血清淀粉样蛋白A对VEGFR2的表达及血管新生影响的研究

发布时间：2018-05-20 06:55

本文选题：血清淀粉样蛋白A + 血管内皮生长因子受体2　；参考：《山东大学》2016年硕士论文

【摘要】：研究背景动脉粥样硬化是冠状动脉和脑血管疾病最重要的病理过程,是引发猝死的主要原因。血管新生是动脉粥样硬化的关键致病因素之一。血管新生是指内皮细胞在原有毛细血管的基础上发生增殖、迁移,形成新的血管的过程。血管新生的作用有其两面性,一方面血管新生可用于治疗缺血性心脏病,这是一种在治疗上需要促进的血管新生；另一方面血管新生可以加速动脉粥样硬化中斑块的进展,导致斑块不稳定性增加,进而发生破裂出血等,这是一种在治疗上需要抑制的血管新生。根据其两面性,可以将血管新生分为生理性和病理性两种。探究病理性血管新生的发生机制可能为控制动脉粥样硬化的进展提供新的靶点。血清淀粉样蛋白A (Serum amyloid A, SAA)是一组极其保守的急性时相反应蛋白家族,有SAA1、SAA2、SAA3及SAA4四个不同的亚型,其中SAA1是最主要的亚型。SAA主要由肝细胞合成分泌,在机体受到炎症刺激时其血清浓度能迅速增加1000倍左右。因此,循环血液中高浓度的SAA是急性和慢性炎症疾病的重要标志物。作为一种急性时相蛋白,SAA在多种疾病(如类风湿关节炎、巨细胞动脉炎等)中可以促进病理性血管新生。因此,探究SAA促进血管新生的机制尤为重要。血管内皮生长因子(Vascular endothelial growth factor, VEGF)是促进内皮细胞血管新生的关键调控因子。血管内皮生长因子受体2 (Vascular endothelial growth factor receptor 2, VEGFR2)是VEGF的受体之一,是VEGF促血管新生信号转导通路的门户。VEGF/VEGFR2信号通路活化后,能够发挥促内皮细胞迁移、增殖和血管新生等作用。VEGFR2主要在血管内皮细胞上表达,但是其表达调控的机制尚待完善。以往研究显示,SAA能够促进人颈动脉内皮细胞(Human carotid artery endothelial,HCtAE)中VEGF的表达；同时,SAA能够通过激活p38 MAPK信号转导通路促进内皮细胞血管新生。因此我们推测SAA可能会影响VEGFR2的表达进而引起血管新生的变化。目前,SAA, VEGFR2与血管新生的研究存在以下亟待解决的问题：①SAA能否影响VEGFR2的表达;②SAA影响VEGFR2表达的相关信号转导通路；③SAA是否能够通过影响VEGFR2的表达引起血管新生的变化。研究目的1.探讨SAA对VEGFR2表达的影响；2.探索SAA影响VEGFR2表达的相关信号转导通路；3.探索SAA引起的VEGFR2表达变化在血管新生中的作用。材料方法1.培养人脐静脉内皮细胞在内皮完全培养基(Endothelial complete medium,ECM)中培养人脐静脉内皮细胞(Human umbilical vein endothelial cells, HUVECs),每2-3天换液一次并置于含有5% CO2的37℃温箱中。待细胞达到合适密度时加入SAA1或待研究信号通路的激动剂以及抑制剂刺激细胞。2.实时定量PCR (Real-time PCR)检测利用Trizol法从HUVECs中提取出mRNA,并获得相应的cDNA。利用GAPDH为内参检测VEGFR2的mRNA表达。3.蛋白印记(Western blot)检测收集内皮细胞,提取蛋白,以GAPDH蛋白表达为内参检测VEGFR2的蛋白表达；以相应总蛋白为内参检测p-ERK1/2, p-JNK及p-p38的蛋白表达。4.成管实验(Tube formation assay)在96孔板中铺入matrigel基质胶,待胶凝固后每孔加入密度为2×104的细胞量,以未加任何刺激的组为对照显微镜下观察各实验组的成管情况。5.统计学分析所有数值均用Prism version 5和SPSS 18.0软件进行统计学分析。用单因素方差分析来确定有无统计学意义。结果用均值±标准差来表示,P0.05代表数据间具有统计学差异。结果1.SAA上调HUVECs细胞中VEGFR2的表达使用不同浓度梯度(0,5,10和50μg/ml)的SAA刺激HUVECs,24小时后收集细胞,提取蛋白和mRNA。通过Western blot及Real-time PCR发现,与对照组相比,SAA呈剂量依赖性的方式上调VEGFR2的表达(P0.01)；以固定浓度的SAA (10μg/ml)刺激细胞,选择不同时间点(0,2,3,6,12和24小时)收集细胞,提取蛋白和mRNA。通过Western blot及Real-time PCR发现,与对照组相比,SAA呈时间依赖性的方式上调VEGFR2的表达(P0.05)。2.膜受体FPRL1能够介导SAA诱导的VEGFR2表达上调用膜受体FPRL1的特异性抑制剂WRW4预刺激细胞,通过Western blot发现,与单独SAA刺激组相比,WRW4能显著抑制SAA诱导的VEGFR2的表达上调(P0.01)。用FPRL1特异性的激动剂WKYMVm刺激细胞,WKYMVm组较对照组能明显上调VEGFR2的表达(P0.01)。这一结果表明,FPRL1能够介导SAA诱导的VEGFR2表达上调。3 HAPKs信号通路能够介导SAA诱导的VEGFR2表达上调使用SAA刺激细胞后,通过Western blot发现,与对照组相比,ERK1/2, JNK及p38的磷酸化水平显著增强且在1小时达到高峰(P0.01)。然后分别使用三者特异性的抑制剂PD98059, SP600125以及SB203580刺激细胞6,12及24小时。Western blot结果显示,加用三种抑制剂组较单独SAA刺激组均能不同程度的降低VEGFR2的表达(P0.05)。这一结果表明,MAPKs信号通路能够介导SAA诱导的VEGFR2表达上调。4.膜受体FPRL1能够调控下游MAPKs信号通路的活化利用受体FPRL1的特异性抑制剂WRW4预处理细胞,Western blot结果显示,WRW4组较单独SAA刺激组能明显抑制MAPKs信号通路的磷酸化(P0.05)。另外,我们用FPRL1的特异性激动剂WKYMVm处理细胞,Western blot结果显示,与对照组相比,用WKYMVm处理细胞组能够显著上调MAPKs信号通路的磷酸化水平(P0.01)。这一结果表明,FPRL1作为上游信号分子能够调控MAPKs信号通路的活化。5.SAA诱导的VEGR2:表达增多能够促进内皮细胞的血管新生利用受体VEGFR2的特异性抑制剂BIBF1120及FPRL1/MAPKs信号通路的激动剂和抑制剂刺激细胞,成管实验结果显示,较单独SAA刺激组相比,加用BIBF1120, WRW4, PD98059, SP600125及SB20358均能够显著抑制内皮细胞成管(P0.01)。而WKYMVm处理组较对照组相比则显著促进内皮细胞成管(P0.01)。这些结果证实SAA诱导的VEGFR2的表达及相关信号通路能够促进内皮细胞的血管新生。结论1.SAA能够上调HUVECs细胞中VEGFR2的表达；2. FPRL1/MAPKs信号通路介导了SAA诱导的VEGFR2表达上调；3.SAA诱导的VEGFR2表达上调能够促进内皮细胞的血管新生。
[Abstract]:Background atherosclerosis is the most important pathological process of coronary and cerebrovascular diseases. It is the main cause of sudden death. Angiogenesis is one of the key pathogenic factors of atherosclerosis. Angiogenesis is the process of proliferation, migration and formation of new blood vessels on the basis of the original capillary. On the one hand, angiogenesis can be used to treat ischemic heart disease. It is a kind of angiogenesis that needs to be promoted in treatment. On the other hand, angiogenesis can accelerate the progress of plaque in atherosclerosis, cause plaque instability to increase, break bleeding