【作者】 曾琦；

【导师】 夏晓波；

【作者基本信息】 中南大学 ， 眼科学， 2010， 博士

【摘要】 目的研究体外培养的大鼠视网膜Muller细胞经条件培养基诱导后是否可以产生视网膜干细胞的特性。方法体外培养出生后7-10天的SD大鼠视网膜Muller细胞,RT-PCR及免疫荧光染色法鉴定Muller细胞纯度。取第3-4代细胞改由含1×N2 supplement、碱性成纤维细胞生长因子(Basic fibroblast growth factor,FGF2)和表皮生长因子(Epidermal growth factor,EGF)的DMEM/F12干细胞条件培养基培养3-5天。采用免疫荧光染色法及RT-PCR法鉴定去分化后的细胞。结果RT-PCR及免疫荧光染色结果显示,分离培养的视网膜Muller细胞纯度高,无其他细胞污染。干细胞条件培养基培养3-5天后,大部分Muller细胞克隆生长形成细胞球,经免疫荧光染色鉴定显示细胞球内95.07±1.35%以上的细胞表达视网膜干细胞特异性标记物Nestin阳性。约10.34±3.26%细胞浆内表达神经胶质细胞特异性标记物GFAP阳性。90.26±4.12%以上细胞表达增殖细胞特异性标记物BrdU阳性。结论新生SD大鼠视网膜Muller细胞经体外条件培养基诱导后可以产生具有增殖能力的视网膜干细胞,这为干细胞研究和视神经再生治疗等提供了新的方法和手段。目的构建PEGFP-N1-Math5真核表达质粒。探讨以Miiller细胞去分化而来的视网膜干细胞作为基因靶细胞,电穿孔法转染Math5基因的可行性。方法构建真核细胞表达质粒PEGFP-N1-Math5。采用酶切及基因测序鉴定。然后选用脂质体转染和电穿孔转染的方法,分别将math5基因转染Muller细胞去分化而来的干细胞,荧光显微镜下观察,并检测转染效率。结果连接产物经EcoRⅠ及BamHⅠ双酶切鉴定,产生约4.7 kb和450bp的两个DNA片段,与连接前载体和插入片段大小相符,证实成功获得了PEGFP-N1-Math5重组真核表达质粒。重组质粒PEGFP-N1-Math5的正、反向序列测定分析显示,Math5的序列与Gene Bank中Math5 cDNA的序列完全一致。阳离子脂质体Lipofectamine2000和电穿孔法介导Math5基因转染Muller细胞去分化而来的干细胞,48h时转染效率均达到最高,分别为0.13±0.17%和15.54±2.43%。电穿孔法较脂质体法转染的细胞内Math5基因有更好的表达,两者比较差异有统计学意义(P<0.01)。结论电穿孔法可使视网膜Muller细胞去分化而来的干细胞得到较高效的基因表达。这将为视神经再生的基因治疗和进一步的干细胞研究奠定基础。目的明确Math5对体外培养的鼠视网膜Muller细胞定向分化为视网膜神经节细胞的调控作用。方法将视网膜Muller细胞去分化来的干细胞随机分为未转染、转染空载体或Math5的三组,分别用电穿孔法将空载体质粒PEGFP-N1或PEGFP-N1-Math5重组质粒转染后两组细胞,随后改由含1%胎牛血清(Fetal bovine serum, FBS)、1 ng/ml脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)和1μM黄酸(Retinoic acid, RA)的DMEM条件培养基中诱导三组干细胞进一步分化,选用神经节细胞特异性标记物Thy1.1行免疫荧光染色,并计数视网膜神经节细胞占分化而来的总细胞数的比例。结果将未转染和转染空载体及Math5的三组视网膜干细胞进行进一步分化培养,5-7天后可见神经球贴壁且分化出大量细胞,免疫荧光染色鉴定发现,Math5转染组分化所得神经节细胞占总细胞数的比例(61.10±1.93%)明显高于其他两组,差异具有统计学意义(P＜0.01)。结论Math5可调控视网膜Muller细胞去分化来的干细胞定向分化为视网膜神经节细胞。这可能为青光眼视网膜神经节细胞再生提供新的来源,从而为青光眼的临床治疗提供一条崭新的基因治疗和视神经再生途径。更多还原

