mmLDL损伤小鼠肠系膜动脉内皮依赖性舒张功能作用及机制
发布时间：2018-11-05 07:57
【摘要】：研究背景与目的弱氧化型低密度脂蛋白（minimally modified low densitylipoprotein，mmLDL）是指仅脂质部分被氧化，载脂蛋白B100结构中赖氨酸残基未被破坏的LDL。离体器官和细胞培养研究发现，mmLDL可以显著上调心脑血管平滑肌内皮素B受体，增强血管的收缩功能；mmLDL还能与巨噬细胞上的TLR4结合，启动免疫反应诱发大量炎症因子的产生，还诱导细胞吞噬功能改变，，加速泡沫细胞的形成。但是，mmLDL是否对整体动物血管舒张功能产生影响还不清楚。本实验将探讨mmLDL对阻力血管肠系膜动脉内皮依赖性舒张功能的影响及其作用机制。为抗心脑血管疾病的研究提供一定的理论指导和实验基础，并为心脑血管疾病的防治提供新的思路和新的药物治疗靶点。 方法（1）小鼠肠系膜动脉mmLDL损伤模型建立：1mg/kg的剂量尾静脉注射mmLDL，从第1次注射后开始计时，之后每隔12h再注射1次，第六次注射后（即在0、12、24、36、48及60h各注射1次），将小鼠脱臼处死，取出含肠系膜动脉组织，显微镜下剥离血管。（2）运用微血管张力描记仪测定mmLDL对小鼠肠系膜动脉收缩功能与舒张功能的影响。（3）运用ELISA技术检测血浆中炎症因子IL-1β、TNF-α的浓度水平。（4）透射电镜观察血管内皮超微结构。（5）RT-PCR技术检测肠系膜动脉中IL-1β、TNF-α、KCa2.3、KCa3.1、 maxi KCa的mRNA表达水平。（6）采用WesternBlot技术检测肠系膜动脉中TLR4、TNF-α、KCa2.3、KCa3.1、 maxi KCa的蛋白表达量。 结果（1）尾静脉注射mmLDL能时间和剂量依赖性的损伤小鼠肠系膜动脉内皮依赖性舒张功能，并且在1mg/kg的注射剂量和72小时作用达到最大。（2）与生理盐水组pIC506.42±0.03和Rmax(63±5)%相比，mmLDL显著损伤EDHF-通路介导的血管内皮依赖性舒张功能[pIC505.67±0.07和Rmax(31±3)%，P0.001，P0.001]；与生理盐水组pIC505.87±0.10和Rmax(47±4)%相比，mmLDL显著损伤NO-通路介导的血管内皮依赖性舒张功能[pIC505.44±0.12和Rmax(31±4)%，P0.01，P0.01]；而对PGI2-通路的损伤无显著影响。（3）与生理盐水组pIC506.44±0.11和Rmax(40±4)%相比，mmLDL显著损伤KCa2.3-通路介导的血管内皮依赖性舒张功能[pIC505.30±0.13和Rmax(18±3)%，P0.001，P0.01]；与生理盐水组pIC505.44±0.18和Rmax(21±4)%相比，mmLDL显著损伤KCa3.1-通路介导的血管内皮依赖性舒张功能[pIC504.72±0.13和Rmax(8±2)%，P0.01，P0.01]；而对maxi KCa-通路的损伤基本无显著影响。（4）mmLDL在局部区域损伤内弹力膜，并且诱发肠系膜动脉内皮细胞剥落和水肿，导致了其对血管内皮依赖性舒张功能的损伤。（5）mmLDL还诱导血管组织中TLR4蛋白表达明显上调（P0.01）。（6）与生理盐水组140.15±22.68pg/mL相比，mmLDL显著上调血清中TNF-α的血清浓度水平（328.95±28.85，P0.001）；与生理盐水组60.73±9.73pg/mL相比，mmLDL显著上调血清中IL-1β的血清浓度水平（132.75±13.37，P0.01）；IL-1β和TNF-α的mRNA水平与血清浓度水平趋势基本一致。 结论mmLDL显著降低血管内皮依赖性舒张功能，其机制与下列因素有关：（1）通过上调TLR4的蛋白表达量，诱发炎症反应，损伤内皮细胞超微结构。（2）抑制NO和EDHF信号通路。（3）下调KCa2.3-通道和KCa3.1-通道的蛋白表达。
[Abstract]:Background and objective weakly oxidized low density lipoprotein (mLDL) refers to LDL whose lipid fraction is oxidized and the lysine residue in apolipoprotein B100 structure is not destroyed. From the study of body organ and cell culture, mLDL can significantly upregulate the endothelin B receptor of cardiovascular and cerebrovascular smooth muscle and enhance the contraction function of blood vessel; mmLDL can also bind TLR4 on macrophages, initiate immune response to induce the generation of a great amount of inflammatory factors, and also induce the change of phagocyte function. the formation of foam cells is accelerated. However, it is not clear whether mmLDL has an impact on the whole animal's vasodilation function. This study will investigate the effect of mmLDL on endothelium-dependent vasodilation function of vascular mesenteric artery and its mechanism of action. To provide some theoretical guidance and experimental basis for the study of cardiovascular and cerebrovascular diseases, and to provide new thought and new drug therapy target for the prevention and treatment of cardiovascular and cerebrovascular diseases. Method (1) Mouse mesenteric artery mLDL injury model was established: 1 mg/ kg dose tail vein injection of mmLDL, the timing after the first injection, followed by injection once every 12h, and after the sixth injection (i.e. 1 times at 0, 12, 24, 36, 48, and 60h), the mice were dislocated. Euthanasia, removal of mesenteric artery tissue, dissection under microscope blood vessel. (2) Measurement of the contractile function and diastolic function of mLDL on mesenteric artery in mice by means of microangiogram (3) To detect the concentration of IL-1, TNF-and IL-1 in plasma by ELISA. Observation of vascular endothelial ultrastructure by transmission electron microscope (TEM) Structure. (5) RT-PCR technique was used to detect the mRNA expression of IL-1, TNF-, KCa2 +, KCa3. 