LED光源对人视网膜色素上皮细胞分泌单核细胞趋化因子-1和白细胞介素-8的影响

目的　观察ＬＥＤ光源对人视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉａｌｃｅｌｌｓ,ＲＰＥ）细胞增生及分泌单核细胞趋化因子-１（ｍｏｎｏｃｙｔｅｃｈｅｍｏｔａｃｔｉｃｐｒｏｔｅｉｎ-１,ＭＣＰ-１）和白细胞介素-８（ｉｎｔｅｒｌｅｕｋｉｎ-８,ＩＬ-８）的影响。方法　传代培养的ＡＲＰＥ细胞分别置于５００ｌｕｘ、１０００ｌｕｘ的ＬＥＤ白光、蓝光、绿光中分别照射６ｈ、１２ｈ、２４ｈ,并分别设置对照组避光培养。采用ＭＴＴ法检测ＲＰＥ细胞的增生值（Ａ４６０）,分别使用Ｒｅａｌ-ｔｉｍｅＰＣＲ和ＥＬＩＳＡ法检测各组ＲＰＥ细胞ＭＣＰ-１和ＩＬ-８的表达水平。结果　ＭＴＴ法检测细胞增生值显示:蓝光、白光各组比较差异均有统计学意义（均为Ｐ＜０．０５）,白光５００ｌｕｘ２４ｈ和１０００ｌｕｘ１２ｈ、２４ｈ,蓝光各照度６ｈ、１２ｈ、２４ｈ,ＲＰＥ细胞增生值均较对照组低,差异均有统计学意义（均为Ｐ＜０．０５）,细胞增生值随着白光和蓝光光照强度及光照时间的增加逐渐下降。ＲＴ-ＰＣＲ结果显示,各组ＲＰＥ细胞中ＭＣＰ-１和ＩＬ-８的ｍＲＮＡ比较差异均有统计学意义（均为Ｐ＜０．０５）。其中白光５００ｌｕｘ２４ｈ和１０００ｌｕｘ２４ｈ,蓝光５００ｌｕｘ１２ｈ、２４ｈ和１０００ｌｕｘ１２ｈ、２４ｈ,绿光５００ｌｕｘ２４ｈ和１０００ｌｕｘ２４ｈ,各组ＲＰＥ细胞中ＭＣＰ-１和ＩＬ-８的ｍＲＮＡ表达水平较对照组高,差异均有统计学意义（均为Ｐ＜０．０５）。ＥＬＩＳＡ结果显示,白光５００ｌｕｘ２４ｈ和１０００ｌｕｘ１２ｈ、２４ｈ,蓝光５００ｌｕｘ１２ｈ、２４ｈ和１０００ｌｕｘ１２ｈ、２４ｈ,绿光５００ｌｕｘ２４ｈ、１０００ｌｕｘ２４ｈ,与对照组相比, ＲＰＥ细胞ＭＣＰ-１、ＩＬ-８分泌量增加,差异均有统计学意义（均为Ｐ＜０．０５）;蓝光在５００ｌｕｘ２４ｈ和１０００ｌｕｘ１２ｈ、２４ｈ,ＭＣＰ-１和ＩＬ-８分泌量较白光、绿光增加,差异均有统计学意义（均为Ｐ＜０．０５）。结论　ＬＥＤ白光、蓝光和绿光能以照度和时间依赖性影响ＡＲＰＥ细胞增生及分泌ＭＣＰ-１和ＩＬ-８,以蓝光更为显著。     

Role and regulation of IL-8 and MCP-1 in LPS-induced uveitis in rabbits

Intravitreal injection of lipopolysaccharide (LPS) induces leukocyte infiltration and protein leakage into the aqueous humor. In the present study, we investigated the role of IL-8 and MCP-1 and regulation of these chemokines by TNFalpha and IL-1 in LPS-induced uveitis in rabbits. After intravitreal injection of LPS, generation of IL-8 in the aqueous humor showed a biphasic pattern with the first peak at 12 hr and the second one at 24 hr, while MCP-1 was produced in a monophasic pattern and peaked at 24 hr. Immunohistochemistry showed that ciliary epithelial cells and infiltrating leukocytes were the producing cells of IL-8 and MCP-1. Administration of anti-IL-8 IgG suppressed by 66% the peak levels of LPS-induced aqueous neutrophil counts at 24 hr but did not suppress aqueous mononuclear cell counts or protein levels. anti-MCP-1 IgG inhibited aqueous mononuclear cell counts by 41% and protein levels by 28%, but did not inhibit aqueous neutrophil counts. The levels of LPS-induced aqueous IL-8 and MCP-1 at 12 hr were inhibited by anti-TNFalpha mAb but not by an IL-1 receptor antagonist (IL-1Ra), while concentrations of the two chemokines at 24 hr were inhibited by both anti-TNFalpha mAb and IL-1Ra. A combination of anti-TNFalpha mAb and rrIL-1Ra had an additive effect on the 24 hr-chemokine levels and inhibited up to 90% chemokine production. Taken together, our results show that IL-8 mediates neutrophil infiltration, while MCP-1 mediates mononuclear cell infiltration and protein leakage in LPS-induced uveitis in rabbits. Levels of aqueous IL-8 and MCP-1 at 12 hr are regulated by TNFalpha, while levels at 24 hr are regulated by TNFalpha and IL-1.     

