关于炎症因子IL-6和血管紧张素II调节内皮酯酶表达的信号传导通路的研究

发布时间：2017-12-31 20:00

本文关键词：关于炎症因子IL-6和血管紧张素II调节内皮酯酶表达的信号传导通路的研究　出处：《山东大学》2013年硕士论文　论文类型：学位论文

【摘要】：背景和目的目前临床上在脂质代谢紊乱方面的治疗还主要集中在降低血浆低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)水平方面。降低LDL-C水平减少罹患心血管疾病风险的观点已经得到广泛认同。然而临床结果显示经过他汀类药物标准治疗后即使LDL-C达标,仍有10.9%的心血管剩留风险。治疗新靶点研究(TNT)显示,经过他汀类药物强化治疗后即使LDL-C水平降至1.99mmol/L,明显低于达标水平,仍有8.7%的绝对冠状动脉事件剩留风险,提示即使经过他汀类药物强化治疗,冠状动脉事件剩留风险仍较高。血脂异常相关的心血管剩留风险与多种因素有关,以低高密度脂蛋白(high-density lipoprotein cholesterol, HDL-C)为特征的血脂异常是经过他汀类药物治疗降低LDL-C水平后最常见的其中一种。如何降低冠状动脉事件剩留风险,减少心血管事件发生率,也是目前研究的焦点之一。HDL-C水平与心血管发生率呈负相关,约有1/3的血脂异常患者通过提高HDL-C水平而受益,有研究表明血浆HDL-C每升高0.03mmol/L(1.0mg/dl)就可以使罹患心血管疾病的风险下降2%-4%。另外LDL-C/HDL-C比值也是一个与心血管事件密切相关而独立于LDL-C和HDL-C的重要指标。因此治疗动脉粥样硬化除了在一定范围内降低LDL-C水平之外如何有效的提高HDL-C水平就成为了重要的研究方向。内皮脂酶(endothelial lipase, EL)是近年新发现的甘油三酯脂肪酶家族新成员,直接由血管内皮细胞分泌,在局部发挥作用。主要具有磷脂酶活性,是代谢HDL-C的关键酶,如何能够通过减低或抑制EL活性,从而减少HDL-C的降解,是治疗动脉粥样硬化的新发展思路。因此研究EL的转录表达调节,如何有效的降低EL就显得极为迫切。血管紧张素Ⅱ(Angiotensin Ⅱ, Ang Ⅱ)和炎症因子白细胞介素-6(interleukin-6,IL-6)是两种非常重要的致动脉粥样硬化危险因素。Ang Ⅱ能通过增加巨噬细胞清道夫受体CD36,促进巨噬细胞摄取氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL),加速泡沫细胞形成；能通过诱导炎症反应及细胞凋亡、产生氧自由基和影响纤溶功能等多方面参与动脉粥样硬化的病理过程;可以增加斑块不稳定因子[7]EMMPRIN和MMPs[8、9]的表达,加重动脉粥样硬化病变。IL-6也是一种重要的致动脉粥样硬化炎症因子,它构成了许多急慢性疾病的病理学基础,能够损伤内皮细胞造成内皮细胞功能障碍,增加单核-内皮细胞的粘附,还可以促进巨噬细胞对脂质的摄取。核因子NF-κB是种重要的核转录调节因子,激活后可启动包括细胞因子、化学因子等多种效应基因的表达。丝裂原活化蛋白激酶MAPKs级联反应是细胞内重要的信号传导系统之一,参与多种胞内信息传递过程,能对广泛的细胞外刺激发生反应,研究表明,NF-κB和p38MAPK在动脉粥样硬化中表达增加，可能是各种危险因子诱发动脉粥样硬化的机制之一,NF-κB和p38MAPK信号传导途径可能在EL的转录表达过程中发挥作用。本课题通过体外培养人脐静脉内皮细胞,以IL-6和Ang Ⅱ刺激内皮细胞,观察IL-6、Ang Ⅱ、NF-κB p65阻断剂PDTC (Pyrrolidinedithioearbamic acid)和SB203580(p38MAPK抑制剂)对EL表达的影响,验证IL-6和AngⅡ是否通过NF-κB和MAPK信号传导途径调节EL的表达。研究方法 1.提取新生儿脐静脉内皮细胞进行原代培养,经鉴定为内皮细胞后,贴壁法传代培养。第四代细胞用于实验。 2.用于实验的人脐静脉内皮细胞(human umbilical vein endothelial cells.HUVKCs)共分六组①AngⅡ刺激组：在培养液中加入AngⅡ,使之终浓度为10umol/L;②四氢化吡咯烷二硫代氨基甲酸酯(PDTC)+AngⅡ刺激组,在培养液中加入PDTC使之终浓度为10mmol/L,预处理1h后加入AngⅡ (10umol/L)刺激;③SB203580+AngⅡ (10umol/L)刺激组,在培养液中加入SB203580使之终浓度为10imol/L,预处理1h后加入AngⅡ(10umol/L)刺激；④IL-6刺激组：在培养液中加入rhIL-6,使之终浓度为10ng/mL,预处理1h后加入AngⅡ(10umol/L)刺激;⑤四氢化吡咯烷二硫代氨基甲酸酯(PDTC)+IL-6刺激组,在培养液中加入PDTC,使之终浓度为10mmol/L,预处理1h后加入rhIL-6(10ng/ml)刺激;⑥SB203580(10umol/L)+IL-6刺激组,在培养液中加入SB203580使之终浓度为10umol/L,预处理1h后加入IL-6(10ng/mL)刺激。各组分别孵育0、2、4、8、12、24h后终止实验,收集细胞。 3.将收集的细胞提取蛋白,使用western blot法检测EL的表达。 4.以上数据使用统计学进行分析。结果 1. AngⅡ可以上调HUVECs EL的表达。HUVECs经AngⅡ刺激后,在4,8,12h时其EL的表达量分别为0.362±0.041,0.738±0.034和0.387±0.051,均较0h(0.254±0.027)时明显增加,均p0.05。 2. PDTC可以抑制由AngⅡ引起的EL表达的增高。HUVECs经PDTC预处理1h后再加入AngⅡ刺激,其EL表达量在作用4,8,12h时分别为0.287±0.037,0.433±0.041,0.303±0.025,与AngⅡ刺激组在相应时间段内EL的表达量相比明显降低,均p0.05。 3.SB203580可以抑制由AngⅡ引起的EL表达的增高。