1．54±0．07)％。与对照组比较,前两组两项指标均明显升高(P<0.01),后一组却与之相近。各组细胞均表达ERK;但pERK水平以SGC上清液+EGF组最高,其次为SGC上清液组,SGC上清液+PD98059组和对照组几乎无表达。结论人MSC、SGC间接共培养可诱导MSC表型转化,ERK通路参与该过程并起着积极作用。
Objective To study the cellular phenotype conversion of human mesenchymal stem cells （MSCs） cocultured with human sweat gland cells （SGCs） and the contribution of extracellular signal-regulated kinase （ERK） pathway in the process. Methods MSC and SGC were isolated, amplified , and identified with two-step immunohistochemistry method. The primary SGCs were heat-shocked at 47 ℃. Then the supernatants were collected immediately and 24hr later. The 3rd passage of MSCs were divided into control, SGC supernatant（ cells were cultured in DMEM/F12 medium containing 30% SGC supernatant） , SGCsupernatant ＋ EGF（cells were cultured in DMEM/F12 medium containing 30% SGC supernatant and 50 μg/L EGF）, and SGC supernatant ＋ PD98059（ cells were cultured in DMEM/F12 medium containing 30% SGC supernatant and 10 μmol/L PD98059）groups. The positive expression of CK7and CEA in MSCs were detected on the 7th post-stimulation day （PSD） by flow cytometry. The expression of ERK and phosphorylated ERK were determined with Western blotting. Results The positive expression rate of CK7 and CEA was （5.76 ±0.10）%,（2.01 ±0.09）% inSGC supernatantgroup; （7.31 ±0.21）% and （7.27 ±0.12）% in SGC supernatant＋EGF group; and （1.63 ±0. 11）%, （1.54 ±0.07）% in SGC supernatant ＋PD98059 group; they were all obviously higher than that in control group（ P 〈 0.01 ）. Moreover, ERK expression was observed in all groups. The expression of pERK in SGC supernatant ＋ EGF group was higher than that in SGC supernatant group, but almost no expression of pERK was found in the SGC supernatant ＋ PD98059 and control groups. Conclusion Indirect coculture of MSCs with SGCs can induce the phenotype conversion of MSCs through ERK pathway.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2006年第5期347-350,共4页
Chinese Journal of Burns
关键词
间质干细胞
细胞外信号调节MAP激酶类
热休克
汗腺细胞
Mesenchymal stem cell
Extracellular signal regulated MAP kinases
Heat shock
Sweat gland cell
