Pannexin1对LXR介导的巨噬细胞吞噬ox-LDL的影响及分子机制的初探

发布时间：2018-03-03 10:09

本文选题：巨噬细胞　切入点：Pannexin1　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：动脉粥样硬化(Atherosclerosis,AS)是一种由多种因素诱导的血管慢性炎症疾病,在AS发生过程中,巨噬细胞吞噬脂质转变为泡沫细胞,泡沫细胞堆积形成脂质条纹,最后发展形成脂质斑块。斑块破裂后形成血栓堵塞血管,导致缺血性中风或心肌梗死,严重威胁人类健康。AS的发生发展过程中,脂质代谢紊乱是重要的环节。肝X受体(Liver X Receptor,LXR)激活后可诱导细胞释放腺嘌呤核苷三磷酸(Adenosine Triphosphate,ATP),且明显减少AS斑块的形成。但详细的分子机制尚不明确。Pannexin1是六聚体形成的膜通道蛋白,其开放后可允许小分子通过,ATP即可经由该通道释放。但Pannexin1通道是否参与巨噬细胞吞噬脂质,从而进一步转化为泡沫细胞未见报道。本研究检测了Pannexin1通道对LXR介导THP-1巨噬细胞摄取氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)作用,并初步对其分子机制进行了探讨。该研究为深入研究AS发生发展机制提供了新的思路。1.采用佛波酯(Phorbol 12-myristate 13-acetate,PMA)诱导人急性白血病单核细胞(THP-1)分化成为THP-1巨噬细胞,并经细胞形态学及CD14m RNA的表达的检测对细胞分化是否成功进行鉴定。2.采用RT-PCR检测THP-1巨噬细胞中Pannexin1的m RNA的表达。3.采用Western Blot技术检测LXR激动剂(GW3965)刺激前后THP-1巨噬细胞中LXRα和LXRβ的表达情况。4.采用荧光显微镜观察LXR激动剂(GW3965)介导THP-1巨噬细胞摄取Dil-ox-LDL。通过加入Pannexin1通道抑制剂(10Panx1)前后观察LXR激动剂(GW3965)介导THP-1巨噬细胞摄取Dil-ox-LDL的变化,使用图像分析软件(Image-pro plus v6.0)对荧光图片进行分析,计算出对应的细胞数,用以衡量细胞摄取Dil-ox-LDL量的变化情况。5.采用萤光素酶法以及RT-PCR技术检测应用Pannexin1通道抑制剂(10Panx1)前后GW3965刺激细胞释放ATP的变化情况以及ATP结合盒转运子A1(ATP-Binding Cassette Transporters A1,ABCA1)的表达。结果:1.THP-1细胞经160n M PMA诱导分化24小时后,细胞形态由分化前的圆形悬浮生长变为梭型,并有大量的伪足出现,且呈贴壁生长。RT-PCR检测结果显示,THP-1经诱导分化为THP-1巨噬细胞后,THP-1巨噬细胞中CD14 m RNA表达水平明显上调,与THP-1细胞诱导前相比有显著性差异(p0.001)。2.Western Blot检测显示LXR激动剂(GW3965)可以明显诱导THP-1巨噬细胞LXRα和LXRβ的表达。使用GW3965处理THP-1巨噬细胞前后差异有统计学意义(p0.001)。3.THP-1巨噬细胞摄取Dil-ox-LDL结果显示:以Pannexin1通道抑制剂10Panx1(200μM)预处理细胞30分钟,再用GW3965刺激THP-1巨噬细胞4小时,结果发现抑制剂组可以减弱细胞对Dil-ox-LDL的摄取,与GW3965刺方法:激组相比有显著性差异(p0.01)。4.THP-1巨噬细胞经GW3965刺激不同时间(0min、5min、10min、20min、30min)后,GW3965刺激组各时间点细胞释放的ATP含量较对照组显著增加(p0.001),并且GW3965刺激10分钟时达到高峰;当使用200μM的Pannexin1通道特异性抑制(10Panx1)预处理细胞30分钟,检测结果显示ATP的释放量较GW3965刺激组明显降低,差异有统计学意义(p0.01)。5.RT-PCR检测显示LXR激动剂(GW3965)可以诱导ABCA1的表达,当使用200μM的Pannexin1通道特异性抑制剂(10Panx1)预处理细胞30分钟,可明显地抑制GW3965诱导ABCA1的表达,差异有统计学意义(p0.01)。结论:1.Pannexin1通道抑制剂(10Panx1)可抑制LXR激动剂(GW3965)介导的THP-1巨噬细胞释放ATP的过程。2.Pannexin1通道抑制剂(10Panx1)对LXR激动剂(GW3965)诱导巨噬细胞摄取ox-LDL的能力起到抑制作用。3.Pannexin1通道抑制剂(10Panx1)对LXR激动剂(GW3965)诱导巨噬细胞ABCA1的表达起到抑制作用
[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

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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