HUVECs经SB203580预处理1h后再加入AngⅡ刺激,其EL表达量在作用4,8,12h时分别为0.258±0.027,0.372±0.038,0.296±0.034,与AngⅡ刺激组在相应时间段内EL的表达量相比明显降低,均p0.05。 4.IL-6可以上调HUVECs EL的表达。HUVECs经IL-6刺激后,在4,8,12h时其EL的表达量分别为0.293±0.062,0.633±0.052,0.462±0.065,均较Oh(0.254±0.027)时明显增加,均p0.05。 5. PDTC可以抑制由IL-6引起的EL表达的增高。HUVECs经PDTC预处理1h后再加入AngⅡ刺激,其EL表达量在作用4,8,12h时分别为0.208±0.056,0.582±0.036,0.428±0.066,与IL-6刺激组在相应时间段内的EL表达量相比明显降低,均p0.05。 6.SB203580可以抑制由IL-6引起的EL表达的增高。HUVECs经SB203580预处理1h后再加入AngⅡ刺激,其EL表达量在作用4,8,12,24h时分别为0.238±0.023,0.508±0.058,0.423±0.052,0.199±0.042,与IL-6刺激组在相应时间段内的EL表达量相比明显降低,均p0.05。结论 1. AngⅡ可能通过NF-κB和p38MAPK信号传导通路促进人脐静脉内皮细胞中内皮脂肪酶的表达。 2.IL-6可能通过NF-κB和p38MAPK信号传导通路促进人脐静脉内皮细胞中内皮脂肪酶的表达。
[Abstract]:Background and purpose
At present the clinical treatment of lipid metabolic disorder in the area is mainly concentrated in the lower plasma low density lipoprotein cholesterol (low-density lipoprotein cholesterol, LDL-C) level. Reduce the level of LDL-C has been widely recognized by the risk of cardiovascular disease. However, the idea of reducing the clinical results after statin drugs after treatment even if the LDL-C standard standard, there are still 10.9% the residual cardiovascular risk. A new target for the treatment of (TNT) showed that after intensive statin therapy. Even after the level of LDL-C to 1.99mmol/L was significantly lower than the standard level, there are still 8.7% of the absolute residual risk of coronary events, suggesting that even after intensive statin therapy, coronary events residual risk is still high. Dyslipidemia related residual cardiovascular risk associated with many factors, with low high density lipoprotein (high-density lipoprotein choleste Rol, HDL-C) is characterized by abnormal blood lipid after statin therapy reduces the level of LDL-C after the most common one. How to reduce the residual risk of coronary events, reduce the incidence of cardiovascular events is currently one of the research focus in the.HDL-C level and cardiovascular incidence were negatively correlated, about 1/3 of patients with dyslipidemia the level of HDL-C and benefit, studies have shown that plasma HDL-C increased 0.03mmol/L (1.0mg/dl) can be an important indicator of the risk of cardiovascular disease by 2%-4%. and ratio of LDL-C/HDL-C is a closely related with cardiovascular events independently of LDL-C and HDL-C. So the treatment of atherosclerosis in addition to reduce the level of LDL-C in a certain range from how to effectively improve the level of HDL-C has become an important research direction.
