本文關(guān)鍵詞：大鼠肺泡巨噬細(xì)胞ERG鉀通道的表達(dá)及其功能的初步研究　出處：《第三軍醫(yī)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】： ERG(ether-a-go-go-related gene)鉀通道屬于電壓依賴性鉀離子通道,在人心肌和腦組織中高表達(dá)。越來越多的報(bào)道發(fā)現(xiàn)該通道與多種病理過程相關(guān),如心律失常、腫瘤等,因此成為近年研究的熱點(diǎn)。新近的研究表明,骨髓來源的中樞系統(tǒng)單核-巨噬類細(xì)胞——小膠質(zhì)細(xì)胞上存在ERG鉀通道。但是,ERG鉀通道是否存在于肺泡單核-巨噬細(xì)胞,若存在表達(dá),其功能如何?與肺泡巨噬細(xì)胞的分泌及吞噬功能有何相關(guān),目前均不清楚。探討肺泡巨噬細(xì)胞ERG鉀通道的表達(dá)及功能,可為急性肺損傷機(jī)制的研究以及治療提供新的思路。 目的: 1.觀察肺泡巨噬細(xì)胞上是否有ERG鉀通道的表達(dá)。 2.初步探討ERG鉀通道與該細(xì)胞的分泌及吞噬功能是否相關(guān)。 方法 1.大鼠肺泡巨噬細(xì)胞ERG鉀通道的表達(dá) 以大鼠肺泡巨噬細(xì)胞系NR8383為研究對象,人肺腺癌細(xì)胞系A(chǔ)549為陰性對照,采用RT-PCR方法檢測NR8383細(xì)胞erg鉀通道m(xù)RNA的表達(dá),RT-PCR產(chǎn)物進(jìn)行cDNA測序驗(yàn)證;免疫細(xì)胞化學(xué)方法和Western blot檢測NR8383細(xì)胞膜上ERG鉀通道蛋白的表達(dá)。 2. LPS刺激后大鼠肺泡巨噬細(xì)胞系NR8383細(xì)胞erg mRNA和蛋白的表達(dá)變化 將培養(yǎng)24h的NR8383細(xì)胞系,加入LPS 1ug/ml刺激(正常對照組細(xì)胞不加),分別于3h、6h、12h、24h提取細(xì)胞總RNA和總蛋白采用RT-PCR法和Western-blot法檢測ERG鉀通道表達(dá)的變化。 3.阻斷ERG鉀通道后大鼠肺泡巨噬細(xì)胞吞噬和分泌功能的變化 將細(xì)胞分為正常對照組、4-AP阻斷組、TEA阻斷組、E-4031阻斷組。后三組先以相應(yīng)濃度(分別為1mM、mM、5μM)作用于細(xì)胞,阻斷鉀通道,2h后和正常對照組一起加入制備好的5%雞紅細(xì)胞,使雞紅細(xì)胞與大鼠肺泡巨噬細(xì)胞的比例大概為100:1,置于培養(yǎng)箱中孵育1h。之后用4%的多聚甲醛固定,再行姬姆薩染色,油鏡下觀察100個(gè)AM,計(jì)算吞噬率。吞噬率=已吞噬的巨噬細(xì)胞數(shù)/巨噬細(xì)胞總數(shù)×100%。放射免疫法檢測阻斷ERG鉀通道后LPS刺激肺泡巨噬細(xì)胞分泌炎癥因子IL-6的變化,將細(xì)胞分為PBS對照組、LPS組、4-AP+LPS組(非特異阻斷組)、TEA+LPS組(非特異阻斷組)、E-4031(1μM)+LPS組(特異阻斷組)、E-4031(5μM)+LPS組(特異阻斷組);每組設(shè)6個(gè)孔。阻斷鉀通道2h后與LPS組一起加LPS 1ug/ml刺激24h。24h后收集上清,采用放射免疫法檢測各組上清液中IL-6的分泌量。 結(jié)果 1. RT-PCR及測序結(jié)果表明,檢測到480bp左右的片段(與預(yù)期的一致),表明NR8383細(xì)胞有erg mRNA表達(dá),陰性對照A549細(xì)胞未見erg mRNA表達(dá)。經(jīng)cDNA測序進(jìn)一步證實(shí)其序列與數(shù)據(jù)庫中序列完全吻合。免疫細(xì)胞化學(xué)染色顯示,大量棕黃色陽性顆粒分布在NR8383細(xì)胞膜上,表明NR8383細(xì)胞膜上有ERG鉀通道蛋白表達(dá),而陰性對照的A549細(xì)胞膜上未見棕黃色顆粒。Western blot可以在NR8383細(xì)胞蛋白提取物中檢測到155kDa和135kDa兩條特異的ERG蛋白條帶,而陰性對照A549細(xì)胞未檢測到相應(yīng)條帶。 2. LPS刺激肺泡巨噬細(xì)胞后,ERG鉀通道m(xù)RNA水平和蛋白水平的表達(dá)變化 (1)半定量RT-PCR檢測不同時(shí)間點(diǎn)LPS刺激后erg mRNA表達(dá)水平的改變,結(jié)果顯示:與未經(jīng)LPS刺激的AM細(xì)胞相比,經(jīng)LPS刺激后6h組erg mRNA表達(dá)明顯增強(qiáng),呈明顯時(shí)間依賴性增加,至所觀察的24h組達(dá)到最高,為對照組的2倍(P0.05)。 (2)通過Western blot檢測LPS刺激后不同時(shí)間ERG鉀通道蛋白表達(dá)水平改變,結(jié)果顯示:與未經(jīng)LPS刺激的AM細(xì)胞相比,LPS刺激6h后顯著增強(qiáng),至所觀察的24h組達(dá)到最高,呈時(shí)間依賴性增加,為對照組的2倍(P0.05)。 3. ERG鉀通道對大鼠肺泡巨噬細(xì)胞吞噬和分泌功能的影響 大鼠肺泡巨噬細(xì)胞吞噬實(shí)驗(yàn)結(jié)果顯示,與不加ERG鉀通道阻斷劑的對照組相比,加入特異性阻斷劑E4031及非特異阻斷劑4AP和TEA的吞噬率顯著下降(P0.01),而特異阻斷組和非特異阻斷組之間的吞噬率無顯著差異,提示ERG鉀通道參與了AM細(xì)胞的吞噬功能。ERG鉀通道對大鼠肺泡巨噬細(xì)胞炎癥因子分泌功能的影響,先用特異阻斷劑和非特異阻斷劑阻斷鉀通道,再用LPS刺激AM一定時(shí)間后收集上清檢測IL-6的分泌功能,結(jié)果顯示:與未加阻斷劑的對照細(xì)胞相比,經(jīng)特異性阻斷劑E4031阻斷ERG鉀通道后,AM細(xì)胞分泌IL-6的量顯著下降(P0.01),但小劑量(1μM)并無顯著影響;而使用非特異性阻斷劑4-AP和TEA阻斷后,同樣能夠抑制LPS刺激的IL-6的分泌,而二者之間沒有顯著差異。以上結(jié)果提示:ERG鉀通道參與了AM細(xì)胞的分泌功能。 結(jié)論 1.大鼠肺泡巨噬細(xì)胞系NR8383存在erg鉀通道m(xù)RNA,蛋白表達(dá)定位于細(xì)胞膜;LPS刺激NR8383細(xì)胞后,erg鉀通道m(xù)RNA和蛋白水平呈時(shí)間依賴性升高。 2. ERG鉀通道可能與NR8383細(xì)胞的吞噬與分泌功能有關(guān),特異性及非特異性阻斷劑可降低LPS引起的IL-6分泌和細(xì)胞吞噬功能。
