摘要
目的探讨人骨髓间充质干细胞（mesenchymal stem cells，MSCs）在体外向角膜上皮细胞及角膜缘干细胞横向分化的可能性。方法从成人骨髓中分离培养出具有MSCs特性的细胞，经过5次传代培养纯化后使用以下5种培养基对MSCs进行诱导分化：1组：DMEM／F12、体积分数10％FBS；2组：DMEM／F12、体积分数10％FBS、5μg·L^-1表皮生长因子（epidermal growth factor，EGF）；3组：DMEM／F12、体积分数10％FBS、10μg·L^-1EGF；4组：DMEM／F12、体积分数10％FBS、15μg·L^-1EGF；5组：DMEM／F12、体积分数10％FBS、体积分数50％条件培养基、10μg·L^-1EGF。其中第1组为对照组，使用的培养基为培养MSCs的培养基。各组细胞培养30d后，取出细胞爬片进行AE1、AK5和P63的免疫组化染色。结果从成人骨髓中分离培养出的细胞绝大部分具有MSCs的特性。各诱导分化组的细胞经过AE1染色后计算的细胞诱导转化率分别为：0、（5．28±0．90）％、（15．65±1．65）％、（27．35±2．36）％、（15．53±1．61）％，第2、3、4组着色细胞的数量和着色深度递增，第5诱导分化组的染色情况和第3组相近．对照组染色为阴性。各组AE5染色均为均匀淡染。各组P63染色均为阴性。结论5～15μg·L^-1EGF能诱导MSCs横向分化为角膜上皮细胞，而且在此浓度范围内EGF的诱导作用随浓度升高逐渐增强。用成人角膜缘干细胞的培养液制成的体积分数50％条件培养基对诱导MSCs横向分化为角膜上皮细胞无明显促进作用，本实验方法不能将MSCs诱导分化为角膜缘干细胞。
Objective To explore the possibility of mesenchymal stem cells （MSCs） transdifferentiating into corneal epithelial cells and limbal stem cell in vitro. Methods Cells possessing characteristics of MSCs were isolated from bone marrow aspiration samples of adult human donors, then they were cultivated and passaged 5 times for ulterior experiment. The passage 5 MSCs were divid- ed into 5 groups,and the following 5 different culture media were added to every group：Group 1：DMEM/F12,10% FBS;Group 2：DMEM/F12,10% FBS,5 μg · L^-1 EGF;Group 3：DMEM/F12,10% FBS, 10 μg · L^-1 EGF;Group 4：DMEM/F12, 10% FBS,15 μg · L^-1 EGF;Group 5：DMEM/F12,10% FBS,50% conditional medium, 10 μg · L^-1 EGF. The first group was the control group. After 30 days,cells were stained for cytokeratin AE1 and cytokeratin AE5 and P63 with immunohisto- chemistry. Results Most cells isolated culture from adult human bone marrow had characteristics of MSCs. Cytokeratin AE1 immunohistochemistry stain shew that the transformation ratio of the 5 groups was respectively：0, （ 5.28 ± 0. 90） %, （15.65 ±1.65）% ,（27.35 ±2.36）% ,（15.53 ± 1.61）% .The quantity and color depth of positive cell increased progressively from group 2 to group 4. Staining results of group 3 was similar with group 5. Positive cell was not found in group 1 that was control group. Results of cytokeratin AE5 immunohistochemistry stain shew cells of every group were lightly stained in cytoplasm. P63 immunohisto chemistry stain was negative in every group. Conclusion EGF with the concentration of 5 μg · L^-1 to 15 μg · L^-1 can induce MSCs to transdifferentiate into corneal epithelial cells, and the effectiveness of its induction enhanced gradually in the wake of its concentration increased. 50% conditional media prepared with cul- tured medium of adult limbal stem cell could not induce MSCs to transdifferentiate into corneal epithelial cells. MSCs could not be induced and transdifferentiate into limbal stem cells with cultured medium in our experiment.
出处
《眼科新进展》
CAS
2008年第7期505-510,共6页
Recent Advances in Ophthalmology
基金
江西省科委重大科技攻关项目基金资助(编号:200301003)~~
关键词
间充质干细胞
角膜上皮细胞
角膜缘干细胞
诱导分化
mesenchymal stem cells
corneal epithelial cells
limbal stem cells
transdifferentiation
