视网膜色素上皮细胞培养基筛选及其相关生长因子的影响(英文)  

Optimization of culture medium for primary retinal pigment epithelium cells and investigation of medium effcts on growth factor expression

作　　者：郝丽娜[1] 何守志[2] 贾志旸[1] 王舸[1] 董素婷[1] 

机构地区：[1]河北省人民医院眼科,中国河北省石家庄市050051 [2]中国人民解放军总医院眼科,中国北京市100853

出　　处：《国际眼科杂志》2009年第2期214-219,共6页International Eye Science

摘　　要：目的:通过研究肿瘤坏死因子(TNF-α),血管内皮生长因子(VEGF)、β成纤维细胞生长因子(βFGF)、转移生长因子β2(TGFβ2)、干扰素-γ(IFN-γ)及半胱氨酸天冬氨酸蛋白酶-3(Casepase-3)在不同浓度胎牛血清(fetal bovine serum,FBS)与胰岛素转铁蛋白亚硒酸钠(insulin-transferrin-sodium selenite,ITS)混合培养基中的表达及其对正常视网膜色素上皮(retinal pigment epithelium,RPE)细胞生长的影响以探索维持正常RPE细胞生长的理想培养基。方法:首先分别在不含RPE细胞和DMEM培养基的20,40,100mL/LFBS和10,20,30g/LITS中检测TNF-α,VEGF、βFGF、TGFβ2、IFN-γ及Casepase-3是否存在。然后取四十只C57BL/6系小鼠眼的RPE细胞分别培养于含20,40,100mL/LFBS以及20,40,100mL/LFBS与10g/LITS分别组合的DMEM培养基中。免疫组织化学染色以及细胞计数鉴定、评估RPE细胞的存在与生长状况。采用原代培养48h的第三代、第四代RPE细胞,分别用反转录聚合酶链反应(RT-PCR)以及酶联免疫吸附(ELISA)法检测RPE细胞和上清液中TNF-α,VEGF、βFGF、TGFβ2、IFN-γ及casepase-3的表达强弱;用蛋白印记(Western blotting)法检测RPE细胞中Casepase-3的表达。结果:在20,40,100mL/LFBS中检测到了TNF-α,VEGF、βFGF、TGFβ2及casepase-3(IFN-γ没有表达)并且随浓度增加而表达上调。在10,20,30g/LITS中没有检测到上述生长因子。RPE细胞培养成功。在含20,40,100mL/L不同浓度的FBS以及20,40,100mL/LFBS与10g/LITS分别组合的DMEM培养基中随浓度增加,TNF-α,VEGF、βFGF、TGFβ2及casepase-3的表达呈上调趋势,各对应组组间的表达强度没有明显差异,但不同浓度组组内的表达强度有明显差异(P<0.01)。IFN-γ没有表达。上述因子在含20mL/LFBS与20mL/LFBS+10g/LITS的培养基中表达最低,但20mL/LFBS+10g/LITS的培养基中RPE细胞生长良好。结论:在RPE细胞和上清液中有TNF-α,VEGF、βFGF、TGFβ2及casepase-3的表达。20mL/LFBS+10g/LITS的混合培养基可能是维AIM. To optimize the conditions for in vitro culture of retinal pigment epithelium （RPE） cells, we characterized expressions of various growth factors in RPE cells, including tumor necrosis factor （ TNF-α ）, vascular endothelial growth factor （VEGF）, β fibroblast growth factor （βFGF）, transforming growth factor β2 （TGFβ2）, and interferon-γ, （IFN-γ）. We also studied expressions of caspase-3 under different concentrations of fetal bovine serum （FBS） with insulin-transferrin-sodium selenite （ITS） supplement. METHODS. First, we investigated if expressions of TNF- α VEGF,βFGF, TGFβ2, IFN-γ, and caspase-3 in FBS and ITS with of concentration. Second, we cultured primary RPE cells from eyes of forty C57 BL/6 mice in standard dulbecco＇s modified eagle＇s medium （DMEM） containing 20,40,100mL/L FBS and 20,40,100mL/L FBS together with 10g/L ITS. Immunohisto-chemical staining and cell counting were performed to verify the existence and growth condition of RPE cells. Expressions of TNF-α, VEGF, βFGF, TGFβ2 and IFN--γ were determined using cells and supernatant from passage-3 to -4 primary RPE cell after 48 hours of culture with RT-PCR and enzyme- linked immunosorbent assays （ELISA）. The expression of casepase-3 was determined via Western blotting. The major outcome measurement is the expression level of growth factors in cultured RPE cells and the experiment design is to expose the RPE cells to different culture medium. RESULTS： TNF-α, VEGF, βFGF, TGFβ2, but not IFN-γ, were expressed and the expressions increased with concentration. No expression of the aforementioned genes was detected in presence of ITS. The primary cultures of RPE cells were successfully established. TNF-α, VEGF, βFGF, TGFβ2 （but no IFN-γ） and the active caspase-3 were detected in 20,40,100mL/L FBS or 20,40,100mL/L FBS combined with 10g/L ITS; the expressions were upregulated with increasing concentration of FBS. There is no significant difference in the expression of growth factors

关 键 词：视网膜色素上皮细胞 生长因子 半胱氨酸天冬氨酸蛋白酶-3 培养 

分 类 号：R774.1[医药卫生—眼科] R392-33[医药卫生—临床医学]