成年小鼠视网膜穆勒细胞向神经干细胞转化的实验研究  

Experimental research of transformation of adult mouse Muller cells into neural stem cells

作　　者：肖宗宇[1] 李文辉[1] 惠超杰 张广华[1] 唐万峰[1] 杨生龙[1] 曹立新[1] 马进海[1] 巨虎 XIAO Zongyu;LI Wenhui;HUI Chaojie;ZHANG Guanghua;TANG Wanfeng;YANG Shenglong;CAO Lixin;MA Jinhai;JYU Hu(Department of Neurosurgery,The Affiliated Hospital of Qinghai University,Xi'ning 810001,China)

机构地区：[1]青海大学附属医院神经外科

出　　处：《西部医学》2020年第2期198-202,共5页Medical Journal of West China

基　　金：青海省科技厅基金资助项目(2016-ZJ-722)

摘　　要：目的探讨Muller细胞在视网膜损伤后能够转化为神经干细胞,并经诱导后可以分化、新生视网膜中多种类型的神经元,研究该培养细胞是否具有多能向分化能力。方法取72只雌性成年SD小鼠建立视神经切断模型,采用酶解法对SD小鼠视网膜Muller细胞进行消化传代提纯。取第4代胰酶消化过的视网膜Muller细胞,用含有碱性成纤维细胞生长因子(FGF-2)、表皮生长因子(EGF)的改良的Eagle培养基(DMEM)-F12培养基培养5d,用含有0.5μmol/L RA、1μg/L BDNF与0.5%胎牛血清的DMEM培养基进行10d诱导分化,采用免疫荧光染色法对去分化及再分化后的细胞进行鉴定。结果免疫荧光染色鉴定3~4代培养的Muller细胞结果显示,特异性标记物谷氨酰胺合成酶(GS)和谷氨酸转运体(GLAST)抗体表达阳性,呈现红色,荧光染色GS和GLAST抗体表达阳性细胞比例分别为(92.76±4.28)%与(95.28±2.79)%。对去分化后的Muller细胞球给予免疫荧光染色鉴定,结果显示特异性标记物巢蛋白(Nestin)表达阳性,呈现红色,荧光染色阳性细胞比例为(95.37±1.46)%。免疫荧光染色鉴定去分化后的Muller细胞球,结果显示神经胶质细胞特异性标记物胶质纤维酸性蛋白(GFAP)表达阳性,呈绿色,荧光染色阳性细胞比例为(10.28±3.37)%。细胞抗5溴-2脱氧尿苷(BrdU)的表达阳性,呈现红色,荧光染色阳性细胞比例为(90.44±4.23)%。免疫荧光染色鉴定诱导分化后的Muller细胞球,结果显示神经节细胞的特异性标记物Thy1.1表达阳性,呈现绿色,主要为轴突和细胞质,荧光染色阳性细胞比例为(21.25±4.62)%。结论视网膜Muller细胞是一种视网膜干细胞潜在来源细胞,细胞因子刺激能够使体外培养的视网膜Muller细胞获得干细胞特征。Objective To explore whether Muller cells can be transformed into neural stem cells after retinal injury,and can differentiate and regenerate many types of neurons in the retina after induction,and to explore whether the cultured cells have the ability of multi differentiation.Methods 72 female adult SD mouse were used to establish an optic nerve severing model.The retinal Muller cells of SD mouse were digested and purified by enzymatic hydrolysis.The 4 th generation trypsin-digested retinal Muller cells were cultured for 5 days in modified Eagle medium(DMEM)-F12 medium containing basic fibroblast growth factor(FGF-2)and epidermal growth factor(EGF).The cells were differentiated by DMEM medium containing 0.5μmol/L RA,1μg/L BDNF and 0.5%fetal bovine serum for 10 days,and the cells after dedifferentiation and re-differentiation were identified by immunofluorescence staining.Results Immunofluorescence staining showed that Muller cells cultured in 3~4 passages showed that the specific markers of glutamine synthetase(GS)and glutamate aspartate transporter(GLAST)were positive,showing red color.The proportion of positive cells expressing fluorescent staining of GS and GLAST antibody was(92.76±4.28)%and(95.28±2.79)%,respectively.Immunofluorescence staining was performed on the dedifferentiated Muller cell sphere.The results showed that the specific marker nestin was positively expressed in red,and the proportion of fluorescent staining positive cells was(95.37±1.46)%.Immunofluorescence staining was used to identify the dedifferentiated Muller cell spheres.The results showed that the glial cell-specific marker glial fibrillary acidic protein(GFAP)was positively expressed in green,and the proportion of fluorescent staining positive cells was(10.28±3.37)%.Some cells were positive for anti-5 bromo-2 deoxyuridine(BrdU)and showed red color.The proportion of positive cells stained with fluorescent staining was(90.44±4.23)%.The Muller cell spheres were induced by immunofluorescence staining.The results showed that the spe

关 键 词：MULLER细胞 神经干细胞 细胞分化 

分 类 号：R775.9[医药卫生—眼科]