氧化低密度脂蛋白对小鼠骨髓源性平滑肌祖细胞CXCR4表达的影响

发布时间：2018-05-08 11:12

本文选题：基质细胞衍生因子受体 + 氧化低密度脂蛋白　；参考：《华中科技大学》2009年硕士论文

【摘要】：背景基质细胞衍生因子(stromal cell derived-factor1,SDF-1)又名CXCL12或前B刺激因子,属于CXC族趋化因子成员,同基因编码两种蛋白:SDF-1α和SDF-1β。SDF-1主要在骨髓基质细胞及骨髓内皮细胞表达,在造血干细胞中也有相应的表达。SDF-1唯一的受体CXCR4是一个有七个跨膜结构域的G蛋白耦联的受体,在造血干/祖细胞表面及内皮祖细胞表面高表达,新近研究表明CXCR4也在骨髓间充质细胞(marrow stromal cells;MSC)上表达。文献报道,在心肌梗死后的心肌中立即出现了SDF-1的上调并导致过表达CXCR4的MSC向损伤心肌迁移。在Zhang等的研究中表明CXCR4+的MSC参与了新生血管的生成并分化为新的心肌细胞,动物实验表明SDF-1/CXCR4轴的相互作用在心肌修复时对MSC的募集起着重要作用,在SDF-1和/或CXCR4敲除老鼠的研究中表明该轴在细胞分化中起着重要作用。MSC是骨髓微环境中重要细胞成分,具有向各种细胞分化的潜能,且有文献报道MSC可向平滑肌祖细胞(Smooth muscle progenitor cell;SPC)分化。骨髓源性MSC或SPC是动脉粥样硬化(atherosclerosis,As)斑块中平滑肌细胞重要来源之一。那么在As的斑块形成及修复过程中SDF-1/CXCR4轴是否能募集MSC或骨髓源性的SPC的归巢致损伤动脉,参与As形成尚未见报道。目的检测As危险因子氧化低密度脂蛋白(Oxidized Low Density Lipoprotein;ox-LDL)对小鼠骨髓源性SPC SDF-1受体CXCR4表达的影响,探讨SDF-1/CXCR4对骨髓源性SPC归巢至受损动脉的作用。方法小鼠骨髓源性SPC与ox-LDL(50μg/mL)共同孵育,用逆转录聚合酶链反应法(Reverse transcription-polymerase chian reaction;RT-PCR)检测SPC CXCR4 mRNA的表达,免疫印迹法(immunoblotting)及免疫荧光染色和共聚焦激光扫描显微镜分析(immunofluorescence staining and confocal laser scanning microscope analysis)检测SPC CXCR4蛋白表达,观察ox-LDL对SPC表达CXCR4的时效关系。结果未给予ox-LDL刺激的SPC有基础水平的CXCR4表达,50μg/mL的ox-LDL刺激SPC 0-72h,随刺激时间的延长,CXCR4表达逐渐增强,36h达峰值,其mRNA和蛋白水平分别为基础水平的5.73倍和5.02倍,差异均有统计学意义(p0.05),随后逐渐下降,但仍高于基础水平。免疫荧光染色和共聚焦激光检测并观察到在0h时即未给予ox-LDL刺激时SPC的胞质和细胞膜表面有少量的CXCR4表达,随着作用时间的延长,CXCR4的表达量逐渐上调且在36h时其在SPC的胞质和细胞膜表面的表达最强,60h后CXCR4的表达量逐渐下降。结论ox-LDL上调小鼠骨髓源性SPC CXCR4的表达,提示ox-LDL有可能通过上调SPC CXCR4的表达致SPC迁移和归巢至病变动脉参与As形成。
[Abstract]:Stromal cell derived-factor1 (SDF-1), also known as CXCL12 or pre B stimulator, is a member of the CXC chemokines, and the same gene encodes two proteins: SDF-1 A and SDF-1 beta.SDF-1 are mainly expressed in bone marrow stromal cells and bone marrow endothelial cells, and there are also.SDF-1 receptor C in the blood making stem cells. XCR4 is a G protein coupled receptor with seven transmembrane domains, which is highly expressed on the surface of hematopoietic stem / progenitor cells and on the surface of endothelial progenitor cells. Recent studies have shown that CXCR4 is also expressed on bone marrow mesenchymal cells (marrow stromal cells; MSC). It is reported that the up regulation of SDF-1 in the myocardium after myocardial infarction and the result of the overexpression are reported. CXCR4 MSC migrated to the injured myocardium. In the study of Zhang, CXCR4+ MSC participated in the formation of new blood vessels and differentiated into new cardiomyocytes. Animal experiments showed that the interaction of SDF-1/CXCR4 axis played an important role in the recruitment of MSC in the repair of myocardium. In the study of SDF-1 and / or CXCR4 knockout mice, the axis was fine. Cell differentiation plays an important role in.MSC, an important cell component in bone marrow microenvironment, and has the potential to differentiate into various cells. And it is reported that MSC can differentiate into smooth muscle progenitor cells (Smooth muscle progenitor cell; SPC). Myelogenic MSC or SPC is an important source of smooth muscle cells in atherosclerotic (atherosclerosis, As) plaques. First, it is not reported that whether the SDF-1/CXCR4 axis can raise the injured arteries of the MSC or bone marrow derived SPC during the plaque formation and repair of As. The formation of As is not yet reported.
Objective to investigate the effect of As risk factor Oxidized Low Density Lipoprotein (ox-LDL) on the CXCR4 expression of bone marrow derived SPC SDF-1 receptor in mice, and to explore the effect of SDF-1/CXCR4 on the SPC homing of bone marrow to the damaged artery.
Methods the mouse bone marrow derived SPC was incubated with ox-LDL (50 mu g/mL), and the expression of SPC CXCR4 mRNA was detected by reverse transcription polymerase chain reaction (Reverse transcription-polymerase Chian reaction; RT-PCR), and immunoblotting (immunoblotting), immunofluorescence staining and confocal laser scanning microscopy were used to analyze the expression of the expression. Nd confocal laser scanning microscope analysis was used to detect SPC CXCR4 protein expression and to observe the time-dependent relationship between ox-LDL and SPC expression.
The results showed that SPC with no ox-LDL stimulation had the basic level of CXCR4 expression, and the ox-LDL of 50 mu g/mL stimulated SPC 0-72h. With the time of stimulation, the expression of CXCR4 increased gradually and the 36h reached the peak value. The mRNA and protein levels were 5.73 times and 5.02 times of the basic level respectively. The difference was statistically significant (P0.05), and then gradually decreased, but still higher than the base water. Immunofluorescence staining and confocal laser detected and observed that there was a small amount of CXCR4 expression in the cytoplasm of SPC and the surface of the cell membrane in the absence of ox-LDL stimulation at 0h. The expression of CXCR4 increased gradually with the prolongation of action time and the expression of CXCR4 was the strongest in the cytoplasm and surface of the cell membrane at the time of 36h, and the amount of CXCR4 expressed gradually after 60H. Drop.
Conclusion ox-LDL up-regulated the expression of bone marrow derived SPC CXCR4 in mice, suggesting that ox-LDL may be involved in SPC migration and homing to the lesion artery to participate in As formation by up regulation of the expression of SPC CXCR4.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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