成人视网膜前体细胞的体外分离培养和鉴定

In Vitro Isolation,Culture, and Identification of Retinal Progenitor Cells from Adult Human Eye

【目的】对成人视网膜前体细胞进行体外分离、培养和鉴定。【方法】成人眼球12只,“机械分离联合酶消化法”分离出睫状上皮层的色素性细胞,10ng/mL碱性成纤维细胞生长因子(bFGF)+10ng/mL表皮生长因子(EGF)的无血清DMEM/F12培养液中培养,并从三方面鉴定其神经干细胞特性:A.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;B.自我更新能力:原代神经球消化传代后,继续培养观察新神经球的形成;C.多向分化潜能:原代细胞培养4～5d,改用含10mL/L胎牛血清的DMEM/F12培养液,继续培养7～10d,分别用抗神经丝蛋白(NF)抗体、抗微管相关蛋白-2(MAP2)抗体、抗胶质纤维酸性蛋白(GFAP)抗体、抗视紫质蛋白抗体、抗β-微管蛋白抗体以及抗无长突细胞特异性抗体作免疫荧光细胞染色。【结果】0.48%±0.08%(1∶208)原代细胞在含bFGF+EGF无血清培养条件下能保持增殖未分化状态,形成神经球样结构,消化传代后90.1%±8.3%的第二代细胞能重新形成新的神经球样结构,具有自我更新能力;原代及传代后的神经球均表达神经干细胞特异性抗原nestin;在含10mL/L胎牛血清的促分化培养液中,细胞分别分化为可表达神经细胞、星形神经胶质细胞和感光细胞等特异性抗原的细胞,表明具有多向分化潜能。【结论】在成人眼睫状体扁平部分离得到具有干细胞特性的视网膜前体细胞,并在体外成功进行培养鉴定。

[Objective] To isolate, culture, and identify retinal progenitor cells from adult human eyes in vitro. [Methods] Pigmented epithelia of ciliary body from 12 eyes of adult persons were separated by the combined ways of mechanical tear and trypsinization, and then cultured in the serum-free medium supplemented with 10 ng/mL FGF-2 and 10 ng/mL EGF. Three aspects of experiments have been carried out to determine their characteristics of neural stem cells： A. Expression of the neural stem cell marker nestin by immunoflurescence staining; B. Selfrenewal ability： primary neural spheres were dissociated into single cells and recultured for the observation of secondary neurosphere formation;C. Multipotential differentiation ability： primary cells were cultured in serum-free medium for 4-5 days, then the culture medium was replaced with 10 mL/L FBS DMEM/F12 for another 7-10 days before immunofluorescence analysis was performed using lineage-specific antibodies： anti-neurofilaments （NF）, anti-microtubule-associated protein-2 （MAP2）, anti-glial fibrillary acidic protein （GFAP）, anti-rhodopsin, anti-β tubulin and anti-syntaxin. [Results] 0.48%±0.08% （1：208） of the primary cells were capable of proliferating to form neurospheres in serum-free medium containing EGF＋bFGF. 90.1%±8.3% of dissociated single cells from the primary spheres gave rise to new neural spheres, showing the potential of self-renewal. Both the primary and secondary neurospheres expressed neural stem cell specific marker nestin. While in the differentiation promoting medium containing 10 mL/L FBS DMEM/F12, cells began to differentiated into neural-like cells expressing multiple neuron-derived markers such as NF, MAP2, GFAP and rhodopsin, which corresponded to neurons, glia, and rod photoreceptors, respectively. This indicated their multipotentiality. [Conclusions] Retinal progenitor ceils with the characteristics of neural stem ceils from adult human ciliary body were isolated and cultured successfully in vitro.