缺氧缺糖诱导小鼠小胶质细胞株BV2细胞释放前B细胞克隆增强因子PBEF

发布时间：2018-06-04 11:01

本文选题：缺糖缺氧 + 小胶质细胞　；参考：《浙江大学》2014年硕士论文

【摘要】：研究背景：前B细胞克隆增强因子(Pre-B-cell colony-enhancing factor, PBEF)又被称为内脏脂肪素(visfatin)和尼克酰胺磷酸核糖转移酶(Nicotinamide Phosphoribosyltransferase, Nampt). PBEF因具有细胞因子、脂肪因子、酶等多种作用参与机体的一系列生命活动过程而受到广泛的关注。研究发现, PBEF在体内广泛表达,并可被多种细胞释放,且PBEF的释放是细胞类型依赖的。在外周,PBEF作为一种新型的炎症介质,被淋巴细胞、单核细胞、巨噬细胞等外周炎症细胞释放。在中枢,PBEF主要表达于神经元和血管内皮细胞,胶质细胞不表达PBEF;大鼠脑缺血后,星形胶质细胞仍无PBEF表达,但在脑缺血亚急性期小胶质细胞可大量表达PBEF。小胶质细胞是脑内介导免疫、炎症反应最主要的效应细胞,其对中枢神经系统起免疫监视的作用,且小胶质细胞具有与单核细胞、巨噬细胞相同的来源。因而,我们推测在一定条件下小胶质细胞可诱导表达并释放PBEF,而小胶质细胞释放的PBEF作为脑内新型的炎症介质可促进和／或维持中枢炎症。然而,目前PBEF的释放机制仍未阐明。研究发现,PBEF缺少一般分泌蛋白具备的N端出胞信号肽序列,其释放不遵循经典的内质网-高尔基体依赖的释放途径。因而,本课题关注以下问题：1)小胶质细胞能否诱导表达并释放PBEF?2)小胶质细胞释放PBEF涉及哪些途径?3)小胶质细胞释放PBEF受哪些因素的调节? 研究目的：观察缺糖缺氧(oxygen-glucose deprivation/recovery, OGD/R)诱导小鼠小胶质细胞株BV2细胞表达并释放新型炎症因子PBEF的作用,并初步探讨BV2细胞释放PBEF的途径及影响因素。研究方法： BV2细胞经缺糖缺氧恢复(oxygen-glucose deprivation/recovery, OGD/R)模拟脑缺血处理后,分别以western blotting和ELISA检测PBEF在BV2细胞内的表达和PBEF的释放,RT-PCR检测PBEF mRNA表达变化；以经典的内质网-高尔基体依赖的蛋白分泌途径抑制剂及非经典的蛋白分泌途径抑制剂预处理BV2细胞后,再以OGD1h/R12h处理细胞,初步分析BV2细胞释放PBEF所涉及的途径；以免疫荧光染色确定PBEF在BV2细胞内的分布,并以同样方法确定表达了荧光蛋白特异标记的溶酶体(Lamp1)、线粒体(MitoRed)、自噬体(LC3)的BV2细胞在OGD/R前后,PBEF与各细胞器的共定位情况,从而进一步确证PBEF的释放途径；最后,分析常见的影响细胞胞吐的因素(ATP、钙离子浓度)对OGD/R诱导BV2细胞释放PBEF的影响。研究结果：第一部分缺糖缺氧可诱导BV2细胞释放PBEF OGD1小时／恢复12-24小时(OGD1h/R12-24h),可诱导BV2细胞释放PBEF明显增高,并伴有细胞内PBEF的蛋白量的下降,PBEF的mRNA表达上调；OGD/R诱导的PBEF的释放不受经典的蛋白分泌途径抑制剂BFA、自噬抑制剂3-MA的影响,而NH4CI处理、ABCA1转运体抑制剂Glyburide及DIDS均可剂量依赖性抑制PBEF的释放；免疫荧光染色结果表明,PBEF主要分布于BV2细胞胞浆,核内也有少量分布,PBEF与溶酶体、线粒体有共定位,OGD/R处理后,PBEF向胞膜聚集；外钙内流及胞外ATP浓度升高能进一步增强OGD/R诱导的PBEF释放,且P2X7受体抑制剂BBG, PPADS可剂量依赖性抑制OGD/R诱导的PBEF释放而ATP降解酶Apyrase可逆转这一过程。第二部分缺糖缺氧诱导BV2细胞释放PBEF的过程中伴有其他细胞因子的释放缺糖缺氧1h/2h/4h恢复不同时间,随着时间延长,BV2细胞释放PBEF增多,并伴随有TNF-α、IL-6等细胞因子的释放。这提示细胞释放的PBEF与其他炎症细胞因子之间存在相互作用的可能。第三部分缺糖缺氧诱导BV2细胞释放的PBEF对BV2细胞本身活性的影响正常条件下,胞外的PBEF对BV2细胞活性无明显影响,但在缺糖缺氧条件下,胞外PBEF浓度升高可逆转缺糖缺氧导致的BV2细胞活性的下降,且PBEF浓度越高保护作用越明显。结论： 1.缺糖缺氧可诱导BV2细胞释放PBEF；BV2细胞释放PBEF部分通过ABCA1转运体、溶酶体途径,外钙内流及胞外ATP浓度增高可增强OGD/R诱导的PBEF释放,且P2X7受体参与调节这一过程。 2.缺糖缺氧诱导BV2细胞释放PBEF及其他炎症因子,提示PBEF与其他炎症因子之间存在相互作用的可能。 3.缺糖缺氧条件下,胞外PBEF对BV2细胞具有保护作用。
[Abstract]:Research background:
Pre-B-cell colony-enhancing factor (PBEF) is also known as visceral fatty acid (visfatin) and nicotinamide phosphate ribose transferase (Nicotinamide Phosphoribosyltransferase, Nampt). PBEF is involved in a series of biological activities such as cytokines, fat factors and enzymes involved in a series of life activities in the body. To a wide range of concerns.
It has been found that PBEF is widely expressed in the body and can be released by a variety of cells, and the release of PBEF is dependent on cell types. In the peripheral, PBEF is released as a new type of inflammatory mediator, and is released by lymphocytes, monocytes, macrophages and other peripheral inflammatory cells. In the center, PBEF is mainly expressed in neurons and vascular endothelial cells and glia cells. There is no expression of PBEF, but there is still no PBEF expression in astrocytes after cerebral ischemia in rats, but in the subacute phase of cerebral ischemia, microglia can express PBEF. microglia as the most important effector cell in the brain, and it plays an immune monitoring role in the central nervous system, and microglia has a monocytic and macrophage. Therefore, we speculate that microglia can induce the expression and release of PBEF under certain conditions, and the PBEF released by microglia can promote and / or maintain central inflammation as a new type of inflammatory mediator in the brain.
However, the release mechanism of PBEF has not yet been clarified. The study found that PBEF lacks the sequence of N endocytosis with the general secretory protein, and its release does not follow the classical endoplasmic reticulum - Golgi dependent release pathway. Therefore, this topic focuses on the following questions: 1) whether microglia can induce expression and release PBEF? 2) microglia release What are the ways to release PBEF? 3) what are the factors regulating the release of PBEF from microglia?
The purpose of the study is:
