大鼠视网膜干细胞转染绿色荧光蛋白的实验研究

The study of transfection protein to rat retinal stem cells

目的观察蛋白质粒转染大鼠视网膜干细胞后蛋白的表达及细胞生长、分化情况。方法应用机械-酶消化法将出生10d大鼠的睫状体组织制成单细胞悬液,并应用含20μg/L表皮生长因子、20μg/L碱性成纤维细胞生长因子和1×B27添加剂在无血清DMEM/F12培养基中培养。取第2代细胞应用免疫细胞化学染色检测神经干细胞特异性抗原神经巢蛋白(nestin)及细胞分裂增殖标志物5-溴脱氧尿核苷(5-bromodeoxyuridine,BrdU)的表达。应用脂质体转染法将含有绿色荧光蛋白(green fluorescent protein,GFP)的质粒pGCsilencerTM U6/Neo/GFP转染到RSCs内,转染后应用荧光显微镜观察细胞生长情况,转染后应用G418筛选,一周后对转染后的细胞应用含50ml/L的胎牛血清的培养基进行诱导分化,并应用免疫细胞化学染色方法对于分化后的细胞进行神经元标志物神经元特异性烯醇酶(neurone specific enolase,NSE)、神经胶质细胞标志物神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP)检测。结果原代细胞培养2d后在培养基内可以观察到悬浮的折光性强的小细胞团,4-5d后细胞团数量增多且体积变大,7-9d后细胞传代,应用免疫细胞化学方法可检测到细胞表达神经标志物nestin及增殖标志BrdU,基因转染1d后即可在细胞内观察到荧光的表达,于转染2-3d后荧光逐渐增强并可维持较长时间。筛选一周时细胞基本均表达GFP。转染后细胞的生长特性未发生明显变化,在以血清为诱导条件下仍可分化出神经样细胞。诱导第7日免疫化学染色分别检测出神经元及胶质细胞标志物NSE及GFAP的表达。结论来源于睫状区的大鼠视网膜干细胞经基因转染后可在一定时间内稳定表达,并且细胞增殖及分化未受到明显影响,实验为下一步进行RSCs内表达或抑制特定的目的基因的实验研究奠定了基础。

Objective To observe the expression of fluorescent protein in rat retinal stem cells and the proliferation and differentiation of cells after transfection.Methods Cells of the ciliary body of postnatal 10d rat were isolated by mechanical and enzymic method,then were cultivated in serum free DMEM/F12 medium with 20 μg/L EGF,20 μg/L bFGF and 1×B27Supplement,RSCs of the second generation were identified by detection of nestin and 5-bromodeoxyuridine（BrdU） with immunocytochemistry staining,pGCsilencerTMU6/Neo/GFP were transfected into RSCs by liposome transfection.The characteristics of proliferation of cells after transfection were observed by fluorescence microscope.Differentiation of RSCs was induced by 50mL/L FBS medium after one week selection by G418.The neuron marker neurone specific enolase（NSE） and glial marer glial fibrillary acidic protein（GFAP） of RSCs were detected by immunocytochemistry staining.Results After two days in the primary cultivation,cell spheres with high refraction can be found.The volume and the amount of cell spheres increased gradually after four days.Cells were transferred to next generation after cultivation for 7-9 days.The marker（nestin and BrdU） of the second generation cells were detected by immunocytochemistry staining.Fluorescence can be observed in RSCs just 1d after gene transfection.It increased in 2-3d and maintained for a long time.After selection by G418 for a week,all cells expressed GFP.The characteristics of RSC had not apparent change after GFP transfection.In the seventh day after differentiation,NSE and GFAP can be detected by immunocytochemistry staining.Conclusion The characteristics of the shape,proliferation and differentiation of RSCs from ciliary body zone had no change after GFP gene transfection,this result provide evidences for further research of special gene modification in RSCs.