体外诱导大鼠脂肪干细胞向光感受器细胞和视网膜色素上皮细胞分化的初步研究
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摘要
第一部分:大鼠脂肪干细胞的分离、培养和鉴定
目的:分离、培养和鉴定大鼠的脂肪干细胞
方法:Ⅰ型胶原酶分离大鼠脂肪干细胞,用贴壁筛选法,在含10%胎牛血清的DMEM/F12培养基中培养。通过连续传代使细胞纯化后,用流式细胞仪检测CD45、CD90、CD49d、CD106鉴定干细胞的表面抗原。
结果:上述方法可以获得大量高纯度的脂肪干细胞。原代脂肪干细胞表型检测:CD45阳性率是1.6%,CD90是71.3%,CD49d是7.8%,CD106是3.5%。从传1代至传5代,CD45阳性率是0.8%～9.3%,CD90是84.7%～94.8%,CD49d是16.8%～31.0%,CD106是3.5%。各代ADSCs中,CD49d的阳性率均高于CD106。
第二部分:大鼠脂肪干细胞经体外诱导向光感受器细胞和视网膜色素上皮细胞的分化
目的:体外诱导脂肪干细胞向光感受器细胞和RPE细胞的分化,并对不同诱导条件进行比较。
方法:采用机械分离法获得视网膜感觉神经上皮细胞,以及Dispase消化法获得RPE细胞,制备视网膜细胞提取液。用EGF、激活素A、牛磺酸、视黄酸和视网膜细胞提取液单独诱导大鼠脂肪干细胞;用EGF+牛磺酸、EGF+视黄酸、牛磺酸+视黄酸、EGF+牛磺酸+视黄酸联合诱导大鼠脂肪干细胞。用免疫荧光法和流式细胞仪鉴定被诱导的细胞的视紫红质、CK和S-100的表达。免疫荧光法显示脂肪干细胞和骨髓间充质干细胞表达视紫红质、CK和S-100。流式细胞仪检测对照组和各种诱导组的视紫红质、CK和S-100的表达,并计算诱导效应。
结果:视网膜细胞提取液诱导的部分细胞,呈卵圆形或梭形,细胞内可见色素。在含有诱导剂的1%FBS/αMEM中,细胞生长速度减慢,细胞呈扁平多角形。脂肪干细胞的视紫红质的诱导效应是17.5%～46.0%,CK的诱导效应是19.7%～79.3%,S-100的诱导效应是27.3%～50.7%。骨髓间充质干细胞的视紫红质的诱导效应是55.9%～76.5%,CK的诱导效应是39.4%～75.9%,S-100的诱导效应是25.4%～41.8%。
结论:改良的原代ADSCs分离和培养方法,可以获得更多的细胞。采用贴壁筛选法,获得高纯度的ADSCs。采用机械分离法获得视网膜感觉神经上皮细胞,以及Dispase消化法获得RPE细胞,可以制备全层视网膜细胞提取液,用于ADSCs诱导实验。用EGF、激活素A、牛磺酸、视黄酸和视网膜细胞提取液单独诱导;用EGF+牛磺酸、EGF+视黄酸、牛磺酸+视黄酸、EGF+牛磺酸+视黄酸联合诱导,ADSCs可以表达光感受器细胞的标记—视紫红质、RPE细胞的标记—CK和S-100。说明ADSCs具有向光感受器细胞和RPE细胞分化的潜能,ADSCs和MSCs的诱导效应相似。视网膜细胞提取液,能够诱导ADSCs和MSCs向光感受器细胞和RPE细胞分化,是一种经济、有效的创新方法。
Part One:Isolation,culture and identification of rat ADSCs
Object:To isolate,cultivate and identify rat ADSCs
Methods:The ADSCs were isolated by TypeⅠCollagenase and cultured by their adherent ability to the wall of culture flask in DMEM/F12 medium supplemented with 10%fetal bovine serum.The ADSCs were purified by the method of continual passage.Surface antigens including CD45、CD90、CD49d、CD106 were indentified by flow cytometry.
Results:Large amout of ADSCs were obtained with high purity.For primary generation,the phenotypes of ADSCs were CD45(1.6%),CD90(71.3%), CD49d(7.8%) and CD106(3.5%).From passage 1 to 5,these phenotypes were CD45(0.8%～9.3%),CD90(84.7%～94.8%),CD49d(16.8%～31.0%) and CD106(8.3%～22.2%).There was a higher CD49d percentage than CD106 in all the passages.
