本文選題：巨噬細(xì)胞　切入點：Pannexin1　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：動脈粥樣硬化(Atherosclerosis,AS)是一種由多種因素誘導(dǎo)的血管慢性炎癥疾病,在AS發(fā)生過程中,巨噬細(xì)胞吞噬脂質(zhì)轉(zhuǎn)變?yōu)榕菽?xì)胞,泡沫細(xì)胞堆積形成脂質(zhì)條紋,最后發(fā)展形成脂質(zhì)斑塊。斑塊破裂后形成血栓堵塞血管,導(dǎo)致缺血性中風(fēng)或心肌梗死,嚴(yán)重威脅人類健康。AS的發(fā)生發(fā)展過程中,脂質(zhì)代謝紊亂是重要的環(huán)節(jié)。肝X受體(Liver X Receptor,LXR)激活后可誘導(dǎo)細(xì)胞釋放腺嘌呤核苷三磷酸(Adenosine Triphosphate,ATP),且明顯減少AS斑塊的形成。但詳細(xì)的分子機制尚不明確。Pannexin1是六聚體形成的膜通道蛋白,其開放后可允許小分子通過,ATP即可經(jīng)由該通道釋放。但Pannexin1通道是否參與巨噬細(xì)胞吞噬脂質(zhì),從而進一步轉(zhuǎn)化為泡沫細(xì)胞未見報道。本研究檢測了Pannexin1通道對LXR介導(dǎo)THP-1巨噬細(xì)胞攝取氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)作用,并初步對其分子機制進行了探討。該研究為深入研究AS發(fā)生發(fā)展機制提供了新的思路。1.采用佛波酯(Phorbol 12-myristate 13-acetate,PMA)誘導(dǎo)人急性白血病單核細(xì)胞(THP-1)分化成為THP-1巨噬細(xì)胞,并經(jīng)細(xì)胞形態(tài)學(xué)及CD14m RNA的表達(dá)的檢測對細(xì)胞分化是否成功進行鑒定。2.采用RT-PCR檢測THP-1巨噬細(xì)胞中Pannexin1的m RNA的表達(dá)。3.采用Western Blot技術(shù)檢測LXR激動劑(GW3965)刺激前后THP-1巨噬細(xì)胞中LXRα和LXRβ的表達(dá)情況。4.采用熒光顯微鏡觀察LXR激動劑(GW3965)介導(dǎo)THP-1巨噬細(xì)胞攝取Dil-ox-LDL。通過加入Pannexin1通道抑制劑(10Panx1)前后觀察LXR激動劑(GW3965)介導(dǎo)THP-1巨噬細(xì)胞攝取Dil-ox-LDL的變化,使用圖像分析軟件(Image-pro plus v6.0)對熒光圖片進行分析,計算出對應(yīng)的細(xì)胞數(shù),用以衡量細(xì)胞攝取Dil-ox-LDL量的變化情況。5.采用螢光素酶法以及RT-PCR技術(shù)檢測應(yīng)用Pannexin1通道抑制劑(10Panx1)前后GW3965刺激細(xì)胞釋放ATP的變化情況以及ATP結(jié)合盒轉(zhuǎn)運子A1(ATP-Binding Cassette Transporters A1,ABCA1)的表達(dá)。結(jié)果:1.THP-1細(xì)胞經(jīng)160n M PMA誘導(dǎo)分化24小時后,細(xì)胞形態(tài)由分化前的圓形懸浮生長變?yōu)樗笮?并有大量的偽足出現(xiàn),且呈貼壁生長。RT-PCR檢測結(jié)果顯示,THP-1經(jīng)誘導(dǎo)分化為THP-1巨噬細(xì)胞后,THP-1巨噬細(xì)胞中CD14 m RNA表達(dá)水平明顯上調(diào),與THP-1細(xì)胞誘導(dǎo)前相比有顯著性差異(p0.001)。2.Western Blot檢測顯示LXR激動劑(GW3965)可以明顯誘導(dǎo)THP-1巨噬細(xì)胞LXRα和LXRβ的表達(dá)。使用GW3965處理THP-1巨噬細(xì)胞前后差異有統(tǒng)計學(xué)意義(p0.001)。3.THP-1巨噬細(xì)胞攝取Dil-ox-LDL結(jié)果顯示:以Pannexin1通道抑制劑10Panx1(200μM)預(yù)處理細(xì)胞30分鐘,再用GW3965刺激THP-1巨噬細(xì)胞4小時,結(jié)果發(fā)現(xiàn)抑制劑組可以減弱細(xì)胞對Dil-ox-LDL的攝取,與GW3965刺方法:激組相比有顯著性差異(p0.01)。4.THP-1巨噬細(xì)胞經(jīng)GW3965刺激不同時間(0min、5min、10min、20min、30min)后,GW3965刺激組各時間點細(xì)胞釋放的ATP含量較對照組顯著增加(p0.001),并且GW3965刺激10分鐘時達(dá)到高峰;當(dāng)使用200μM的Pannexin1通道特異性抑制(10Panx1)預(yù)處理細(xì)胞30分鐘,檢測結(jié)果顯示ATP的釋放量較GW3965刺激組明顯降低,差異有統(tǒng)計學(xué)意義(p0.01)。5.RT-PCR檢測顯示LXR激動劑(GW3965)可以誘導(dǎo)ABCA1的表達(dá),當(dāng)使用200μM的Pannexin1通道特異性抑制劑(10Panx1)預(yù)處理細(xì)胞30分鐘,可明顯地抑制GW3965誘導(dǎo)ABCA1的表達(dá),差異有統(tǒng)計學(xué)意義(p0.01)。結(jié)論:1.Pannexin1通道抑制劑(10Panx1)可抑制LXR激動劑(GW3965)介導(dǎo)的THP-1巨噬細(xì)胞釋放ATP的過程。2.Pannexin1通道抑制劑(10Panx1)對LXR激動劑(GW3965)誘導(dǎo)巨噬細(xì)胞攝取ox-LDL的能力起到抑制作用。3.Pannexin1通道抑制劑(10Panx1)對LXR激動劑(GW3965)誘導(dǎo)巨噬細(xì)胞ABCA1的表達(dá)起到抑制作用
[Abstract]:Atherosclerosis (Atherosclerosis, AS) is induced by a variety of factors of vascular chronic inflammatory diseases in the pathogenesis of AS, macrophage lipid into foam cells and foam cells accumulated lipid stripes, and finally form lipid plaque rupture after thrombosis. Plaque clogging blood vessels, leading to ischemic stroke or myocardial infarction, a serious threat human health in the occurrence and development of.AS in the process of lipid metabolism disorder is an important link. The liver X receptor (Liver X Receptor, LXR) after activation can induce cells to release adenosine phosphate (Adenosine three Triphosphate, ATP), and significantly reduce the formation of AS plaque. But the detailed molecular mechanism is not clear.Pannexin1 is six the membrane channel protein body formation, the opening can allow small molecules to pass through the channel, ATP can be released. But the Pannexin1 channel is involved in macrophage lipid Quality, thus further into foam cells has not been reported. This study examined the Pannexin1 channel guide THP-1 macrophage uptake of oxidized low density lipoprotein on LXR (oxidized low density lipoprotein, ox-LDL), and its preliminary molecular