Yang Jian-min, Hao Wen-jie, Liang Yu-rn, Wang Geng-yin, Li Jun-xia
Department of Burns and Plastic Surgery, Bethune International Peace Hospital of PLA, Shijiazhuang 050082, China.
Zhonghua Shao Shang Za Zhi. 2011 Aug;27(4):265-8.
To study the differentiation potential of human umbilical cord-derived mesenchymal stem cells (UCMSC) into human sweat gland cells (hSGC) and the role of extracellular signal-regulated kinase (ERK) pathway.
UCMSC and hSGC were isolated and cultured in vitro. The former was identified with expression of CD14, CD29, CD34, CD44, CD45, CD105, cytokeratin 7 (CK7), CK19, and carcinoembryonic antigen (CEA), while the latter was identified with expression of CK19 and CEA. UCMSC with density of 5 x 10(4) cells per well placed in lower compartment of Transwell chamber were divided into control group (C, cultured with nutrient solution without any stimulation), thermal injury group (TI, treated with heat-shocked hSGC with density of 1 x 10(4) cells per well inoculated into the upper compartment of Transwell chamber for indirect co-culture), thermal injury + EGF group (TIE, treated with indirect co-culture as used in TI group, with addition of 50 ng/mL EGF), thermal injury + PD98059 group (TIP, treated with indirect co-culture as used in TI group, with addition of 10 nmol/mL ERK specific inhibitor PD98059) according to the random number table. One week after culture, the positive expression rates of CK7 and CK19 in UCMSC were detected by flow cytometry, the expression of CK19 and CEA in UCMSC were examined with immunohistochemical staining and the positive expression rate of CEA was calculated, and the expression level of phosphorylated ERK (pERK) was determined by Western blotting. Data were processed with one-way analysis of variance.
(1) CD29, CD44, and CD105 were highly expressed in UCMSC, accompanied by low or negative expression of CD14, CD34, CD45, CK7, CK19, and CEA. The expression of CK19 and CEA were positive in hSGC. The two results showed that UCMSC and hSGC were pure. (2) Compared with those of C group [(2.2 +/- 1.5)%, (2.2 +/- 0.7)%, (3.3 +/- 0.7)%, 0.640 +/- 0.026], the expression levels of CK7, CK19, CEA, and pERK in UCSMC of TI group [(6.4 +/- 0.7)%, (5.7 +/- 0.3)%, (7.4 +/- 1.0)%, 0.790 +/- 0.049] and TIE group [(14.3 +/- 1.0)%, (12.6 +/- 1.1)%, (17.6 +/- 2.3)%, 1.200 +/- 0.032] were significantly increased (with F value respectively 78.49, 139.36, 87.13, and 191.74, P values all below 0.01), and those of TIE group were higher than those of TI group (with F value from 50.14 to 145.47, P values all below 0.01). There were no obvious difference in the 4 indexes between TIP group and C group (with F value from 0.00 to 0.13, P values all above 0.05).
UCMSC co-cultured with heat-shocked hSGC can differentiate into hSGC, and ERK signal pathway participates in the process of differentiation of UCMSC into hSGC.
研究人脐带间充质干细胞（UCMSC）向人汗腺细胞（hSGC）的分化潜能及细胞外信号调节激酶（ERK）通路的作用。
体外分离培养UCMSC和hSGC。前者通过检测CD14、CD29、CD34、CD44、CD45、CD105、细胞角蛋白7（CK7）、CK19和癌胚抗原（CEA）的表达进行鉴定，后者通过检测CK19和CEA的表达进行鉴定。将密度为5×10⁴个/孔的UCMSC接种于Transwell小室下层，分为对照组（C组，用无任何刺激的培养液培养）、热损伤组（TI组，将密度为1×10⁴个/孔的热休克hSGC接种于Transwell小室上层进行间接共培养）、热损伤+表皮生长因子（EGF）组（TIE组，同TI组进行间接共培养并加入50 ng/mL EGF）、热损伤+PD98059组（TIP组，同TI组进行间接共培养并加入10 nmol/mL ERK特异性抑制剂PD98059），按随机数字表法分组。培养1周后，采用流式细胞术检测UCMSC中CK7和CK19的阳性表达率，免疫组织化学染色检测UCMSC中CK19和CEA的表达并计算CEA阳性表达率，蛋白质印迹法检测磷酸化ERK（pERK）表达水平。数据采用单因素方差分析处理。
（1）UCMSC中CD29、CD44和CD105高表达，CD14、CD34、CD45、CK7、CK19和CEA低表达或阴性表达。hSGC中CK19和CEA表达阳性。这两项结果表明UCMSC和hSGC均为纯细胞。（2）与C组[（2.2±1.5）%，（2.2±0.7）%，（3.3±0.7）%，0.640±0.026]比较，TI组[（6.4±0.7）%，（5.7±0.3）%，（7.4±1.0）%，0.790±0.049]和TIE组[（14.3±1.0）%，（12.6±1.1）%，（17.6±2.3）%，1.200±0.032]的UCMSC中CK7、CK19、CEA和pERK表达水平均显著升高（F值分别为78.49、139.36、87.13和191.74，P值均<0.01），且TIE组高于TI组（F值为50.14145.47，P值均<0.01）。TIP组与C组上述4项指标比较差异无统计学意义（F值为0.000.13，P值均>0.05）。
与热休克hSGC共培养的UCMSC可分化为hSGC，ERK信号通路参与UCMSC向hSGC的分化过程。
