ZNF580在ROS介导的内皮细胞炎症反应中的作用研究

发布时间：2018-05-26 12:22

本文选题：ZNF580 + H_2O_2　；参考：《河北医科大学》2011年硕士论文

【摘要】：ZNF580是由本课题组克隆的一种新的锌指蛋白基因,该基因定位于人19号染色体19q13.42,最初是以LDL诱导人脐静脉内皮细胞系ECV304,筛选人主动脉cDNA文库得到的新基因(Genbank注册号:AF184939),国际人类基因命名委员会命名为ZNF580。ZNF580与Krüppel样锌指转录因子(Krüppel-like factors, Klfs)家族成员类似,其功能仍在进一步研究中,本实验旨在探讨ZNF580在ROS介导的内皮细胞炎症反应中的信号转导机制。目的: 以人脐静脉内皮细胞株(EA.hy926)为体外模型,在典型的活性氧(reactive oxygen species, ROS)——H_2O_2的作用下,研究人C2H2型锌脂蛋白新基因ZNF580表达情况,分析ZNF580基因表达与氧化应激相关的核转录因子NF-κB转录激活之间的关系,以及ZNF580基因过表达对炎症因子IL-8的释放和单核细胞迁移趋化的影响,进一步揭示ZNF580在ROS介导的内皮细胞炎症反应中的作用。方法: 1人脐静脉内皮细胞株(EA.hy926),用含10%胎牛血清的高糖DMEM培养基在37℃、5% CO_2、饱和湿度下培养。 2 H_2O_2是一个细胞损伤的刺激因素,而LDH(乳酸脱氢酶)是反映细胞膜完整性的重要检测指标。为确定H_2O_2对内皮细胞的损伤情况,不同浓度H_2O_2(0-600μM)作用于EA.hy926细胞6h后,提取细胞上清,采用LDH活性检测分析H_2O_2对内皮细胞的损伤作用。 3胰酶消化收集对数生长期EA.hy926细胞,将其悬浮于高糖DMEM细胞培养液中,浓度约为5-6×106/ml,接种到六孔板中2 ml/孔,37℃,5%CO_2孵箱培养。在各孔内加H_2O_2 0μM、50μM、100μM、200μM、300μM、400μM,每组设三个平行孔。1h后收集细胞,提取总RNA和蛋白,利用半定量RT-PCR、Western blot方法检测不同浓度H_2O_2对ZNF580表达的影响。 4在各孔中加入浓度为100μM的H_2O_2,每组设三个平行孔,于0h、0.5h、1h、2h、4h、6h后收集细胞,提取总RNA和蛋白,同样利用半定量RT-PCR、Western blot方法检测不同作用时间的H_2O_2对ZNF580表达的影响。 5内皮细胞经不同处理后,实验分为对照组、H_2O_2处理组、PDTC干预组和DMSO对照组。各组细胞爬片后固定,免疫细胞化学检测H_2O_2和特异性抑制剂PDTC对NF-κB亚基p65激活表达的影响。 6同样EA.hy926细胞分为不同的处理方式,抽提RNA以及蛋白,利用半定量RT-PCR扩增和Western blot方法检测H_2O_2是否通过NF-κB信号通路影响和调节ZNF580的表达。 7利用pEGFP-ZNF580和pEGFP-C1瞬时转染EA.hy926内皮细胞,获得瞬时过表达ZNF580的EA.hy926细胞。运用倒置荧光显微镜观察转染效率,RT-PCR以及western-blot技术鉴定转染后ZNF580的表达情况。 8运用real-time PCR技术从转录水平检测构建的瞬时高表达ZNF580内皮细胞和正常内皮细胞中白介素-8的表达差异。以ELISA方法从蛋白表达水平检测构建的瞬时高表达ZNF580内皮细胞和正常内皮细胞中白介素-8的表达差异。 9人急性单核细胞白血病细胞株(THP-1),用含10%胎牛血清的RPMI-1640培养基在37℃、5% CO_2、饱和湿度下培养。迁移趋化实验分析瞬时高表达ZNF580内皮细胞和正常内皮细胞中IL-8对单核细胞株THP-1迁移率的影响。结果: 1不同浓度H_2O_2作用于EA.hy926细胞, LDH测定结果显示,H_2O_2浓度为400μM时,LDH的释放量最大。 2不同浓度H_2O_2作用EA.hy926内皮细胞1h后,提取RNA扩增ZNF580片段,以及western-blot观察ZNF580的蛋白表达水平,结果证实,ZNF580在mRNA转录水平和蛋白水平的表达均有增加,并呈现浓度依赖的趋势。(P0.05)。在浓度为100μM时,ZNF580的表达最高。 3浓度为100μM的H_2O_2作用EA.hy926内皮细胞不同时间后,由RT-PCR和western-blot检测可知ZNF580 mRNA和蛋白水平的表达增加(P0.05)。在刺激时间为1h时,ZNF580的表达最高。 4 H_2O_2作用可激活NF-κB亚基p65由细胞质转移至细胞核,而特异性抑制剂PDTC存在时,可抑制NF-κB亚基p65在EA.hy926细胞内的核转位。阴性对照DMSO未引起p65核转位。对照组可见棕色颗粒位于细胞质中,细胞核中未见。 5 NF-κB信号通路抑制剂PDTC和H_2O_2共同处理可抑制ZNF580 mRNA及蛋白的上调表达,而单独H_2O_2的处理可明显上调ZNF580 mRNA及蛋白的表达。 6脂质体瞬时转染ZNF580质粒于EA.hy926细胞中,成功构建瞬时高表达ZNF580的EA.hy926细荧光显微镜鉴定转染效率为50%左右,RT-PCR和western-blot鉴定结果显示:与转染pEGFP-C1的细胞相比,转染pEGFP-ZNF580的细胞ZNF580表达量显著升高(P0.01)。 7 Real-time PCR及ELISA分析结果表明,高表达ZNF580的EA.hy926细胞中IL-8的mRNA转录和蛋白表达显著增高。高表达ZNF580的EA.hy926细胞表达的IL-8显著高于正常细胞(P0.01)。 8 Transwell实验检测分析结果表明,高表达ZNF580的EA.hy926细胞对单核细胞THP-1迁移趋化作用有明显的促进作用,与正常组比较结果有统计学意义(P0.01)。结论: H_2O_2通过NF-κB信号通路影响ZNF580的表达,进一步促进其下游的炎症因子IL-8的释放,ZNF580在内皮细胞炎症反应中起着重要的作用。该研究阐明了一条与炎症发生相关的信号传导途径,为探讨人C2H2型锌脂蛋白新基因ZNF580功能及其与炎症发生之间的关系提供科学依据。
