本文選題：缺糖缺氧 + 小膠質(zhì)細(xì)胞��； 參考：《浙江大學(xué)》2014年碩士論文

【摘要】：研究背景： 前B細(xì)胞克隆增強(qiáng)因子(Pre-B-cell colony-enhancing factor, PBEF)又被稱為內(nèi)臟脂肪素(visfatin)和尼克酰胺磷酸核糖轉(zhuǎn)移酶(Nicotinamide Phosphoribosyltransferase, Nampt). PBEF因具有細(xì)胞因子、脂肪因子、酶等多種作用參與機(jī)體的一系列生命活動(dòng)過程而受到廣泛的關(guān)注。 研究發(fā)現(xiàn), PBEF在體內(nèi)廣泛表達(dá),并可被多種細(xì)胞釋放,且PBEF的釋放是細(xì)胞類型依賴的。在外周,PBEF作為一種新型的炎癥介質(zhì),被淋巴細(xì)胞、單核細(xì)胞、巨噬細(xì)胞等外周炎癥細(xì)胞釋放。在中樞,PBEF主要表達(dá)于神經(jīng)元和血管內(nèi)皮細(xì)胞,膠質(zhì)細(xì)胞不表達(dá)PBEF;大鼠腦缺血后,星形膠質(zhì)細(xì)胞仍無PBEF表達(dá),但在腦缺血亞急性期小膠質(zhì)細(xì)胞可大量表達(dá)PBEF。小膠質(zhì)細(xì)胞是腦內(nèi)介導(dǎo)免疫、炎癥反應(yīng)最主要的效應(yīng)細(xì)胞,其對(duì)中樞神經(jīng)系統(tǒng)起免疫監(jiān)視的作用,且小膠質(zhì)細(xì)胞具有與單核細(xì)胞、巨噬細(xì)胞相同的來源。因而,我們推測(cè)在一定條件下小膠質(zhì)細(xì)胞可誘導(dǎo)表達(dá)并釋放PBEF,而小膠質(zhì)細(xì)胞釋放的PBEF作為腦內(nèi)新型的炎癥介質(zhì)可促進(jìn)和／或維持中樞炎癥。 然而,目前PBEF的釋放機(jī)制仍未闡明。研究發(fā)現(xiàn),PBEF缺少一般分泌蛋白具備的N端出胞信號(hào)肽序列,其釋放不遵循經(jīng)典的內(nèi)質(zhì)網(wǎng)-高爾基體依賴的釋放途徑。因而,本課題關(guān)注以下問題：1)小膠質(zhì)細(xì)胞能否誘導(dǎo)表達(dá)并釋放PBEF?2)小膠質(zhì)細(xì)胞釋放PBEF涉及哪些途徑?3)小膠質(zhì)細(xì)胞釋放PBEF受哪些因素的調(diào)節(jié)? 研究目的： 觀察缺糖缺氧(oxygen-glucose deprivation/recovery, OGD/R)誘導(dǎo)小鼠小膠質(zhì)細(xì)胞株BV2細(xì)胞表達(dá)并釋放新型炎癥因子PBEF的作用,并初步探討B(tài)V2細(xì)胞釋放PBEF的途徑及影響因素。 研究方法： BV2細(xì)胞經(jīng)缺糖缺氧恢復(fù)(oxygen-glucose deprivation/recovery, OGD/R)模擬腦缺血處理后,分別以western blotting和ELISA檢測(cè)PBEF在BV2細(xì)胞內(nèi)的表達(dá)和PBEF的釋放,RT-PCR檢測(cè)PBEF