and so on. This is a kind of treatment. Angiogenesis may be divided into two physiological and pathological types based on its dual nature. Exploring the mechanism of pathological angiogenesis may provide a new target for controlling the progress of atherosclerosis. Serum amyloid A (Serum amyloid A, SAA) is an extremely conservative group of acute phase reactive protein families There are four different subtypes of SAA1, SAA2, SAA3 and SAA4, of which SAA1 is the main subtype.SAA mainly synthesized and secreted by liver cells, and the serum concentration can be increased by about 1000 times when the body is stimulated by inflammation. Therefore, the high concentration of SAA in the circulating blood is an important marker of acute and slow inflammatory diseases. Phase protein, SAA can promote pathological angiogenesis in a variety of diseases, such as rheumatoid arthritis, giant cell arteritis and so on. Therefore, it is particularly important to explore the mechanism of SAA to promote angiogenesis. Vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) is a key regulator of endothelial cell angiogenesis. Growth factor receptor 2 (Vascular endothelial growth factor receptor 2, VEGFR2) is one of the receptors of VEGF. It is the activation of the portal.VEGF/VEGFR2 signaling pathway of angiogenesis pathway of VEGF promoting angiogenesis. It can express endothelial cell migration, proliferation and angiogenesis, but it is mainly expressed on vascular endothelial cells by.VEGFR2, but it is mainly expressed on vascular endothelial cells. The mechanism of expression regulation remains to be improved. Previous studies have shown that SAA can promote the expression of VEGF in human Human carotid artery endothelial (HCtAE), and SAA can promote the angiogenesis of endothelial cells by activating the p38 MAPK signal transduction pathway. Therefore, we speculate that SAA may affect the expression of VEGFR2 to induce the expression of VEGFR2. Changes in angiogenesis. At present, there are some problems to be solved in SAA, VEGFR2 and angiogenesis: (1) whether SAA can affect the expression of VEGFR2; (2) the correlation signal transduction pathway that SAA affects the expression of VEGFR2; (3) whether SAA can affect the changes of blood vessels by affecting the expression of VEGFR2. Purpose 1. to explore the expression of SAA to VEGFR2 2. to explore the correlation signal transduction pathway that SAA affects VEGFR2 expression; 3. explore the role of SAA induced VEGFR2 expression in angiogenesis. Material method 1. cultured human umbilical vein endothelial cells cultured human umbilical vein endothelial cells (Human umbilical vein endoth) in the endothelial complete medium (Endothelial complete medium, ECM) Elial cells, HUVECs), once every 2-3 days, the liquid was replaced once every 2-3 days and placed in a temperature box containing 5% CO2. When the cell reached the appropriate density, the SAA1 or the excitant to study the signal pathway and the real-time quantitative PCR (Real-time PCR) of the inhibitor stimulated cell.2. were detected by Trizol method from HUVECs, and the corresponding utilization was obtained. The mRNA expression of.3. protein imprint (Western blot) was used to detect the endothelial cells and extract the protein. The protein expression was detected by the expression of GAPDH protein as the internal parameter, and the protein expression was expressed as the internal parameter of the.3.. The protein expression of the corresponding total protein was used as the internal parameter for the detection of p-ERK1/2, and the protein expression of p-JNK and p-p38 was tested in the 96 hole plate. After the gel was solidified, the cell volume of each hole was 2 * 104, and the control