mechanism were discussed. The research provides a new idea of.1. by phorbol ester for further study on the mechanism of the occurrence and development of AS (Phorbol 12-myristate 13-acetate, PMA) in human acute monocytic leukemia cell (THP-1) differentiation into THP-1 macrophages, and detect the expression of cell morphology and CD14m RNA on the success of the expression of.3. cell differentiation and identification of.2. by M RNA Pannexin1 RT-PCR to detect THP-1 in macrophages by Western Blot detection technology LXR agonist (GW3965) before and after stimulation of THP-1 macrophages in LXR alpha and LXR beta.4. expression by fluorescence microscopy LXR agonists (GW39 65) THP-1 mediated by macrophage uptake of Dil-ox-LDL. by adding the Pannexin1 channel inhibitor (10Panx1) were observed before and after LXR agonist (GW3965) mediated changes in THP-1 macrophage uptake of Dil-ox-LDL, using image analysis software (Image-pro plus V6.0) on the fluorescence image analysis, calculate the number of cells corresponding, using luciferase method and RT-PCR technology the application of Pannexin1 inhibitors for detecting changes of.5. to measure the cellular uptake amount of Dil-ox-LDL (10Panx1) before and after the change of stimulated GW3965 cells to release ATP and ATP binding cassette transporter A1 (ATP-Binding Cassette Transporters A1, ABCA1). Results: the expression of 1.THP-1 cells by 160n M PMA induced differentiation after 24 hours, the cell morphology by circular the growth of suspension before differentiation into the spindle, and a large number of pseudopodia, and were adherent.RT-PCR test results show that the differentiation of THP-1 THP-1 was significantly up-regulated after macrophage, CD14 m and RNA THP-1 expression in macrophages, there was significant difference compared with THP-1 cells before induction (p0.001).2.Western Blot assay showed that LXR agonist (GW3965) can induce THP-1 expression of macrophage LXR alpha and LXR beta. THP-1 macrophages treated with GW3965 before and after the difference was statistically significant (p0.001).3.THP-1 macrophages Dil-ox-LDL results showed that the 10Panx1 inhibitor Pannexin1 (200 M) cells pretreated with 30 minutes, 4 hours of stimulation with GW3965 THP-1 macrophages, the inhibitor group can reduce the cellular uptake of Dil-ox-LDL, GW3965 and needling method: shock group showed a significant difference (P0.01) in.4.THP-1 macrophages by different the stimulation of GW3965 (0min, 5min, time 10min, 20min, 30min), ATP content in GW3965 stimulation group at each time point of the release of cells increased significantly compared with the control group (P0 .001), and GW3965 reached the peak at 10 minutes after Pannexin1 stimulation; the channel specificity using 200 M suppression (10Panx1) cells pretreated with 30 minutes, test results showed that the release amount of ATP with GW3965 stimulation group decreased significantly, the difference was statistically significant (P0.01).5.RT-PCR test showed LXR agonist (GW3965) expression can be induced by ABCA1, a specific inhibitor of Pannexin1 channel when using 200 M (10Panx1) cells were pretreated for 30 minutes, the expression inhibited GW3965 induced by ABCA1, the difference was statistically significant (P0.01). Conclusion: 1.Pannexin1 channel inhibitor (10Panx1) can inhibit LXR agonist (GW3965) inhibitors of.2.Pannexin1 channel mediated process the THP-1 macrophage ATP release (10Panx1) of LXR agonist (GW3965) induced by macrophage uptake of ox-LDL to inhibit the.3.Pannexin1 channel inhibitor (10Panx1) on LXR (GW3965) induced by agonist The expression of ABCA1 in macrophage plays an inhibitory role

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R543.5

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本文編號：1560582