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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持正常RPE细胞生长的一种理想培养基。
OBJECTIVE: To investigate the effects of tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), β-fibroblast growth factor (TGFβ2), interferon-γ Caspase-3 was incubated in a mixed medium of fetal bovine serum (FBS) and insulin-transferrin-sodium selenite (ITS) And its effect on the growth of retinal pigment epithelium (RPE) cells in order to explore the ideal medium for maintaining the growth of normal RPE cells. Methods: Firstly, the presence of TNF-α, VEGF, TGFβ2, IFN-γ and Casepase-3 were detected in 20,40,100mL / LFBS and 10,20,30g / LITS without RPE cells and DMEM medium respectively. Forty RPE cells from the eyes of C57BL / 6 mice were then cultured in DMEM medium containing 20, 40 and 100 mL / L LFBS and 20, 40, and 100 mL / L respectively. Immunohistochemical staining and cell counting were performed to assess the presence and growth of RPE cells. The third generation and fourth generation of RPE cells cultured in primary culture for 48h were used to detect the expression of TNF-α in RPE cells and supernatant by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) , VEGF, TGFβ2, IFN-γ and casepase-3 were detected by Western blotting. The expression of Casepase-3 in RPE cells was detected by Western blotting. Results: TNF-α, VEGF, βFGF, TGFβ2 and casepase-3 were detected in 20,40,100 mL / LFBS (IFN-γ was not expressed) and upregulated with increasing concentration. No above-mentioned growth factors were detected at 10, 20 and 30 g / LITS. RPE cells were cultured successfully. The expression of TNF-α, VEGF, βFGF, TGFβ2 and casepase-3 in DMEM medium containing 20,40,100mL / L FBS and 20,40,100mL / LFBS and 10g / There was no significant difference in the expression intensity among the corresponding groups, but there was a significant difference in the expression intensity between different groups (P <0.01). IFN-γ is not expressed. The above factors were the lowest in medium containing 20mL / LFBS and 20mL / LFBS + 10g / LITS, but RPE cells grew well in 20mL / LFBS + 10g / LITS medium. CONCLUSIONS: The expression of TNF-α, VEGF, βFGF, TGFβ2 and casepase-3 in RPE cells and supernatants. Mixed medium of 20mL / LFBS + 10g / LITS may be an ideal medium to maintain the growth of normal RPE cells.