改良胎鼠视网膜干细胞的分离培养方法

Isolation and cultivation of retinal stem cells of embryonic rat with improved method

目的对大鼠胚胎视网膜干细胞的培养方法进行改良,并观察其生长及分化特性,以期寻求有效的视网膜干细胞培养方法。方法取18d孕龄的SD大鼠视网膜睫状缘(CMZ)色素上皮层组织,通过剪切、吹打、胰酶消化、过滤、离心后,在DMEM/F12培养基中加入碱性成纤维细胞生长因子(bFGF),用无血清培养技术扩增视网膜干细胞;传代后离心收集培养细胞,以5%胎牛血清诱导细胞分化;用免疫细胞化学技术对干细胞及诱导分化的细胞进行鉴定,确定所培养细胞是否为干细胞且具有分化潜能。结果从SD胚胎大鼠视网膜中分离的细胞在体外悬浮生长,形成致密的球形细胞团,传代后能够生成新的细胞团,免疫细胞化学染色显示原代和传代细胞中Nestin和掺入BrDU均呈阳性表达。诱导分化后的细胞经鉴定证明部分表达胸腺细胞表面糖蛋白(Thy1.1)。结论SD胎鼠视网膜内存在视网膜干细胞,通过改良方法可以进行体外培养和扩增,产生分化,部分分化细胞具有视网膜神经细胞特性,包括分化形成节细胞。

Objective To isolate and cultivate the retinal stem cells （RSCs） with improved method, identify the RSCs from embryonic rats, explore the RSCs＇ features in growth and differentiation, and research for an effective method to cultivate retinal stem cell in vitro from rat ocular tissues. Methods RSCs were isolated from rodents and humans and characterized in vitro. RSCs were isolated from SD rat embryonic retina （gestational age 18d） by shearing, blowing, digesting, centrifuging and filtering, and amplified by using serum-free DMEM/F12 medium containing basic fibroblast growth factor （bFGF）, and then the cells were cultured and sub-cultivated by floating growth in vitro. A/ter sub-cultivation, the RSCs were gathered by centrifuging, differentiation was induced with 5% fetal bovine serum （FBS）, and the differentiated cells grew and adhered to the culture medium. Immunocytochemistry was employed with specific antibodies to identify RSCs and their potentiality of differentiation. The potentiality of RSCs to replace retinal cells in retinal degenerative diseases was confirmed by transplantation test. Results The primarily cultured retinal cells could grow in a floating state to, form compact cellular spheres, and they had the potential to proliferate in vitro in subsequent cultures to form new cellular spheres. After incubation in the medium with bromodeoxyuridine （BrDU）, most of both primarily cultured and sub-cultivated cells expressed the neuroectodermal marker Nestin and BrDU. Differentiated RSCs expressed CD90. 1 （Thyl. 1） after being induced. Conclusion The isolated retinal stem cells from SD em- bryonic rats, which are undifferentiated, multipotent and may self-renew, have the ability of proliferation and differentiation in vitro, and the differentiated RSCs possess the characteristics of retinal neural cells, including differentiation to retinal ganglion ceils.