不同来源人角膜缘干细胞的体外培养研究  

Ex Vivo Culture of Fetal and Adult Limbal Stem Cells

作　　者：易敬林[1] 钟文贤[1] 

机构地区：[1]江西省人民医院眼科,江西南昌330006

出　　处：《江西医学院学报》2005年第6期16-20,31,共6页Acta Academiae Medicinae Jiangxi

基　　金：[江西省科委社会发展攻关资助项目]

摘　　要：目的探讨胎儿和成人角膜缘干细胞的体外培养方法并观察其生物学特性.方法应用消化培养法对胎儿和成人角膜缘上皮组织分别进行体外培养,观察记录培养细胞的生长特性并计算其克隆形成率(colony-forming efficiency, CFE).对胎儿和成人角膜缘组织及培养细胞采用一系列单克隆抗体进行免疫酶细胞化学染色检测;并用扫描电镜观察原代细胞的表面状况.结果胎儿角膜缘干细胞原代培养时生长旺盛,8～10 d达到生长高峰,CFE为(26.18±4.75)%,传代时保持高增殖速率;成人角膜缘干细胞原代培养时生长不及胎儿细胞旺盛,6～8 d达到生长高峰,CFE为(15.45±3.88)%,随着传代次数增加其增殖能力明显下降;两种细胞原代培养时CFE值差异有显著性(t＝6.78, P＜0.001).免疫酶细胞化学染色见培养前胎儿角膜缘组织基底层有多量AE5阴性、AE1和PCNA阳性细胞,并见较多HLA-DR阳性细胞;胎儿原代培养细胞绝大部分AE5和HLA-DR染色阴性,AE1和PCNA阳性;传代培养至第3代时AE5阴性细胞仍占大部分;成人角膜缘组织及培养细胞染色结果则见上述干细胞特征细胞数量比例较低,而HLA-DR染色结果相似.扫描电镜观察胎儿原代细胞以球形或短圆柱形为主,表面多突起和微绒毛,成人原代细胞形态相似,但直径较大.结论采用消化培养法可成功培养出具有高增殖力等干细胞特性的胎儿角膜缘干细胞,其抗原性较培养前明显降低,且其生物学特性显著优于成人角膜缘干细胞,是一种新的优越的角膜缘干细胞来源,将在临床眼表重建中展现广阔的应用前景.Objective To investigate ex vivo cells and observe their biological characteristics. were cultured in vitro respectively by enzymatic culture technique of fetal and adult limbal stem Methods Fetal and adult limbal epithelial tissues digestion culture method, then the morphological characteristics and the colonyorming efficiency（CFE） of cultured cells were recorded. The expression of 64KD keratin（AES）, acidic keratin of low molecular weight（AE1）, proliferating cell nuclear antigen（PCNA） and HLA-DR antigen in the limbus and cultured cells of fetus and adult were detected by enzyme immunocytochemistry staining. The surface characteristics of primary culture cells were observed by scanning electron microscope. Results Fetal limbal stem cells grew swiftly and reached growth peak in 8～10 days in primary culture when their CFE was （26.18± 4.75） %. They could keep high proliferating rate in subculture. Adult limbal stem cells grew less swiftly than fetal cells and reached growth peak in 6～8 days in primary culture when their CFE was （15. 45±3.88）%. Moreover, their proliferating ability declined distinctly along with the passage times increasing. The difference of the CFE value of the two kinds of primary culture cells was statistically significant （t = 6. 78, P〈0. 001）. In the results of enzyme immunocytochemistry staining, many cells in basal layer of fetal limbus were negative for AE5 and positive for AE1, PCNA and HLA-DR determinations. A absolute majority of primary culture fetal cells were negative for AE5 and HLA-DR, and positive for AEI and PCNA. Moreover, a majority of fetal cells in the third passage were AE5 negative. By compariso, to the staining results of fetal limbus and cultured cells, the proportion of the cells with staining characteristics of above-mentioned stem cells was lower but the staining results of HLA-DR were similar in the staining results of adult limbus and cultured cells. The morphological characteristics of primary culture cells of fetus and adult were m

关 键 词：胎儿 角膜缘 干细胞 培养 生物学特性 

分 类 号：Q813.11[生物学—生物工程]