Part Two:In vitro differentiation of rat ADSCs into photoreceptor cells and RPE cells
Object:To explore and compare the induced differentiation of rat ADSCs into Photoreceptor cells and RPE cells.
Methods:Retinal neuroepithelial cells were isolated and obtained by mechanic measures and furthure digested by Dispase for RPE cells as well as extracted liquid of retina.ADSCs were induced for differentiation by EGF,activin A, Taurine,retinoic acid(RA) and extracted liquid of retina respectively. Meanwhile,ADSCs were combinedly induced by EGF+Taurine,EGF+RA, Taurine + RA,EGF + Taurine + RA respectively.Immunofluorescence was introduced for detecting the expression of rhodopsin,CK and S-100 and flow cytometry for quantification.Induction efficacy was calculated by contrasting control groups and induced groups.MSCs underwent the same procedures and served as reference.
Results:A portion of the induced cells in the extracted liquid of retina were oval or fusiform,with pigment inside plasm.The induced cells in the 1%FBS/αMEM with induction agents presented a slower growth and flat polygonal shape.The induction efficacy of ADSCs was 17.5%～46.0%for rhodopsin,19.7%～79.3%for CK and 27.3%～50.7%for S-100.The induction efficacy of MSCs was 55.9%～76.5%for rhodopsin,39.4%～75.9%for CK and 25.4%～41.8% for S-100.
Conclusion:More ADSCs will be otabined by modified isolation and culture of the primary generation.The characteristic adherence to the wall of culture flask facilitates high purity.Mechanic measures in the isolation of retinal neuroepithelial cells and Dispase digestion for RPE cells successfully prepares extracted liquid of retina which induced differentiation of ADSCs.After either single induction or combined induction,the ADSCs expressed the specific markers of photoceptors(rhodopsin) and RPE markers(CK and S-100),which indicated potential differentiation into photoceptors and RPE cells.ADSCs present a similar induction efficacy to MSCs.The novel method of extracted liquid of retina proves to be economical and effective in induced differentiation of ADSCs and MSCs.
目的:分离、培养和鉴定大鼠的脂肪干细胞
方法:Ⅰ型胶原酶分离大鼠脂肪干细胞,用贴壁筛选法,在含10%胎牛血清的DMEM/F12培养基中培养。通过连续传代使细胞纯化后,用流式细胞仪检测CD45、CD90、CD49d、CD106鉴定干细胞的表面抗原。
结果:上述方法可以获得大量高纯度的脂肪干细胞。原代脂肪干细胞表型检测:CD45阳性率是1.6%,CD90是71.3%,CD49d是7.8%,CD106是3.5%。从传1代至传5代,CD45阳性率是0.8%～9.3%,CD90是84.7%～94.8%,CD49d是16.8%～31.0%,CD106是3.5%。各代ADSCs中,CD49d的阳性率均高于CD106。
第二部分:大鼠脂肪干细胞经体外诱导向光感受器细胞和视网膜色素上皮细胞的分化
目的:体外诱导脂肪干细胞向光感受器细胞和RPE细胞的分化,并对不同诱导条件进行比较。
方法:采用机械分离法获得视网膜感觉神经上皮细胞,以及Dispase消化法获得RPE细胞,制备视网膜细胞提取液。用EGF、激活素A、牛磺酸、视黄酸和视网膜细胞提取液单独诱导大鼠脂肪干细胞;用EGF+牛磺酸、EGF+视黄酸、牛磺酸+视黄酸、EGF+牛磺酸+视黄酸联合诱导大鼠脂肪干细胞。用免疫荧光法和流式细胞仪鉴定被诱导的细胞的视紫红质、CK和S-100的表达。免疫荧光法显示脂肪干细胞和骨髓间充质干细胞表达视紫红质、CK和S-100。流式细胞仪检测对照组和各种诱导组的视紫红质、CK和S-100的表达,并计算诱导效应。
结果:视网膜细胞提取液诱导的部分细胞,呈卵圆形或梭形,细胞内可见色素。在含有诱导剂的1%FBS/αMEM中,细胞生长速度减慢,细胞呈扁平多角形。脂肪干细胞的视紫红质的诱导效应是17.5%～46.0%,CK的诱导效应是19.7%～79.3%,S-100的诱导效应是27.3%～50.7%。骨髓间充质干细胞的视紫红质的诱导效应是55.9%～76.5%,CK的诱导效应是39.4%～75.9%,S-100的诱导效应是25.4%～41.8%。