【Abstract】 Objective:The purpose of this study was to certify the ability of Muller cells for producing retina progenitor cells in vitro.Methods:Muller cells were isolated from rat retina,and proliferating cells were expanded in serum-containing medium.The third or fourth passage of cells were identified by RT-PCR and Immunocytochemistry analysis.For dedifferentiation, the cultured cells were transferred to the sphere-culture medium composed of DMEM/F-12 supplemented with 1×N2、bFGF and EGF for 3-5days. At last, Cells in this stages were identified by immunocytochemical analysis and RT-PCR.Results:Approximately 95.31±2.68%and 93.04±4.15%of cells in the culture were Muller cells as revealed by expressing glutamate-aspartate transporters (GLAST) and glutamine synthetase (GS) immunoreactivities. RT-PCR analysis also revealed that the culture was enriched for Miiller cells and not contaminated with other retinal cells.After 3-5 days cultuerd in the the sphere-culture medium, the Miiller cells became round and differentiate to neurospheres.95.07±1.35%of cells in the neurosphere were positively reacted for Nestin,10.34±3.26% for GFAP and 90.26±4.12%for BrdU.Conclusion:New born SD rat Muller cells can generate clonal neurospheres,which consist of proliferating and multipotent cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina. Objective:The purpose of this study was to certify the ability of Muller cells for producing neural stem cells in vitro and to evaluate the possibility of transferring math5 gene to the stem cells which produces by Miiller cells via electroporation.Methods:Firstly we created the PEGFP-N1-Math5 plasmid. Secondly the neurospheres were transfected either by electroporation or by lipofection using the PEGFP-N1-Math5 plasmid.At last,the cells were analyzed 24h,48 h,3d,4d,1w and 2w after transfection and detect the effectiveness of transfection by fluorescence microscopy.Results:The recombinant transformers were digested with EcoR I and BamH I. The fragment was about 450 bp, indicating that Math5 fragment had been inserted into the vector. Moreover, The result of DNA sequencing also confirmed that the recombinant plasmid of Math5 was constructed correctly.The PEGFP-N1-Math5 plasmid were transfected into the neurospheres.The transfection efficiency was superior with electroporation (15.54±2.43%) as compared to lipofection (0.13±0.17%) at forty-eight hours after transfection.Conclusion:Electrotransfection is a method which can induce stem cells express gene effectively.This study may lay a fundation for further study on stem cells and gene therapy for neural regeneration in retina. Objective:The purpose of this study was to certify the ability of math5 on the differentiation of retinal Muller cells into retinal ganglion cells.Method:The neurospheres dedifferentiated from Miiller cells were divided into three groups:(A) neurospheres transfected by PEGFP-N1-Math5; (B) neurospheres transfected by PEGFP-N1; and (C) neurospheres without transfection.Then they were transferred to the culture medium, which was composed of Brain-derived neurotrophic factor (BDNF; 1 ng/ml),Retinoic acid (RA; 1μM) and 1% Fetal bovine serum at 37℃in 5%CO2. After 7 days,cells were fixed using cold 4% paraformaldehyde for immunocytochemical analysis.Result:After plating, cells in the spheres migrated outwards and began to differentiate. It was found that the percentage of RGCs in group transfected by PEGFP-N1-Math5(61.10±1.93%) was higher than that of the group transfected by PEGFP-N1 and the group without transfection.They were statistically significant(P<0.01).Conclusion:Math5 can promote the differentiation of retinal Miiller cells into retinal ganglion cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina.更多还原