摘要
目的观察人脐带问充质干细胞（UCMSC）向汗腺细胞（SGC）分化的能力以及细胞外信号调节激酶（ERK）信号通路在分化过程中的作用。方法（1）体外分离培养UCMSC和SGC，通过检测CD14、CD29、CD34、CD44、CD45、CD105、细胞角蛋白7（CK7）、CK19、癌胚抗原（CEA）表达情况鉴定UCMSC，检测CK19、CEA表达情况鉴定SGC。（2）制作热损伤SGC模型，按随机数字表法将铺于Transwell培养板下层的UCMSC分为4组，均用汗腺培养液培养：对照组，培养液中不添加刺激因素；热损伤组，将热损伤SGC（每孔1×10^4个）接种于Transwell培养板小室与UCMSC间接共培养；热损伤＋EGF组．在热损伤组处理条件基础上，培养液中添加50ng／mLEGF；热损伤＋PD98059组，在热损伤组处理条件基础上，培养液中添加10nmol／mL ERK信号通路特异性抑制剂PD98059。1周后，流式细胞仪检测各组UCMSC中CK7、CK19表达率，免疫组织化学法检测CK19、CEA表达情况并计算CEA表达牢，蛋白质印迹法检测磷酸化ERK（pERK）表达水平。对数据行多组间单因素方差分析。结果（1）UCMSC高表达CD29、CD44、CD105，少揎表达或不表达CD14、CD34、CD45、CK7、CK19、CEA；SGC中CEA和CK19均呈阳性表达，证实获得的2种细胞均为纯化细胞。（2）诱导培养1周后，热损伤组与热损伤＋EGF组UCMSC巾CK7、CK19、CEA阳性表达率及pERK表达水平分别为（6．4±0．7）％、（5．7±0．3）％、（7．4±1．0）70、0．790±0．049与（14．3±1．0）％、（12．6±1．1）％、（17．6±2．3）％、1．200±0．032，均显著高于对照组的（2．2±1．5）％、（2．2±0．7）％、（3．3±0．7）％、0．640±0．026，F值分别为78．49、139．36、87．13、191．74，P值均小于0．01；热损伤＋EGF组UCMSC各指标水平照著高于热损伤组（F值为50．14～145．47，P值均小于0．01）；热损伤＋PD98059组与对照组UCMSC各指标水平相近（F值为0、00～0．13，P值均大于0．05）。结论UCMSC在热损伤SGC的微环境巾能够分化为SGC，ERK通路参与了UCMSC向汗腺细胞分化的过程。
Objective 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 signalregulated kinase （ERK） pathway. Methods 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 ） , CKI9, and careinoemhryonic antigen （CEA）, while the latter was identified with expression of CK19 and CEA. UCMSC with density of 5 × 10^4 cells per well placed in lower compartment of Transwell chamber were divided into ctrul group （C, cuhured with nutrient solution withou! any stimnlation） , thermal injury group （ TI, treated with heat-shocked hSGC with density of 1 ×10^4 cells per well inoculated into the upper compart- ment 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 CKI9 in UCMSC were detected by flow cytometry, the expression of CK19 and CEA in UCMSC were examined with immunohistochemieal 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. Results （ 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±0.0491 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 ）. Conclusions 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.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2011年第4期265-268,共4页
Chinese Journal of Burns
