摘要
背景：目前同种异体角膜移植是角膜病的主要治疗手段，但该法存在免疫排斥反应、供体来源短缺、角膜内皮细胞慢性丧失功能等难以克服的缺点。目的：探讨人骨髓间充质干细胞在体外向角膜上皮细胞横向分化的可能性。设计、时间及地点：细胞学体外对比观察，于2004-06／2005-06在江西省人民医院中心实验室江西省干细胞重点实验室完成。材料：成人角膜缘上皮组织由江西省眼库提供，南志愿者捐献。经肝素抗凝的成人骨髓7份，来自汀西省人民医院骨髓穿刺室同期收治的需要行骨髓细胞学榆查的患者，均排除血液系统恶性疾病。方法：无菌条件下将人角膜缘上皮组织剪碎，胰蛋白酶消化，过滤离心，加入含20％FBS的DMEM／F12培养液，制作条件培养基，置于-20℃备刷。采用淋巴细胞分离液密度梯度法从人骨髓液中分离出单个核细胞层，贴蹙法培养扩增，取传至第5代的骨髓间充质干细胞，按2×10^4个/孔密度接种，待细胞完全贴壁后吸除培养液，设立5组：第1组为空白对照，仅加入含10％FBS的DMEM／F12培养基继续培养；其余4组在空白对照的基础上分别加入5，10，15μg/L表皮生长因子、10μg／L表皮生长因子＋50％条件培养基，每3d换液1次，培养30d。主要观察指标：通过细胞角蛋白抗体AE1、AE5免疫组织化学染色结果检测细胞横向诱导转化率。结果：AE1免疫组化染色后与空白对照组比较，5，10，15μg／L表皮生长因子组、10μg／L表皮生长因子＋50％条件培养基组的细胞显色率均明显升高（F=415．39，P〈0．01）；各诱导组间两两比较差异均有显著性意义（F=373．96，q=38．65，18．16，20．49，P〈0，01）：10μg／L表皮生长因子组与10μg／L表皮生长因子＋50％条件培养基组细胞显色率基本相似（t=-0．12，P〉0．05）。AE5免疫组化染色后，各组细胞的胞浆都能被DAB均匀淡染。结论：5～15μg/L表皮生长因子能诱导人骨髓间充质干细胞横向分化为角膜上皮细胞，且此浓度范围内的表皮生长因子诱导作用逐渐增强。用成人角膜缘干细胞培养液制成的50％条件培养基对诱导人骨髓间充质干细胞横向分化为角膜上皮细胞无明显促进作用。
BACKGROUND： Corneal allotransplantation is a main therapy for keratopathy. However, immunological rejection, lacks of donor source, chronic functional loss of the graft are still unsolved. OBJECTIVE： To explore the possibility of bone marrow mesenchymal stem cells （MSCs） transdifferentiating into corneal epithelial cells in vitro. DESIGN, TIME AND SETTING： In vitro contrast of cytology was performed at the Key Laboratory of Stem Cells of Jiangxi Province, and Central Laboratory of Jiangxi Provincial People＇s Hospital between June 2004 and June 2005. MATERIALS： Adult corneal limbus epithelium was provided by Eye Bank of Jiangxi Province. Seven samples of heparinized adult bone marrow were provided by patients undergoing bone marrow cell examination through bone marrow puncture in Jiangxi Provincial People＇s Hospital. The patients had no malignant hematological system disease. METHODS： Corneal limbus epithelium was cut into pieces and digested with trypsin, centrifuged, and added fetal bovine serum （FBS, 20%）-conditioned DMEM/F12 Cells to prepare culture medium, which was stored at -20 ℃. Mononuclear cells were isolated from bone marrow aspiration samples using lymphocyte isolation solution and density gradient method, cultivated and passaged. The fifth passage MSCs were seeded with density of 2× 10^4 in each plate well. After cell attachment, culture solution was removed, and cells were divided into 5 groups： Cells of blank control group were cultured in DMEM/F12 containing 10% FBS, and cells in the other groups were cultured in.DMEM/F12 containing 10% FBS and 5, 10, 15 μ g/L epidermal growth factor （EGF） or 50% conditioned medium and 10 μ g/L EGF for 30 days. The culture media were changed every three days. MAIN OUTCOME MEASURES： Cell transdifferentiation was detected using cytokeratin AE1 and cytokeratin AE5 immunohistochemical staining. RESULTS： Cytokeratin AE1 immunohistochemistry staining showed that the quantity of positive cells in blank control group was significantly lower than other groups （F=415.39, P 〈 0.01）, and there were significant differences between any two induction groups （F=373.96, q=38.65, 18.16, 20.49, P 〈 0.01）. The quantity of positive cells was similar in 10 μ g/L EGF group and 50% conditioned mediumand 10 μ g/LEGF group（t=-0.12,P〉0.05）.Results of cytokeratin AE5 immunohistochemistry stain showed that cells of every group were lightly stained in cytoplasm. CONCLUSION： EGF with the concentration of 5 μ g/L to 15 μ g/L can induce MSCs to transdifferentiate into corneal epithelial cells, and the effectiveness of its induction enhances gradually with increase of its concentration. 50% conditioned media prepared with cultured medium of adult limbal stem cell could not induce MSCs to transdifferentiate into corneal epithelial cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第38期7491-7494,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江西省科委重大科技攻关项目(200301003)~~
