A类清道夫受体在巨噬细胞凋亡中的作用研究

发布时间：2018-04-04 23:21

本文选题：A类清道夫受体　切入点：fucoidan　出处：《南京医科大学》2009年硕士论文

【摘要】：目的: 关于A类清道夫受体(scavenger receptor A, SR-A)在巨噬细胞凋亡中的作用现今尚无一致意见。本课题应用SR-A的配体岩藻聚糖硫酸酯(fucoidan)作为工具药,深入研究了SR-A对于巨噬细胞凋亡的影响、作用机制和下游信号通路,旨在阐明fucoidan在内质网应激(ER stress)诱导的巨噬细胞凋亡中所起的作用及其分子机制,为阻止巨噬细胞凋亡、防治动脉粥样硬化斑块进展提供新的途径。方法: 本研究首先用SR-A敲除和野生型的小鼠腹腔巨噬细胞复制ER stress,在此基础上观察给予或不给予fucoidan对细胞凋亡的影响,细胞凋亡用Annexin V/PI染色和PI染色流式检测法进行检测。接着分别用毒胡萝卜素(TG)或乙酰化LDL加酰基辅酶A:胆固醇酰基转移酶的抑制剂58035(造成细胞游离胆固醇蓄积,FC loading)处理小鼠RAW264.7巨噬细胞,使之产生ER stress,观察细胞的凋亡情况以及给予fucoidan后细胞凋亡率的改变,同时用MTT法检测细胞活性的变化情况。有关fucoidan对巨噬细胞凋亡影响的作用机制研究主要依靠Western blot检测法来进行。首先观察fucoidan作用引起的细胞ERK1/2信号分子磷酸化活性的改变,并利用SR-A敲除和野生型小鼠腹腔巨噬细胞确定其作用是否通过SR-A发挥。接着运用ERK1/2的阻断剂PD98059,观察当ERK1/2磷酸化活性被抑制后,fucoidan对ER stress诱导凋亡的抑制作用是否仍存在,确认ERK1/2的磷酸化激活在fucoidan抑制ER stress诱导的巨噬细胞凋亡中所起的作用。结果: 用TG处理小鼠腹腔巨噬细胞可引起ER stress,Annexin V/PI染色实验结果提示,同时还发生了细胞凋亡,在处理后12 h即出现较高比例晚期凋亡细胞(Annexin V和PI染色均呈阳性),在SR-A敲除和野生型的巨噬细胞中无明显差异。如果在诱导ER stress同时加入fucoidan共孵育,则可明显降低晚期凋亡率,凋亡细胞多呈现早期凋亡状态(Annexin V染色阳性,PI染色阴性)。这种现象仅见于SR-A野生型巨噬细胞,而在SR-A-/-巨噬细胞中,细胞早晚期凋亡率与ER stress组相比无明显差异。PI染色流式检测细胞凋亡的结果与Annexin V染色的结果相一致,提示fucoidan可能通过SR-A发挥抑制ER stress诱导的巨噬细胞晚期凋亡的作用。用小鼠RAW264.7巨噬细胞系进行重复实验,发现用TG或FC loading诱导的细胞凋亡均可被fucoidan所抑制,细胞凋亡被抑制率分别可达到86%和90%左右,MTT结果显示fucoidan甚至能部分恢复细胞活性,可以使TG或FC loading导致的细胞生长抑制分别减少约19%和15%。进一步研究fucoidan的细胞活性保护机制,发现fucoidan处理细胞可以激活MAPK家族的数种主要信号分子的磷酸化活性,尤其能长时间持续激活ERK1/2磷酸化活性。当敲除SR-A基因后,fucoidan丧失对ERK1/2磷酸化的激活作用。用ERK1/2磷酸化活性抑制剂PD98059进行实验,发现当抑制ERK1/2的磷酸化活性后,fucoidan对ER stress所诱导的细胞凋亡的抑制作用也消失,其拮抗ER stress所起的细胞活性保护作用也终止,提示fucoidan抑制巨噬细胞凋亡、保护细胞活性的作用可能由ERK1/2信号分子所介导。结论: 作为A类清道夫受体的配体,fucoidan与SR-A结合以后,通过激活ERK1/2的磷酸化活性,抑制了由ER stress所诱导的巨噬细胞的晚期凋亡,提高细胞活性。
[Abstract]:Purpose :

The effect of SR - A on the apoptosis of macrophages , the mechanism of action and the downstream signaling pathway were studied . The purpose of this study was to clarify the role and molecular mechanism of SR - A in macrophage apoptosis induced by ER stress , and to provide a new approach to prevent the apoptosis of macrophages and prevent the progression of atherosclerotic plaque .

Method :

In this study , we first observed the effect of the inhibition on apoptosis of mouse RAW264.7 macrophages by using SR - A knock - out and wild - type mouse peritoneal macrophages .

Results :

At the same time , apoptosis was observed in mice peritoneal macrophages treated with TG , and apoptosis was observed . At 12 h after treatment , there was no significant difference in apoptosis cells ( both annexin V and PI staining ) . This phenomenon was found only in SR - A wild type macrophages , whereas in SR - A - / - macrophages , the early and late apoptotic rate of the cells was not significantly different from that of ER stress group . The results of PI staining flow cytometry showed that SR - A could play a role in inhibiting the late apoptosis of macrophages induced by ER stress .

The inhibition rate of cell apoptosis induced by TG or FC loading could be up to 86 % and 90 % respectively . The results showed that the cell growth inhibition induced by TG or FC loading could be reduced by about 19 % and 15 % , respectively .

Further research on the mechanism of cell activity protection of the MAPK family has been investigated . It is found that the phosphorylation activity of the MAPK family can be activated for a long period of time , especially for a long period of time . When the phosphorylation activity of ERK 1 / 2 is inhibited , the inhibitory effect of the phosphorylation of ERK 1 / 2 on the phosphorylation of ERK 1 / 2 is also eliminated . The effect of the inhibition of ER stress on the apoptosis of the cells is also terminated . It is suggested that the effect of the inhibitory effect on the apoptosis of macrophages and the protection of cell activity may be mediated by the 1 / 2 signal molecule .

Conclusion :

As a ligand for the class A scavenger receptor , after binding with SR - A , the late apoptosis of macrophages induced by ER stress is inhibited and cell activity is increased by activating the phosphorylation activity of 1 / 2 .

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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