KCNQ4钾离子通道在腹主动脉瘤形成中的作用初探

发布时间：2018-06-05 14:12

本文选题：离子通道 + 腹主动脉瘤　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:腹主动脉瘤(abdominal aortic aneurysms,AAA)是一种血管炎症性疾病,KCNQ4离子通道高表达于血管平滑肌细胞中,本实验采用微渗泵在体灌注血管紧张素II(angiotensin II,Ang II)的方法建立小鼠AAA模型,观察KCNQ4~(-/-)小鼠和WT小鼠动脉瘤的发病率及相关形态学的改变,探讨KCNQ4离子通道在腹主动脉瘤病理过程中的作用。方法:1小鼠腹主动脉瘤模型建立随机选取10周龄左右雄性小鼠,提取耳片组织基因组DNA,采用聚合酶链反应(polymerase chain reaction,PCR)技术进行小鼠基因型鉴定。按每只小鼠1000 ng/min/kg的用量经皮下植入含有Ang II的微渗泵。于植泵后0、7、14、21、28天检测小鼠收缩压(systolic blood pressure,SBP)和舒张压(diastolic blood pressure,DBP);在麻醉状态下行小鼠腹主动脉超声检测血管直径。2血管形态观察及免疫化学和免疫荧光染色小鼠植泵28天后,麻醉处死小鼠,取胸腹主动脉,采集血管大体图像,制备石蜡组织切片后进行HE染色和EVG染色,观察血管形态和弹力层变化;免疫组织化学染色,观察小鼠血管中KCNQ4、CD68、IL-6、VCAM-1、ICAM-1的表达变化。制备主动脉冰冻切片,免疫荧光染色观察每组小鼠血管中I型胶原、III型胶原、MMPs、VCAM-1、ICAM-1、CD68、IL-6的表达情况。3统计学处理所有实验数据均采用SPSS 20.0统计软件进行处理。计量资料两样本均数比较采用t检验,多样本均数比较采用单因素方差分析(one way ANOVA)。计数资料以百分率表示,采用χ2检验。P0.05为差异有统计学意义。结果:1 KCNQ4~(-/-)小鼠基础血压高于WT小鼠。在Ang II灌注0、7、14天时KCNQ4~(-/-)小鼠的收缩压均明显高出WT小鼠基础收缩压10 mm Hg左右,有明显统计学差异(P0.05),而二者的基础舒张压无显著统计学差异(P0.05)。但在植泵后第21天,28天,KCNQ4~(-/-)小鼠与WT小鼠的血压差异消失(P0.05)。为进一步确定KCNQ4通道在调节血压中的作用,我们选用KCNQ4通道特异性开放剂RTG,观察其对血压的影响。结果显示:RTG能够引起WT小鼠基础收缩压及注入Ang II形成高血压后收缩压显著降低约10mm Hg,然而RTG对KCNQ4~(-/-)小鼠的基础收缩压及注入Ang II形成高血压后收缩压无显著性影响,RTG对WT小鼠及KCNQ4~(-/-)小鼠基础舒张压及注入Ang II形成高血压后舒张压均无显著性影响。结果提示KCNQ4敲除后可引起小鼠血压升高,而KCNQ4通道开放剂能够显著降低小鼠血压。2敲除KCNQ4可抑制Ang II诱发AAA。植泵后28天,发现Ang II灌注WT组小鼠AAA发病率为30%,明显高于Ang II灌注KCNQ4~(-/-)组和生理盐水组。小鼠超声检测和HE染色结果显示:Ang II灌注组小鼠腹主动脉血管直径明显大于生理盐水组,并且Ang II灌注组中WT小鼠的血管管腔较KCNQ4~(-/-)小鼠显著增加(P0.05),且管壁增加。此外,EVG和荧光染色结果均显示:灌注Ang II形成动脉瘤的WT小鼠血管弹力层断裂明显,而KCNQ4~(-/-)小鼠未见任何血管弹力层断裂现象。免疫荧光染色显示:灌注Ang II形成动脉瘤的WT小鼠I型胶原和III型胶原均较KCNQ4~(-/-)小鼠明显减少(P0.05)。3灌注Ang II后,形成动脉瘤小鼠血管KCNQ4蛋白表达水平高于未形成动脉瘤小鼠。免疫组织化学染色显示:灌注Ang II后形成AAA的WT小鼠血管中KCNQ4蛋白表达水平明显高于未形成AAA的WT小鼠(P0.05)。4 KCNQ4缺陷可抑制Ang II诱导血管炎症引发AAA的形成。采用免疫组织化学染色和免疫组织荧光染色技术检测CD68、IL-6、VCAM-1、ICAM-1、MMP2、MMP9蛋白表达情况。结果显示:灌注Ang II后,形成动脉瘤的WT组小鼠血管组织中CD68、IL-6、VCAM-1、ICAM-1、MMP2、MMP9蛋白表达均明显高于KCNQ4~(-/-)小鼠(P0.05)。结论:1 KCNQ4敲除可引起小鼠血压升高,并且给予KCNQ4通道开放剂能够降低血压。2 KCNQ4敲除可降低Ang II诱发AAA的发生率。3 KCNQ4敲除可抑制血管炎性因子和粘附分子的表达,减轻血管炎性反应。
[Abstract]:Objective: abdominal aortic aneurysms (AAA) is an vasculitis disease. The KCNQ4 ion channel is highly expressed in vascular smooth muscle cells. This experiment uses microosmotic pump in vivo perfusion of angiotensin II (angiotensin II, Ang II) to establish the AAA model of mice and observe the pathogenesis of the aneurysm in mice and mice. The role of KCNQ4 ion channel in the pathological process of abdominal aortic aneurysm was investigated. Methods: 1 mouse abdominal aortic aneurysm model was set up to select the 10 weeks old male mice randomly and extract the genomic DNA of the ear tissue, and the polymerase chain reaction (PCR) technique was used to identify the genotypes of the mice. The microosmotic pump containing Ang II was implanted subcutaneously at the dosage of 1000 ng/min/kg in each mouse. The systolic pressure (systolic blood pressure, SBP) and diastolic pressure (diastolic blood pressure, DBP) were detected on 0,7,14,21,28 days after the implantation of the pump, and the vascular morphology and immuno chemistry of the abdominal aorta in the abdominal aorta of mice were observed and immuno chemical and immunocytochemistry under anesthesia. 