To observe the effect of oxygen-glucose deprivation/recovery (OGD/R) on the expression of BV2 cells in mouse microglia and release of a new type of inflammatory factor PBEF, and to explore the pathway and influencing factors of PBEF release from BV2 cells.
Research methods:
After the simulated ischemic treatment of BV2 cells (oxygen-glucose deprivation/recovery, OGD/R), Western blotting and ELISA were used to detect the expression of PBEF in BV2 cells and the release of PBEF, RT-PCR detection of the expression of PBEF, and the classical endoplasmic reticulum - high matrix dependent protein secretion pathway inhibitors and non - Classics After pretreating BV2 cells by the protein secretory pathway inhibitor, the cells were treated with OGD1h/R12h, and the pathway involved in the release of PBEF by BV2 cells was preliminarily analyzed. The distribution of PBEF in BV2 cells was determined by immunofluorescence staining, and the lysosomes (Lamp1), mitochondria (MitoRed), autophagosomes (L), and autophagosomes (L), were determined by the same method. The co localization of BV2 cells before and after OGD/R, the co localization of PBEF and various organelles, further confirms the release pathway of PBEF; finally, analysis of the common factors affecting cell exocytosis (ATP, calcium concentration) influence the release of PBEF in BV2 cells induced by OGD/R.
The results of the study:
The first part is lack of glucose and hypoxia to induce BV2 cells to release PBEF.
OGD1 hours / recovery 12-24 hours (OGD1h/R12-24h) could induce a significant increase in the release of PBEF in BV2 cells, accompanied by a decrease in the protein content of PBEF in the cell and the up-regulated expression of mRNA in PBEF, and the release of PBEF induced by OGD/R is not affected by the classical protein secretory inhibitor BFA and the 3-MA of the autophagy inhibitor. Yburide and DIDS could inhibit the release of PBEF in a dose-dependent manner, and the results of immunofluorescence staining showed that PBEF was mainly distributed in the cytoplasm of BV2 cells, and there was a small amount of distribution in the nucleus. PBEF and lysosomes were Co located. After OGD/R treatment, PBEF was aggregated to the membrane, and the increase of external calcium influx and extracellular ATP concentration could further enhance OGD/R induced PBEF release. Moreover, P2X7 receptor inhibitor BBG and PPADS can inhibit OGD/R induced PBEF release in a dose-dependent manner. ATP degradation enzyme Apyrase can reverse this process.
The second part is the release of PBEF from BV2 cells induced by lack of glucose and hypoxia, accompanied by the release of other cytokines.
As time prolongs, the release of PBEF in BV2 cells increases with time, and the release of cytokines, such as TNF- a, IL-6, and other cytokines, may be associated with the interaction between PBEF and other inflammatory cytokines released by the cells.
The third part is the effect of glucose deprivation induced PBEF release from BV2 cells on the activity of BV2 cells.
Under normal conditions, the extracellular PBEF has no obvious effect on the activity of BV2 cells. But under the condition of lack of glucose and hypoxia, the elevation of the extracellular PBEF concentration can reverse the decrease of BV2 cell activity caused by the lack of glucose and hypoxia, and the higher the concentration of PBEF, the more protective effect is.
Conclusion:
1. anoxic oxygen deficiency can induce BV2 cells to release PBEF; BV2 cells release PBEF part through ABCA1 transporter, lysosome pathway, external calcium influx and increased extracellular ATP concentration can enhance the OGD/R induced PBEF release, and P2X7 receptor participates in the regulation of this process.
2. glucose deprivation induced BV2 cells to release PBEF and other inflammatory factors, suggesting that PBEF may interact with other inflammatory factors.
3. under the condition of lack of glucose and hypoxia, extracellular PBEF plays a protective role in BV2 cells.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【共引文献】