Endothelial lipase (endothelial lipase EL) is a new member of the family recently discovered triglyceride lipase, secreted by endothelial cells, play a role in the local. The main with phospholipase activity, is a key enzyme in the metabolism of HDL-C, how to reduce or inhibit the activity of EL, thereby reducing the degradation of HDL-C, is a new development way of treating atherosclerosis the expression of EL. So the study of transcriptional regulation, how to effectively reduce the EL is extremely urgent.
Angiotensin II (Angiotensin II, Ang II) and inflammatory cytokines interleukin -6 (interleukin-6, IL-6) are two important risk factors of atherosclerosis.Ang II by increasing macrophage scavenger receptor CD36, promote macrophage uptake of oxidized low density lipoprotein (oxidized low density lipoprotein, ox-LDL), the formation of acceleration foam cells; can induce inflammatory reaction and apoptosis, the pathological process of oxygen free radicals and influence of fibrinolytic function and other aspects involved in atherosclerosis; plaque instability could increase the expression of factor [7]EMMPRIN and MMPs[8,9], increased atherosclerosis in.IL-6 is also an important factor of atherosclerosis, which constitutes the pathology of many acute and chronic diseases the science foundation, can damage endothelial cells caused by endothelial dysfunction, increased monocyte and endothelial cells The adhesion, can also promote the uptake of lipid in macrophage. Nuclear factor kappa B NF- is an important regulator of nuclear transcription factor, activator can activate the expression of a variety of effects including cytokines, chemical factors genes. MAPKs mitogen activated protein kinase cascade is one of the important intracellular signal transduction systems, participate in a variety of intracellular information the transfer process, can on a wide range of extracellular stimuli, studies show that NF- kappa B and increase the expression of p38MAPK in atherosclerosis, may be one of the mechanisms of various risk factors for atherosclerosis, NF- kappa B and p38MAPK signaling pathway may play a role in the process of expression in the transcription of EL. The in vitro cultured human umbilical the venous endothelial cells with IL-6 and Ang II stimulated endothelial cells, observed IL-6, Ang II, NF- kappa B p65 inhibitor PDTC (Pyrrolidinedithioearbamic acid) and SB203580 (p38M The effect of APK inhibitor on the expression of EL verifies whether IL-6 and Ang II regulate the expression of EL by means of NF- - kappa B and MAPK signal transduction pathway.
research method
1. the umbilical vein endothelial cells of the newborn were extracted and cultured. After being identified as endothelial cells, the endothelial cells were cultured and cultured. The fourth generation cells were used for the experiment.