[Abstract]:ZNF580 is a new zinc finger protein gene cloned by our group. This gene is located on human chromosome 19 19q13.42. It was originally a new gene (Genbank registration number: AF184939) obtained by LDL induction of human umbilical vein endothelial cell line ECV304 (Genbank registration number: AF184939). The national human gene naming Committee was named ZNF580.ZNF580 and Kr u. The ppel like zinc finger transcription factor (Kr u ppel-like factors, Klfs) family is similar, and its function is still in further study. The aim of this experiment is to explore the signal transduction mechanism of ZNF580 in ROS mediated endothelial cell inflammatory response.
Objective:
The human umbilical vein endothelial cell line (EA.hy926) was used as an in vitro model to study the expression of ZNF580 in human C2H2 type zinc lipoprotein (reactive oxygen species, ROS), H_2O_2, and to analyze the relationship between the ZNF580 gene expression and the activation of the nuclear transcription factor NF- kappa B transcription related to oxidative stress, as well as ZNF580. The effect of gene overexpression on the release of inflammatory factor IL-8 and the migration and chemotaxis of monocyte, and further reveal the role of ZNF580 in ROS mediated endothelial cell inflammatory response.
Method:
1 human umbilical vein endothelial cell line (EA.hy926) was cultured with high glucose DMEM medium containing 10% fetal bovine serum at 37 degree, 5% CO_2, and saturated humidity.
2 H_2O_2 is a stimulating factor of cell damage, and LDH (lactate dehydrogenase) is an important indicator to reflect the integrity of cell membrane. In order to determine the damage of H_2O_2 to endothelial cells, different concentrations of H_2O_2 (0-600 mu M) act on EA.hy926 cell 6h, extract cell supernatant, and use LDH activity to detect the damage of H_2O_2 to endothelial cells. Use.
3 trypsin digestion and collection of EA.hy926 cells in the logarithmic growth period were suspended in high glucose DMEM cell culture solution, the concentration was about 5-6 x 106/ml, inoculated to 2 ml/ holes in the six pore plate, 37 C and incubated in 5%CO_2 incubator. The cells were added in each hole with H_2O_2 0 mu M, 50 Mu M, 100 u M, 200 mu M, 300 mu M, 400 mu M, and each set of three parallel holes collected cells and extracted total proteins and proteins. Semi quantitative RT-PCR and Western blot were used to detect the effect of different concentrations of H_2O_2 on ZNF580 expression.
4 H_2O_2 with a concentration of 100 M was added to each hole. Each group was set up with three parallel holes. After 0h, 0.5h, 1H, 2h, 4h, and 6h, the cells were collected, and the total RNA and protein were extracted.
