与人汗腺细胞共培养的人骨髓间充质干细胞表型转化中细胞外信号调节激酶通路的作用

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。

The involvement of ERK pathway in the cellular phenotype conversion in human mesenchymal stem cells cocultured with human sweat gland cells

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 degrees C. 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), SGC supernatant + EGF (cells were cultured in DMEM/F12 medium containing 30% SGC supernatant and 50 microg/L EGF), and SGC supernatant + PD98059 (cells were cultured in DMEM/F12 medium containing 30% SGC supernatant and 10 micromol/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)% in SGC supernatant group; (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.