2. experiments on human umbilical vein endothelial cells (human umbilical vein endothelial cells.HUVKCs) were divided into six groups: group Ang II stimulation was added into the culture fluid of Ang II, the final concentration of 10umol/L; the four hydrogenated pyrrolidine two thiocarbamate (PDTC) stimulated by +Ang in the group, added to the culture medium PDTC to the final concentration of 10mmol/L, pretreatment of 1h after adding Ang II (10umol/L) stimulation; the SB203580+Ang II (10umol/L) stimulation group, in the culture medium added SB203580 to a final concentration of 10imol/L, pretreatment of 1h after adding Ang II (10umol/L) and IL-6 stimulation; stimulation group: rhIL-6 in cultures in the end, the concentration of 10ng/mL, pretreatment of 1h after adding Ang II (10umol/L) stimulation; the four hydrogenated pyrrolidine two thiocarbamate (PDTC) +IL-6 stimulation group, PDTC added to the culture medium, so that the final concentration is 10mmol/L, pretreatment of 1h after adding rhIL-6 (10ng/ml SB203580 (10umol/L) stimulation; 6) +IL-6 stimulation group, in the culture medium added SB203580 to a final concentration of 10umol/L, pretreatment of 1h after adding IL-6 (10ng/mL). All groups were incubated to stimulate the termination of the experiment, with 0,2,4,8,12,24h cells were collected.
3. the collected cells were extracted and the protein was extracted and the expression of EL was detected by Western blot.
More than 4. of the data were analyzed by statistics.
Result
1. Ang II can upregulate the expression of HUVECs EL. The expression of EL after.HUVECs stimulation at 4,8,12h is 0.362 + 0.041,0.738 + 0.034 and 0.387 + 0.051, which is significantly increased compared with 0h (0.254 + 0.027), all p0.05..
2. PDTC can inhibit the expression of EL induced by Ang II increased.HUVECs pretreatment with PDTC 1h after adding Ang II stimulation, the EL expression in 4,8,12h was 0.287 + 0.037,0.433 + 0.041,0.303 + 0.025, and the expression of Ang II stimulation group in the corresponding period of EL was lower than that of p0.05.
3.SB203580 can inhibit the expression of EL induced by Ang II increased.HUVECs pretreatment with SB203580 1h after adding Ang II stimulation, the EL expression in 4,8,12h was 0.258 + 0.027,0.372 + 0.038,0.296 + 0.034, and the expression of Ang II stimulation group in the corresponding period of EL was lower than that of p0.05.
4.IL-6 could upregulate the expression of HUVECs EL. The expression level of EL in.HUVECs stimulated by IL-6 was 0.293 + 0.062,0.633 + (0.052,0.462 + 0.065) at 4,8,12h, which was significantly increased compared with Oh (0.254 + 0.027).
5. PDTC can be inhibited by IL-6 induced expression of EL.HUVECs increased by pretreatment with PDTC 1h after adding Ang II stimulation, the EL expression in 4,8,12h was 0.208 + 0.056,0.582 + 0.036,0.428 + 0.066, and IL-6 stimulation group in the corresponding period of time compared to the amount of EL expression decreased obviously, p0.05.
6.SB203580 can be inhibited by IL-6 induced expression of EL.HUVECs increased by pretreatment with SB203580 1h after adding Ang II stimulation, the EL expression in 4,8,12,24h was 0.238 + 0.023,0.508 + 0.058,0.423 + 0.052,0.199 + 0.042, and IL-6 stimulation group in the corresponding period of EL expression was obviously lower than p0.05.
conclusion
1. Ang II may promote the expression of endothelial lipase in human umbilical vein endothelial cells through NF- kappa B and p38MAPK signal transduction pathway.
2.IL-6 may promote the expression of endothelial lipase in human umbilical vein endothelial cells through NF- kappa B and p38MAPK signal transduction pathway.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363.2

【参考文献】