Oxidant-mediated Akt activation in human RPE cells

PURPOSE: To determine whether a model oxidant, hydrogen peroxide (H2O2), influences Akt activation and, if so, whether Akt activation promotes retinal pigment epithelial (RPE) cell survival. METHODS: Cultured human RPE cells were pretreated with medium alone, with LY294002 (LY), an inhibitor of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt, or with Akt/protein kinase B signaling inhibitor (API)-2, a specific Akt inhibitor, and then were stimulated with H2O2 at different doses for various times. Akt phosphorylation was evaluated by Western blot using antibody against phosphorylated Akt (Ser473). The effect of Akt blockade on RPE cell viability was assessed by tetrazolium salt (WST-1) assay and a lactate dehydrognease (LDH) release assay. Caspase-mediated cytokeratin cleavage, an early apoptosis marker, was assessed by M30 antibody staining. Caspase-independent apoptosis was determined by nuclear translocation of apoptosis-inducing factor (AIF). RPE cell morphology was evaluated by electron microscopy. The effect of H2O2 on downstream Akt targets was examined by Western blot using antibody against phosphorylated forkhead in rhabdomyosarcoma (FKHR) and phosphorylated glycogen synthase kinase (GSK)-3beta. RESULTS: H2O2 induced Akt phosphorylation in a dose-dependent manner and also induced the phosphorylation of downstream effectors FKHR and GSK-3beta. LY markedly inhibited H2O2-mediated Akt phosphorylation and significantly enhanced caspase-associated and caspase-independent RPE cell death. CONCLUSIONS: A model oxidant, H2O2, induces PI3K and thereby activates Akt. Akt activation enhances RPE cell survival and thus may protect RPE cells from oxidant-induced cell death under normal circumstances and in disease states such as age-related macular degeneration (AMD).     

人视网膜色素上皮细胞NF-kB的表达

目的探讨核因子闎(NF-кB)在体外培养的人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞中的基础表达以及吡咯二硫代氨基甲酸乙酯(pyrollidi ne dithiocarbamate,PDTC)、IL-1β对NF-кB表达的影响. 方法体外培养的hRPE细胞经同步化后分2组分别加药(1)无PDTC组分别加入IL-1β、生理盐水(用于检测NF-кB在hRRE中的基础表达);(2)PDTC组分别加入IL-1β、生理盐水(用于检测NF-кB在PDTC预处理后hRPE中的表达).免疫荧光抗体染色、流式细胞计数法(flow cytometry, FCM)测定上述2组标本中hRPE的NF-кB的表达率. 结果 NF-кB在hRPE中的基础表达率为8.05 %,IL-1β作用后表达率升高到30.26%; hRPE细胞经PDTC预处理后,NF-кB的表达率下降为3.74%,加入IL-1(10 υg·L-1)作用后表达率为3.66%. 结论 IL-1β可以显著提高NF-кB在hRPE细胞中的表达;PDTC可以显著降低体外培养的hRPE中的NF-кB表达率,PDTC亦可显著抑制由IL-1β诱导的NF-кB的活化过程.     