5 the endothelial cells were treated with different treatments, including the control group, the H_2O_2 treatment group, the PDTC intervention group and the DMSO control group. The cells were fixed in each group after climbing the tablet. The effects of H_2O_2 and specific inhibitor PDTC on the activation and expression of NF- kappa B subunit p65 were detected by immunocytochemistry.
6 the same EA.hy926 cells were divided into different treatments, RNA and protein were extracted. Semi quantitative RT-PCR amplification and Western blot were used to detect whether H_2O_2 could affect and regulate the expression of ZNF580 through the NF- kappa B signaling pathway.
7 transient transfection of EA.hy926 endothelial cells by pEGFP-ZNF580 and pEGFP-C1 was used to obtain transient EA.hy926 cells expressing ZNF580. The transfection efficiency was observed by inverted fluorescence microscopy, and the expression of ZNF580 was identified by RT-PCR and Western-blot technology.
8 real-time PCR technique was used to detect the difference in the expression of interleukin -8 in transient high expression ZNF580 endothelial cells and normal endothelial cells from transcriptional level. The difference in the expression of interleukin -8 in transient high expression ZNF580 endothelial cells and normal endothelial cells was detected by ELISA method from protein expression level.
9 people with acute monocytic leukemia cell line (THP-1) were cultured with RPMI-1640 medium containing 10% fetal bovine serum at 37, 5% CO_2 and saturated humidity. Migration and chemotaxis experiments were conducted to analyze the effect of IL-8 on the mobility of THP-1 in the mononuclear cell line of transient high expression of ZNF580 endothelial cells and normal endothelial cells.
Result:
1 different concentrations of H_2O_2 acted on EA.hy926 cells. The results of LDH showed that LDH release was the largest when H_2O_2 concentration was 400 M.
2 after EA.hy926 endothelial cell 1H was treated with different concentrations of H_2O_2, the ZNF580 fragment was amplified by RNA and the protein expression level of ZNF580 was observed by Western-blot. The results showed that the expression of ZNF580 at mRNA transcriptional level and protein level increased, and showed a tendency of concentration dependence. (P0.05), the expression of ZNF580 was the highest when the concentration was 100 mu M.
The expression of ZNF580 mRNA and protein was increased by RT-PCR and Western-blot (P0.05) after 3 EA.hy926 endothelial cells with a concentration of 100 M, and the expression of ZNF580 mRNA and protein was increased (P0.05). The expression of ZNF580 was the highest when the time of stimulation was 1H.
The effect of 4 H_2O_2 can activate the NF- kappa B subunit p65 from the cytoplasm to the nucleus, while the specific inhibitor PDTC can inhibit the nuclear transposition of NF- kappa B subunit p65 in the EA.hy926 cell. Negative control DMSO does not cause the p65 nucleus transposition. The control group can not be found in the cytoplasm of the cytoplasm and the nucleus of the cell nucleus.
5 NF- kappa B signaling pathway inhibitor, PDTC and H_2O_2, can inhibit the up-regulated expression of ZNF580 mRNA and protein, while H_2O_2 alone can obviously increase the expression of ZNF580 mRNA and protein.
6 liposomes were transiently transfected into the ZNF580 plasmid in EA.hy926 cells. The EA.hy926 fine fluorescence microscope was successfully constructed to identify the transient high expression ZNF580. The transfection efficiency was about 50%. The results of RT-PCR and Western-blot identification showed that the expression of pEGFP-ZNF580 in pEGFP-ZNF580 was significantly higher than that of the transfected pEGFP-C1 cells (P0.01).
The results of 7 Real-time PCR and ELISA analysis showed that the mRNA transcriptional and protein expression of IL-8 in the EA.hy926 cells with high expression of ZNF580 was significantly higher. The IL-8 of EA.hy926 cells expressing ZNF580 was significantly higher than that of normal cells (P0.01).
The results of 8 Transwell test showed that the EA.hy926 cells with high expression of ZNF580 had a significant effect on the chemotaxis of THP-1 migration in mononuclear cells, and the results were statistically significant (P0.01) compared with the normal group.
Conclusion:
H_2O_2 affects the expression of ZNF580 through the NF- kappa B signaling pathway and further promotes the release of the inflammatory factor IL-8 in the lower reaches. ZNF580 plays an important role in the inflammatory response of endothelial cells. This study elucidated a signal transduction pathway associated with the occurrence of inflammation, in order to explore the ZNF580 function of human C2H2 type zinc lipoprotein (znlipoprotein) and its inflammation and inflammation. The relationship between occurrence provides a scientific basis.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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