摘要
目的观察角化细胞生长因子（KGF）在人脐带间充质于细胞（hUC—MSCs）向汗腺样细胞（SGCs）分化过程中的表达活性及其促分化的效应。方法利用流式细胞术检测MSCs的标志物抗原CD29、CD90、CD45；成骨及成脂分化诱导培养基培养21d，检测其成骨、成脂分化潜能。免疫细胞化学法检测hUC．MSC、SGCs的汗腺标志物癌胚抗原（CEA）、角蛋白（CK）14、CK19水平；酶联免疫吸附试验（ELISA）、聚合酶链反应（PCR）、Western blot法检测分化期间7、14、21d的上清液和细胞中的汗腺发育基因KGF及其受体成纤维细胞生长因子受体2（FGFR2）的表达特性。实验分组：以普通汗腺培养基、干细胞培养基以及热休克汗腺上清液＋普通汗腺培养基构成的分化诱导培养基（MIX）为对照组，重组人角化细胞生长因子（rhKGF）＋普通汗腺培养基构成分化诱导培养基（KGF）为实验组；对不同分组中hUC—MSCs分化为SGCs的效能进行评价。结果hUC—MSCs表达CD29、CD90的阳性率分别为92．1％、98．1％；阴性表达CIM5为0．2％；可分化为ALP染色阳性的成骨细胞和油红-O染色阳性的成脂细胞。经过分化的SGCs（SGCs—MIX、SGCs-KGF）均具有类似正常汗腺（SGs）形态；免疫细胞化学检测结果显示，SGCs—MIX、SGCs-KGF可以阳性表达CEA、CK14、CK19，而未分化的hUC—MSCs则未表达。PCR及Western blot检测结果显示，不同分组中的SGCs均可表达KGF及受体FGFR2，活性明显高于hUC—MSCs组（P〈0．05）。ELISA检测结果显示，rhKGF在SGCs分化过程中7、14、21d的表达量分别为63．1、74．6、84．2ng／L，明显高于hUC-MSCs对照组中的2．3、4．1、7．3ng／L（P〈0．05）。结论在hUC—MSCs分化为SGCs过程中具有分泌KGF的活性，KGF具有促进hUC—MSCs向分化为SGCs的潜能。
Objective To analyze the expression and inductive role of keratinocyte growth factor （KGF） during trans- differentiation of sweat gland cells （SGCs） from human umbilical cord derived- mesenchymal stem cells （hUC - MSCs） . Methods The phenotype of hUC - MSCs were examined for stem cells markers （CD29, CD90, CIM5 ）by flow cytometry analysis, while SGCs differentiated from hUC- MSCs were analyzed by immunocytochemistry for sweat gland specific markers （CEA, CK14, CK19）. Samples supernatants were tested for sweat gland development genes KGF/fibroblast growth facor - 2 （FGFR2） through ways of enzyme linked immunosorbent assay （ ELISA）, polymerase chain reaction （PCR）, Western blotting during SGCs induction process of 7, 14, 21 d. The inductive role of KGF were evaluated among groups of rhKGF plus normal sweat gland medium （ SGCs - KGF） , normal mesen- chymal stem cell medium, normal sweat gland medium, and positive control group of heated - shocked sweat gland supernatant induction medium （SGCs -MIX） through positive staining for sweat gland markers. Results hUC - MSCs were positive for CD29, cDgo （92. 1%, 98.1%, respectively） ; and negative for CD45 （0. 2% ）. The SGCs -KGF from rhKGF plus normal sweat gland medium were positive for sweat gland markers （CEA, CK14, CK19）, as well as SGCs -MIX. KGF and its receptor FGFR2 were both positively expressed in SGCs groups similar as mature SGs group, compared with negative expressions of hUC - MSCs group. The novel sweat gland induction medium could also promote differentiation of SGCs from hUC - MSCs with positive staining of sweat gland specific markers. During the induction process, the result of ELISA showed that rhKGF were significantly secreted in induction medium of SGCs （63.1,74. 6, 84.2 ng/L, respectively ） at 7, 14, 2l d after induction with a higher level than that of original hUC - MSCs group （2. 3,4. 1, 7. 3 ug/L, respectively ; P 〈 0.05 ）. Conclusion Sweat gland development gene KGF were significantly
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第1期63-67,共5页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（81201478、81372079、81571916）
教育部博士点基金（20120101120035）
浙江省医药卫生科技计划项目（2013KYA098）
关键词
汗腺
脐带间充质干细胞
角化细胞生长因子
转分化
烧伤
Sweat gland
Umbilical cord derived -mesenchymal stem cells
Keratinocyte growth factor
Trans - differentiation
Burns