视网膜色素上皮细胞中蛋白酶体活性的下降促进IL-6表达的研究

目的　探讨当视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ,ＲＰＥ）细胞的蛋白酶体活性下降时促进白细胞介素-６（ｉｎｔｅｒｌｅｕｋｉｎ-６,ＩＬ-６）表达的机制。方法　将人ＲＰＥ细胞系分成两组培养,第一组只加ＤＭＥＭ,第二组加入ＤＭＥＭ（内含成分同上）和１０μｍｏｌ?Ｌ－１的蛋白酶体抑制剂ＭＧ１３２,首先收集１ｈ、４ｈ、８ｈ等时间点的ＲＰＥ细胞,测ＩＬ-６ｍＲＮＡ含量。再分别在２ｈ、４ｈ、６ｈ、８ｈ、１０ｈ、１２ｈ等时间点,收集上清液,测ＩＬ-６和单核细胞趋化蛋白-１（ｍｏｎｏ-ｃｙｔｅｃｈｅｍｏ-ａｔｔｒａｃｔａｎｔｐｒｏｔｅｉｎ-１,ＭＣＰ-１）的含量。然后测定ＭＧ１３２是否可以激活调控ＩＬ-６分泌的两条主要细胞信号通路———Ｐ３８-丝裂原激活的蛋白激酶（Ｐ３８ｍｉｔｏｇｅｎ-ａｃｔｉ-ｖａｔｅｄｐｒｏｔｅｉｎｋｉｎａｓｅ,Ｐ３８ＭＡＰＫ）途径和ｃ-Ｊｕｎ氨基端激酶（ｃ-ＪｕｎＮ-ｔｅｒｍｉｎａｌｋｉｎａｓｅ,ＪＮＫ）途径。再在细胞培养液中加入这些细胞信号通路的抑制剂,测上清中ＩＬ-６的含量,观察哪个信号通路的抑制剂可以抵消ＭＧ１３２促进ＩＬ-６分泌的作用。结果　ＭＧ１３２增加了ＲＰＥ细胞ＩＬ-６的ｍＲＮＡ水平和蛋白质水平的表达,却降低了ＭＣＰ-１的分泌。ＭＧ１３２可以激活调控ＩＬ-６分泌的两条主要细胞信号通路———Ｐ３８ＭＡＰＫ和ＪＮＫ。Ｐ３８ＭＡＰＫ的抑制剂ＳＢ２０３５８０加入ＲＰＥ培养液后不仅可以降低ＲＰＥ细胞产生ＩＬ-６的基础分泌,而且可以抵消ＭＧ１３２促进ＩＬ-６分泌的作用;而当加入ＪＮＫ的抑制剂ＳＰ６００１２５后,虽然也可以降低ＩＬ-６的基础分泌,但却不能抵消ＭＧ１３２促进ＩＬ-６分泌的作用。结论　在ＲＰＥ中,蛋白酶体活性的下降可以激活Ｐ３８ＭＡＰＫ细胞信号通路,从而促进ＩＬ-６的产生。     

Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.     

Regulatory role of IL-1β in the expression of IL-6 and IL-8 in human corneal epithelial cells during Pseudomonas aeruginosa colonization

Pseudomonas aeruginosa is a virulent pathogen and is frequently associated with bacterial keratitis. Recent studies have shown that high levels of interleukin (IL)‐1β and macrophage inflammatory protein‐2 are associated with the severity of corneal infection. Interleukin‐1β is a principal inflammatory mediator. Understanding the regulatory role of IL‐1β would provide better understanding of host responses during P. aeruginosa corneal infection. A human corneal epithelial (HCE) cell line and three P. aeruginosa strains were used in this experiment. Confluent HCE cells were challenged with P. aeruginosa and monoclonal antihuman IL‐1β antibody (IL‐1β mAb). The culture supernatants were collected for measuring cytotoxicity and protein levels of IL‐1β, IL‐8 and IL‐6 by enzyme‐linked immunosorbent assay. Results showed that HCE cells expressed low levels of IL‐1β and high levels of IL‐6 and IL‐8 during P. aeruginosa colonization. Paer1‐colonized HCE cells produced higher levels of IL‐1β, IL‐6 and IL‐8 protein compared to those produced by 6206‐ and 6294‐ colonized HCE cells. Administration of IL‐1β mAb decreased the production of IL‐8 and IL‐6. In conclusion, P. aeruginosa‐colonized HCE cells produced low levels of IL‐1β and high levels of IL‐6 and IL‐8. Neutralizing IL‐1β protein significantly downregulated the production of IL‐8 and IL‐6.     

人视网膜色素上皮细胞NF-кB的表达

目的探讨核因子闎(NF-кB)在体外培养的人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞中的基础表达以及吡咯二硫代氨基甲酸乙酯(pyrollidi ne dithiocarbamate,PDTC)、IL-1β对NF-кB表达的影响. 方法体外培养的hRPE细胞经同步化后分2组分别加药:(1)无PDTC组:分别加入IL-1β、生理盐水(用于检测NF-кB在hRRE中的基础表达);(2)PDTC组:分别加入IL-1β、生理盐水(用于检测NF-кB在PDTC预处理后hRPE中的表达).免疫荧光抗体染色、流式细胞计数法(flow cytometry, FCM)测定上述2组标本中hRPE的NF-кB的表达率. 结果 NF-кB在hRPE中的基础表达率为8.05 %,IL-1β作用后表达率升高到30.26%; hRPE细胞经PDTC预处理后,NF-кB的表达率下降为3.74%,加入IL-1(10 υg·L-1)作用后表达率为3.66%. 结论 IL-1β可以显著提高NF-кB在hRPE细胞中的表达;PDTC可以显著降低体外培养的hRPE中的NF-кB表达率,PDTC亦可显著抑制由IL-1β诱导的NF-кB的活化过程.