结论:改良的原代ADSCs分离和培养方法,可以获得更多的细胞。采用贴壁筛选法,获得高纯度的ADSCs。采用机械分离法获得视网膜感觉神经上皮细胞,以及Dispase消化法获得RPE细胞,可以制备全层视网膜细胞提取液,用于ADSCs诱导实验。用EGF、激活素A、牛磺酸、视黄酸和视网膜细胞提取液单独诱导;用EGF+牛磺酸、EGF+视黄酸、牛磺酸+视黄酸、EGF+牛磺酸+视黄酸联合诱导,ADSCs可以表达光感受器细胞的标记—视紫红质、RPE细胞的标记—CK和S-100。说明ADSCs具有向光感受器细胞和RPE细胞分化的潜能,ADSCs和MSCs的诱导效应相似。视网膜细胞提取液,能够诱导ADSCs和MSCs向光感受器细胞和RPE细胞分化,是一种经济、有效的创新方法。
Part One:Isolation,culture and identification of rat ADSCs
Object:To isolate,cultivate and identify rat ADSCs
Methods:The ADSCs were isolated by TypeⅠCollagenase and cultured by their adherent ability to the wall of culture flask in DMEM/F12 medium supplemented with 10%fetal bovine serum.The ADSCs were purified by the method of continual passage.Surface antigens including CD45、CD90、CD49d、CD106 were indentified by flow cytometry.
Results:Large amout of ADSCs were obtained with high purity.For primary generation,the phenotypes of ADSCs were CD45(1.6%),CD90(71.3%), CD49d(7.8%) and CD106(3.5%).From passage 1 to 5,these phenotypes were CD45(0.8%～9.3%),CD90(84.7%～94.8%),CD49d(16.8%～31.0%) and CD106(8.3%～22.2%).There was a higher CD49d percentage than CD106 in all the passages.
Part Two:In vitro differentiation of rat ADSCs into photoreceptor cells and RPE cells
Object:To explore and compare the induced differentiation of rat ADSCs into Photoreceptor cells and RPE cells.
Methods:Retinal neuroepithelial cells were isolated and obtained by mechanic measures and furthure digested by Dispase for RPE cells as well as extracted liquid of retina.ADSCs were induced for differentiation by EGF,activin A, Taurine,retinoic acid(RA) and extracted liquid of retina respectively. Meanwhile,ADSCs were combinedly induced by EGF+Taurine,EGF+RA, Taurine + RA,EGF + Taurine + RA respectively.Immunofluorescence was introduced for detecting the expression of rhodopsin,CK and S-100 and flow cytometry for quantification.Induction efficacy was calculated by contrasting control groups and induced groups.MSCs underwent the same procedures and served as reference.
Results:A portion of the induced cells in the extracted liquid of retina were oval or fusiform,with pigment inside plasm.The induced cells in the 1%FBS/αMEM with induction agents presented a slower growth and flat polygonal shape.The induction efficacy of ADSCs was 17.5%～46.0%for rhodopsin,19.7%～79.3%for CK and 27.3%～50.7%for S-100.The induction efficacy of MSCs was 55.9%～76.5%for rhodopsin,39.4%～75.9%for CK and 25.4%～41.8% for S-100.
Conclusion:More ADSCs will be otabined by modified isolation and culture of the primary generation.The characteristic adherence to the wall of culture flask facilitates high purity.Mechanic measures in the isolation of retinal neuroepithelial cells and Dispase digestion for RPE cells successfully prepares extracted liquid of retina which induced differentiation of ADSCs.After either single induction or combined induction,the ADSCs expressed the specific markers of photoceptors(rhodopsin) and RPE markers(CK and S-100),which indicated potential differentiation into photoceptors and RPE cells.ADSCs present a similar induction efficacy to MSCs.The novel method of extracted liquid of retina proves to be economical and effective in induced differentiation of ADSCs and MSCs.
引文
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