摘要
目的观察去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)向上皮细胞的分化,并探讨其机制。方法从胎儿四肢长骨分离BMSCs,扩增后采用流式细胞术分析第3代(P3)细胞表面抗原(CD29、CD34、CD71和HLA-DR)的表达,并用4,6-乙酰基-2-苯基吲哚(DAPI)标记第4代BMSCs(P4-BMSCs)。机械法去除正常胎盘羊膜上皮,制成去上皮羊膜,并制备去上皮羊膜浸提液。将DAPI标记的BMSCs接种于羊膜上,设加或不加表皮细胞生长因子(Epidermal growth factor,EGF)、类胰岛素1号生长因子(Insulin-like growth factor 1,IGF-1)、羊膜浸提液诱导组及细胞爬片对照组,体外诱导培养后,采用免疫荧光组织(细胞)化学染色学法检测各组细胞角蛋白(Cytokeratin,CK)、EGF-R和IGF-1-R的表达,并于诱导后第10天计算CK阳性细胞率。结果原代BMSCs呈典型旋涡状生长,P3细胞表达CD29和CD71,不表达CD34和HLA-DR。羊膜组和细胞爬片组BMSCs在加入EGF或IGF-1诱导后,表达EGF-R和IGF-1-R的时间较未加生长因子的对照组提前2～4 d,表达CK的时间提前2～6 d,单用羊膜组或羊膜浸提液组的表达时间差异无统计学意义(P>0.05);诱导第10天,单用羊膜或羊膜浸提液诱导组的CK阳性细胞表达率明显高于细胞爬片对照组(P<0.05);羊膜与EGF、IGF-1联合诱导组高于单用羊膜组(P<0.05);EGF诱导组高于IGF-1诱导组(P<0.05)。结论羊膜及羊膜浸提液、外源性EGF和IGF-1在体外均可诱导BMSCs向上皮细胞分化,羊膜可能主要通过其所含的细胞因子诱导BMSCs向上皮分化。
Objective To observe the induction of differentiation of bone marrow mesenchymal stem cells（BMSCs） by epithelium-removed amnion and the its extract and investigate the relevant mechanism.Methods BMSCs were prepared and propagated,of which the passage 3（P3） were analyzed for expressions of surface antigens CD29,CD34,CD71 and HLA-DR,and the passage 4 were labeled with DAPI.Amnion without epithelial cells was prepared,and the medium in which the amnion was incubated was collected as the extract.DAPI-labeled BMSCs were divided into nine groups.The BMSCs in groups 1 ～ 4 were inoculated onto the surface of amnion and cultured with DMEM / F12,while those in groups 1 ～ 3 were added with epidermal growth factor（EGF）,insulin-like growth factor-1（IGF-1） and EGF ＋ IGF-1 respectively.However,the BMSCs in groups 5 ～ 9 were inoculated onto corval glass and cultured with DMEM / F12,while those in groups 5,6,7 and 9 were added with EGF,IGF-1,EGF ＋ IGF-1 and amnion extract respectively.The expressions of cytokeratin（CK）,EGF-R and IGF-1-R in cells of various groups were determined by immune fluorescent tissue（cell） chemical staining method,and the percentage of BMSCs positive for CK was calculated on day 10 after induction.Results Typical whirlpool growth was observed in primary BMSCs.The expressions of CD29 and CD71 were observed in P3-BMSCs,but no expression of CD34 or HLA-DR.The expressions of EGF-R and IGF-1-R in BMSCs in groups 1 ～ 3 and 5 ～ 7 appeared 2 ～ 4 d,while that of CK 2 ～ 6 d,earlier than those in groups 4 and 8,respectively.However,the time when expression appeared showed no significant difference in the BMSCs in groups 4 and 9（P〉0.05）.On day 10 after induction,the percentages of BMSCs positive for CK were significantly higher in groups 1 and 3 than in groups 2,4,5,6,8 and 9（P 〈0.05）,in groups 4 and 9 than in group 8（P〈0.05）,in groups 1 and 2 than in groups 5 and 6（P〈 0.05）,in group 3 than in groups 5（P〈0.05）,4,6,8 and 9（P〈0.01）,while showed no significant differences in other groups（P〉0.05）.Conclusion Amnion and its extract as well as exogenous EGF and IGF-1 induced the differentiation of BMSCs to epithelium cells in vitro,of which the mechanism might be involved in the cell factors in amnion.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第9期1147-1151,共5页
Chinese Journal of Biologicals
基金
重庆医科大学创新基金(CX200307)
关键词
羊膜
骨髓间充质干细胞
诱导
细胞分化
Amnion
Bone marrow mesenchymal stem cells（BMSCs）
Induction
Cell differentiation