group without any stimulation was used as the control microscope to observe the tube formation of each experiment group.5.. All the values were statistically analyzed with Prism version 5 and SPSS 18 software. Mean mean standard deviation, P0.05 represents the statistical difference between the data. Results 1.SAA up-regulated the expression of VEGFR2 in HUVECs cells using SAA stimulated HUVECs with different concentration gradient (0,5,10 and 50 u g/ml) and collected cells after 24 hours. The extraction of protein and mRNA. was found by Western blot and Real-time. The expression of VEGFR2 (P0.01) was up-regulated in the manner of the dependability; the cells were stimulated by a fixed concentration of SAA (10 mu g/ml), and the cells were collected at different time points (0,2,3,6,12 and 24 hours). The extraction of protein and mRNA. was found by Western blot and Real-time PCR. PRL1 can mediate SAA induced VEGFR2 expression by invoking a specific inhibitor of the membrane receptor FPRL1, WRW4 prestimulating cells. Through Western blot, it is found that WRW4 significantly inhibits SAA induced VEGFR2 expression up-regulation (P0.01), compared with the individual SAA stimulation group. VEGFR2 expression (P0.01). This result shows that FPRL1 can mediate SAA induced VEGFR2 expression up regulation of.3 HAPKs signaling pathway can mediate SAA induced VEGFR2 expression up-regulated the use of SAA stimulation cells, by Western blot, compared with the control group, the level of phosphorylation is significantly enhanced and reached the peak at 1 hours. 01). Then using three specific inhibitors PD98059, SP600125 and SB203580 stimulating cells 6,12 and 24 hours.Western blot, the results showed that the addition of three inhibitor groups could reduce the expression of VEGFR2 in varying degrees (P0.05). This result shows that MAPKs signaling pathway can mediate SAA VEGFR2 table. Up regulation of the.4. membrane receptor FPRL1 can regulate the activation of the downstream MAPKs signaling pathway by activating the receptor FPRL1 specific inhibitor WRW4 pretreated cells. Western blot results show that WRW4 group can significantly inhibit the phosphorylation of MAPKs signaling pathway (P0.05) than the single SAA stimulus group. Furthermore, we use the specific agonist of FPRL1 to treat cells. RN blot results showed that compared with the control group, the WKYMVm treated cell group could significantly increase the phosphorylation level of the MAPKs signaling pathway (P0.01). This result shows that FPRL1 as a upstream signal molecule can regulate the increase of VEGR2: expression induced by the activated.5.SAA of the MAPKs signaling pathway and can promote the angiogenesis of endothelial cell receptor VEGFR. 2 of the stimulants and inhibitors of the specific inhibitor BIBF1120 and FPRL1/MAPKs signaling stimulated the cells. The results of the tube test showed that the addition of BIBF1120, WRW4, PD98059, SP600125 and SB20358 to the individual SAA stimulation group could significantly inhibit the endothelial cell formation (P0.01). The WKYMVm treatment group significantly promoted the endothelium compared with the control group. Cell formation (P0.01). These results confirm that the expression of SAA induced VEGFR2 and the related signaling pathway can promote the angiogenesis of endothelial cells. Conclusion 1.SAA can up regulate the expression of VEGFR2 in HUVECs cells; 2. FPRL1/MAPKs signal pathway mediates the up regulation of VEGFR2 expression induced by SAA, and the up regulation of VEGFR2 expression induced by 3.SAA can be promoted. The vascular neovascularization of the skin cells.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.5

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