目的:应用组织块培养大鼠视网膜干细胞的有效性。方法:机械分离出生10d大鼠睫状体组织,剪成细小组织块,置入20μg/L碱性成纤维细胞生长因子、20μg/L表皮生长因子及1×B27添加剂的无血清DMEM/F12培养液中培养,同时取大鼠胚胎脑组织,应用常规机械-酶消化法进行神经干细胞的分离培养作为阳性对照。应用免疫荧光染色检测神经干细胞特异性抗原神经巢蛋白(nestin)的表达,应用含100mL/L的胎牛血清的培养基促进其分化以确定其神经干细胞特性。结果:原代培养48h后在组织块边缘开始出现细胞团,折光性强,呈球形或者桑葚状悬浮生长。培养4～5d后,悬浮生长的细胞团数量增多,出现较大的细胞团,原代培养7d后传代,传代后细胞能重新形成细胞团。应用免疫荧光方法可检测到细胞内nestin阳性表达,且视网膜干细胞可在以血清为诱导条件下变为贴壁生长,并分化出具有神经细胞形态的细胞。其形态、增殖及可分化特性与作为阳性对照的神经干细胞相似。结论:组织块培养法对细胞损伤小,操作简便,可成功对大鼠视网膜干细胞进行分离培养。

AIM:To observe the efficiency of cultivation of retinal stem cells (RSC)in vitro by tissue cultivation method. ·METHODS:The tissue of ciliary body of postnatal 10 days rat was isolated and divided into small bits,then cultivated with non-serum DMEM/F12 medium,20μg/L bFGF,20μg/L EGF and 1×B27 supplement at the same time,the embryonic brain derived neural stem cells (NSC) were isolated and cultivated with routine method as positive control. These two kinds of cells were identified by detection of nestin with immunofluorescence method,which was known as a marker of neural stem cells. The characteristic of differentiation of RSC was identified in the induced condition of 100mL/L FBS medium. ·RESULTS:In the primary cultivation,cell spheres with high refraction were observed 48 hours after isolation and cultivation. The cell spheres enlarged and the amount increased gradually in the fourth or fifth day of cultivation. The passage of the cells occurred in the seventh day of cultivation,and the cells of the next passage can also formed the sphere shape. The marker nestin was detected both in RSC and NSC by immunofluorescence method. The RSC became adherent with the bottom of the culture dish in the induced condition with serum,and some of the cells differentiated into cells with neural cell shapes. The characteristics of the shape,proliferation and differentiation of RSC were similar with NSC. ·CONCLUSION:Tissue cultivation has the characteristics of less damage and convenience,which can be used in the isolation and cultivation of RSC successfully.

R329

中国吉林省科技发展计划青年基金资助项目(No.20080136)~~