[Abstract]:ERG (ether-a-go-go-related gene) potassium channels belong to voltage dependent potassium channel, high expression in human myocardium and brain tissues. More and more reports found the channel with a variety of pathological processes, such as arrhythmia, tumor, therefore become a research hotspot in recent years. Research on recent shows that ERG potassium channel exists in central nervous system bone marrow derived mononuclear macrophage cells, microglia. However, the existence of ERG potassium channel in alveolar macrophages, if there is expression, its function and secretion and phagocytosis? What can the relevant alveolar macrophages, are currently unknown. To investigate the expression and function of ERG potassium in alveolar macrophages the channel, to provide new ideas for the research on the mechanism of acute lung injury and treatment.
Objective:
1. the expression of ERG potassium channel was observed on alveolar macrophages.
2. preliminary study on whether the potassium channel of ERG is related to the secretion and phagocytosis of the cell.
Method
Expression of ERG potassium channel in alveolar macrophages of 1. rats
The rat alveolar macrophage cell line NR8383 as the research object, the human lung adenocarcinoma cell line A549 as negative control, RT-PCR was used to detect the expression of NR8383 cell ERG potassium channel mRNA, RT-PCR products were verified by cDNA sequencing; expression of ERG potassium channel protein of NR8383 cell membrane immunocytochemical method and Western blot detection.
Changes in expression of ERG mRNA and protein in NR8383 cells of alveolar macrophage system of rats after 2. LPS stimulation
The NR8383 cell line of 24h was added to LPS 1ug/ml stimulation (normal control group). The total RNA and total protein were extracted from 3h, 6h, 12h and 24h respectively. The expression of potassium channel was detected by RT-PCR method and Western-blot method.
3. changes of phagocytic and secretory function of alveolar macrophages in rats after blocking ERG potassium channel
灝嗙粏鑳?yōu)鍒嗕负姝ｅ父瀵圭収缁?4-AP闃繪柇緇，

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