1 and maxi KCa in mesenteric artery. Levels of TLR4, TNF-CoV, KCa2 + 3, KCa3. 1 and maxi KCa in mesenteric artery were detected by using Blot technique. Results (1) After intravenous injection of mLDL energy and dose-dependent injury to the mesenteric artery endothelium-dependent relaxation function, and in an injection amount of 1 mg/ kg and 72 hours Compared with normal saline group, pIC504.42 and Rmax (63/ 5)%, mmLDL was significantly injured by EDHF-pathway mediated vasodilation function[pIC505. 67, 0. 07 and Rmax (31% 3)%, P0.001, P0. 001]; compared with saline group, pIC505. 87, 0. 10 and Rmax (47%). Compared with 4%, mLDL was significantly injured by NO-pathway-mediated vasodilation function[pIC505. 44, 0. 12 and Rmax (31/ 4)%, P0.05). There was no significant effect. (3) Compared with saline group, pIC504.44, 0.011 and Rmax (40% 4)%, mmLDL significantly injured KCa2 +-pathway-mediated vasodilation function[pIC505. 30, 0. 13 and Rmax (18, 3)%, P0. 001, P0.01]; and pIC505. 44, 0. 18 and Rmax (21) in physiological saline group. Compared with 4%, mmLDL significantly damaged the endothelium-dependent vasodilation function of KCa3. 1-pathway[pIC504. 72, 0. 13 and Rmax (8, 2)%, P0. 01, P0.01], and the damage to maxi KCa-pathway was basic. There is no significant effect. (4) mLDL is an elastic membrane in local area injury and induces exfoliation and edema of mesenteric artery endothelial cells, leading to its endothelium-dependent relaxation. Functional damage. (5) mmLDL also induces significant upregulation of TLR4 protein expression in vascular tissues (P mLDL significantly upregulated the level of serum concentration (328,95, 28,85, P0.001) in serum as compared to the saline group of 140. 15 vs 22. 68pg/ mL; mLDL significantly upregulated the serum concentration level of IL-1 in serum (132. 75, 13.37, P 0. 01) Levels of mRNA and serum concentration of IL-1 and TNF-and IL-1 in serum Conclusion mmLDL significantly reduces the endothelium-dependent vasodilation function, and its mechanism is related to the following factors: (1) by upregulating the expression of TLR4 protein, inducing inflammatory response, injury, Ultrastructure of endothelial cells. (2) Inhibition of NO and EDHF signaling pathway. (3) down-regulation of KCa2 + 3-channel and KCa3. 1
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R54;R743.3
本文编号：2311411
[Abstract]:Background and objective weakly oxidized low density lipoprotein (mLDL) refers to LDL whose lipid fraction is oxidized and the lysine residue in apolipoprotein B100 structure is not destroyed. From the study of body organ and cell culture, mLDL can significantly upregulate the endothelin B receptor of cardiovascular and cerebrovascular smooth muscle and enhance the contraction function of blood vessel; mmLDL can also bind TLR4 on macrophages, initiate immune response to induce the generation of a great amount of inflammatory factors, and also induce the change of phagocyte function. the formation of foam cells is accelerated. However, it is not clear whether mmLDL has an impact on the whole animal's vasodilation function. This study will investigate the effect of mmLDL on endothelium-dependent vasodilation function of vascular mesenteric artery and its mechanism of action. To provide some theoretical guidance and experimental basis for the study of cardiovascular and cerebrovascular diseases, and to provide new thought