28 days after immunofluorescence staining in mice, the mice were killed and the abdominal aorta was taken to collect the general images of the blood vessels. The paraffin tissue sections were prepared by HE staining and EVG staining. The changes of vascular morphology and elastic layer were observed. The changes in the expression of KCNQ4, CD68, IL-6, VCAM-1 and ICAM-1 in the blood vessels of mice were observed by immunohistochemistry. The aorta was prepared. Frozen section, immunofluorescence staining was used to observe the expression of type I collagen, type III collagen, MMPs, VCAM-1, ICAM-1, CD68, IL-6 in each group of mice by immunofluorescence. All the experimental data were treated with SPSS 20 statistical software. The average number of two samples was compared with t test, and a single factor analysis of variance was used. One way ANOVA). The count data were expressed as a percentage, and the x 2 test of.P0.05 was statistically significant. Results: the basal blood pressure of 1 KCNQ4~ (- / -) mice was higher than that of WT mice. The systolic pressure of KCNQ4~ (- / -) mice was significantly higher than that of WT mice at 0,7,14 days when Ang II was perfused. There was no significant difference in basic diastolic blood pressure (P0.05). But the blood pressure difference between KCNQ4~ (- / -) mice and WT mice disappeared (P0.05) at twenty-first days after the implantation of the pump. To further determine the role of the KCNQ4 channel in regulating blood pressure, we selected the KCNQ4 channel specific opening agent RTG to observe its effect on blood pressure. The results showed that RTG could cause WT. The systolic pressure and the injection of Ang II in mice were significantly reduced by 10mm Hg, but RTG had no significant effect on the systolic pressure of KCNQ4~ (- / -) mice and the injection of Ang II in the formation of hypertension. The diastolic pressure of WT mice and KCNQ4~ (- / -) mice and the formation of the diastolic pressure after hypertension were not significant. The results suggested that the KCNQ4 knockout could cause the increase of blood pressure in mice, while the KCNQ4 channel opener could significantly reduce the blood pressure of the mice with the.2 knockout KCNQ4 and the Ang II induced AAA. implantation for 28 days. It was found that the AAA incidence of Ang II perfusion WT group was 30%, which was significantly higher than that in the Ang perfusion (- / -) group and the saline group. The staining results showed that the vascular diameter of abdominal aorta in Ang II perfusion group was significantly greater than that of normal saline group, and the vascular lumen of WT mice in Ang II perfusion group increased significantly (P0.05), and the tube wall increased. In addition, EVG and fluorescence staining results showed that the WT mouse vascular elastic layer fractured with Ang II formed an aneurysm. The KCNQ4~ (- / -) mice did not show any vascular elastic layer rupture. Immunofluorescence staining showed that the I collagen and III collagen in the WT mice perfused with the aneurysm of II were significantly lower than the KCNQ4~ (/ -) mice (P0.05) and.3 perfusion Ang II, and the expression level of the blood tube KCNQ4 protein in the aneurysm mice was higher than that of the non formed aneurysm mice. Histochemical staining showed that the expression of KCNQ4 protein in the blood vessels of WT mice that formed AAA after Ang II was significantly higher than that of WT mice without AAA (P0.05).4 KCNQ4 defects could inhibit the formation of Ang II induced vascular inflammation. The expression of MP9 protein showed that after perfusion of Ang II, the expression of CD68, IL-6, VCAM-1, ICAM-1, MMP2, MMP9 protein in the vascular tissue of the WT group that formed the aneurysm was significantly higher than that in KCNQ4~ (- / -) mice (P0.05). Conclusion: the 1 knockout can cause the increase of blood pressure in mice and the lowering of blood pressure in mice. Low Ang II induced the incidence of AAA..3 KCNQ4 knockout can inhibit the expression of vascular inflammatory factors and adhesion molecules, and alleviate the inflammatory reaction.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.16

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