mRNA表達(dá)變化；以經(jīng)典的內(nèi)質(zhì)網(wǎng)-高爾基體依賴的蛋白分泌途徑抑制劑及非經(jīng)典的蛋白分泌途徑抑制劑預(yù)處理BV2細(xì)胞后,再以O(shè)GD1h/R12h處理細(xì)胞,初步分析BV2細(xì)胞釋放PBEF所涉及的途徑；以免疫熒光染色確定PBEF在BV2細(xì)胞內(nèi)的分布,并以同樣方法確定表達(dá)了熒光蛋白特異標(biāo)記的溶酶體(Lamp1)、線粒體(MitoRed)、自噬體(LC3)的BV2細(xì)胞在OGD/R前后,PBEF與各細(xì)胞器的共定位情況,從而進(jìn)一步確證PBEF的釋放途徑；最后,分析常見的影響細(xì)胞胞吐的因素(ATP、鈣離子濃度)對(duì)OGD/R誘導(dǎo)BV2細(xì)胞釋放PBEF的影響。 研究結(jié)果： 第一部分缺糖缺氧可誘導(dǎo)BV2細(xì)胞釋放PBEF OGD1小時(shí)／恢復(fù)12-24小時(shí)(OGD1h/R12-24h),可誘導(dǎo)BV2細(xì)胞釋放PBEF明顯增高,并伴有細(xì)胞內(nèi)PBEF的蛋白量的下降,PBEF的mRNA表達(dá)上調(diào)；OGD/R誘導(dǎo)的PBEF的釋放不受經(jīng)典的蛋白分泌途徑抑制劑BFA、自噬抑制劑3-MA的影響,而NH4CI處理、ABCA1轉(zhuǎn)運(yùn)體抑制劑Glyburide及DIDS均可劑量依賴性抑制PBEF的釋放；免疫熒光染色結(jié)果表明,PBEF主要分布于BV2細(xì)胞胞漿,核內(nèi)也有少量分布,PBEF與溶酶體、線粒體有共定位,OGD/R處理后,PBEF向胞膜聚集；外鈣內(nèi)流及胞外ATP濃度升高能進(jìn)一步增強(qiáng)OGD/R誘導(dǎo)的PBEF釋放,且P2X7受體抑制劑BBG, PPADS可劑量依賴性抑制OGD/R誘導(dǎo)的PBEF釋放而ATP降解酶Apyrase可逆轉(zhuǎn)這一過程。 第二部分缺糖缺氧誘導(dǎo)BV2細(xì)胞釋放PBEF的過程中伴有其他細(xì)胞因子的釋放 缺糖缺氧1h/2h/4h恢復(fù)不同時(shí)間,隨著時(shí)間延長,BV2細(xì)胞釋放PBEF增多,并伴隨有TNF-α、IL-6等細(xì)胞因子的釋放。這提示細(xì)胞釋放的PBEF與其他炎癥細(xì)胞因子之間存在相互作用的可能。 第三部分缺糖缺氧誘導(dǎo)BV2細(xì)胞釋放的PBEF對(duì)BV2細(xì)胞本身活性的影響 正常條件下,胞外的PBEF對(duì)BV2細(xì)胞活性無明顯影響,但在缺糖缺氧條件下,胞外PBEF濃度升高可逆轉(zhuǎn)缺糖缺氧導(dǎo)致的BV2細(xì)胞活性的下降,且PBEF濃度越高保護(hù)作用越明顯。 結(jié)論： 1.缺糖缺氧可誘導(dǎo)BV2細(xì)胞釋放PBEF；BV2細(xì)胞釋放PBEF部分通過ABCA1轉(zhuǎn)運(yùn)體、溶酶體途徑,外鈣內(nèi)流及胞外ATP濃度增高可增強(qiáng)OGD/R誘導(dǎo)的PBEF釋放,且P2X7受體參與調(diào)節(jié)這一過程。 2.缺糖缺氧誘導(dǎo)BV2細(xì)胞釋放PBEF及其他炎癥因子,提示PBEF與其他炎癥因子之間存在相互作用的可能。 3.缺糖缺氧條件下,胞外PBEF對(duì)BV2細(xì)胞具有保護(hù)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳軍;王愛桃;閔U，

本文編號(hào)：1977119