and new drug therapy target for the prevention and treatment of cardiovascular and cerebrovascular diseases. Method (1) Mouse mesenteric artery mLDL injury model was established: 1 mg/ kg dose tail vein injection of mmLDL, the timing after the first injection, followed by injection once every 12h, and after the sixth injection (i.e. 1 times at 0, 12, 24, 36, 48, and 60h), the mice were dislocated. Euthanasia, removal of mesenteric artery tissue, dissection under microscope blood vessel. (2) Measurement of the contractile function and diastolic function of mLDL on mesenteric artery in mice by means of microangiogram (3) To detect the concentration of IL-1, TNF-and IL-1 in plasma by ELISA. Observation of vascular endothelial ultrastructure by transmission electron microscope (TEM) Structure. (5) RT-PCR technique was used to detect the mRNA expression of IL-1, TNF-, KCa2 +, KCa3. 1 and maxi KCa in mesenteric artery. Levels of TLR4, TNF-CoV, KCa2 + 3, KCa3. 1 and maxi KCa in mesenteric artery were detected by using Blot technique. Results (1) After intravenous injection of mLDL energy and dose-dependent injury to the mesenteric artery endothelium-dependent relaxation function, and in an injection amount of 1 mg/ kg and 72 hours Compared with normal saline group, pIC504.42 and Rmax (63/ 5)%, mmLDL was significantly injured by EDHF-pathway mediated vasodilation function[pIC505. 67, 0. 07 and Rmax (31% 3)%, P0.001, P0. 001]; compared with saline group, pIC505. 87, 0. 10 and Rmax (47%). Compared with 4%, mLDL was significantly injured by NO-pathway-mediated vasodilation function[pIC505. 44, 0. 12 and Rmax (31/ 4)%, P0.05). There was no significant effect. (3) Compared with saline group, pIC504.44, 0.011 and Rmax (40% 4)%, mmLDL significantly injured KCa2 +-pathway-mediated vasodilation function[pIC505. 30, 0. 13 and Rmax (18, 3)%, P0. 001, P0.01]; and pIC505. 44, 0. 18 and Rmax (21) in physiological saline group. Compared with 4%, mmLDL significantly damaged the endothelium-dependent vasodilation function of KCa3. 1-pathway[pIC504. 72, 0. 13 and Rmax (8, 2)%, P0. 01, P0.01], and the damage to maxi KCa-pathway was basic. There is no significant effect. (4) mLDL is an elastic membrane in local area injury and induces exfoliation and edema of mesenteric artery endothelial cells, leading to its endothelium-dependent relaxation. Functional damage. (5) mmLDL also induces significant upregulation of TLR4 protein expression in vascular tissues (P mLDL significantly upregulated the level of serum concentration (328,95, 28,85, P0.001) in serum as compared to the saline group of 140. 15 vs 22. 68pg/ mL; mLDL significantly upregulated the serum concentration level of IL-1 in serum (132. 75, 13.37, P 0. 01) Levels of mRNA and serum concentration of IL-1 and TNF-and IL-1 in serum Conclusion mmLDL significantly reduces the endothelium-dependent vasodilation function, and its mechanism is related to the following factors: (1) by upregulating the expression of TLR4 protein, inducing inflammatory response, injury, Ultrastructure of endothelial cells. (2) Inhibition of NO and EDHF signaling pathway. (3) down-regulation of KCa2 + 3-channel and KCa3. 1
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R54;R743.3
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相关期刊论文 前2条
1 赵丽梅;王燕;马晓真;王亚文;邓秀玲;;氧化应激损伤中电导和小电导钙激活钾通道介导的糖尿病大鼠肠系膜动脉舒张[J];南方医科大学学报;2013年07期
2 范一菲;孔德虎;陈志武;高杉;;血管内皮依赖性超极化因子的研究进展[J];中国临床药理学与治疗学;2006年10期
本文编号：2311411
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