發(fā)布時(shí)間：2018-06-05 14:12

  本文選題：離子通道 + 腹主動脈瘤　； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:腹主動脈瘤(abdominal aortic aneurysms,AAA)是一種血管炎癥性疾病,KCNQ4離子通道高表達(dá)于血管平滑肌細(xì)胞中,本實(shí)驗(yàn)采用微滲泵在體灌注血管緊張素II(angiotensin II,Ang II)的方法建立小鼠AAA模型,觀察KCNQ4~(-/-)小鼠和WT小鼠動脈瘤的發(fā)病率及相關(guān)形態(tài)學(xué)的改變,探討KCNQ4離子通道在腹主動脈瘤病理過程中的作用。方法:1小鼠腹主動脈瘤模型建立隨機(jī)選取10周齡左右雄性小鼠,提取耳片組織基因組DNA,采用聚合酶鏈反應(yīng)(polymerase chain reaction,PCR)技術(shù)進(jìn)行小鼠基因型鑒定。按每只小鼠1000 ng/min/kg的用量經(jīng)皮下植入含有Ang II的微滲泵。于植泵后0、7、14、21、28天檢測小鼠收縮壓(systolic blood pressure,SBP)和舒張壓(diastolic blood pressure,DBP);在麻醉狀態(tài)下行小鼠腹主動脈超聲檢測血管直徑。2血管形態(tài)觀察及免疫化學(xué)和免疫熒光染色小鼠植泵28天后,麻醉處死小鼠,取胸腹主動脈,采集血管大體圖像,制備石蠟組織切片后進(jìn)行HE染色和EVG染色,觀察血管形態(tài)和彈力層變化;免疫組織化學(xué)染色,觀察小鼠血管中KCNQ4、CD68、IL-6、VCAM-1、ICAM-1的表達(dá)變化。制備主動脈冰凍切片,免疫熒光染色觀察每組小鼠血管中I型膠原、III型膠原、MMPs、VCAM-1、ICAM-1、CD68、IL-6的表達(dá)情況。3統(tǒng)計(jì)學(xué)處理所有實(shí)驗(yàn)數(shù)據(jù)均采用SPSS 20.0統(tǒng)計(jì)軟件進(jìn)行處理。計(jì)量資料兩樣本均數(shù)比較采用t檢驗(yàn),多樣本均數(shù)比較采用單因素方差分析(one way ANOVA)。計(jì)數(shù)資料以百分率表示,采用χ2檢驗(yàn)。P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1 KCNQ4~(-/-)小鼠基礎(chǔ)血壓高于WT小鼠。在Ang II灌注0、7、14天時(shí)KCNQ4~(-/-)小鼠的收縮壓均明顯高出WT小鼠基礎(chǔ)收縮壓10 mm Hg左右,有明顯統(tǒng)計(jì)學(xué)差異(P0.05),而二者的基礎(chǔ)舒張壓無顯著統(tǒng)計(jì)學(xué)差異(P0.05)。但在植泵后第21天,28天,KCNQ4~(-/-)小鼠與WT小鼠的血壓差異消失(P0.05)。為進(jìn)一步確定KCNQ4通道在調(diào)節(jié)血壓中的作用,我們選用KCNQ4通道特異性開放劑RTG,觀察其對血壓的影響。結(jié)果顯示:RTG能夠引起WT小鼠基礎(chǔ)收縮壓及注入Ang II形成高血壓后收縮壓顯著降低約10mm Hg,然而RTG對KCNQ4~(-/-)小鼠的基礎(chǔ)收縮壓及注入Ang II形成高血壓后收縮壓無顯著性影響,RTG對WT小鼠及KCNQ4~(-/-)小鼠基礎(chǔ)舒張壓及注入Ang II形成高血壓后舒張壓均無顯著性影響。結(jié)果提示KCNQ4敲除后可引起小鼠血壓升高,而KCNQ4通道開放劑能夠顯著降低小鼠血壓。2敲除KCNQ4可抑制Ang II誘發(fā)AAA。植泵后28天,發(fā)現(xiàn)Ang II灌注WT組小鼠AAA發(fā)病率為30%,明顯高于Ang II灌注KCNQ4~(-/-)組和生理鹽水組。小鼠超聲檢測和HE染色結(jié)果顯示:Ang II灌注組小鼠腹主動脈血管直徑明顯大于生理鹽水組,并且Ang II灌注組中WT小鼠的血管管腔較KCNQ4~(-/-)小鼠顯著增加(P0.05),且管壁增加。此外,EVG和熒光染色結(jié)果均顯示:灌注Ang II形成動脈瘤的WT小鼠血管彈力層斷裂明顯,而KCNQ4~(-/-)小鼠未見任何血管彈力層斷裂現(xiàn)象。免疫熒光染色顯示:灌注Ang II形成動脈瘤的WT小鼠I型膠原和III型膠原均較KCNQ4~(-/-)小鼠明顯減少(P0.05)。3灌注Ang II后,形成動脈瘤小鼠血管KCNQ4蛋白表達(dá)水平高于未形成動脈瘤小鼠。免疫組織化學(xué)染色顯示:灌注Ang II后形成AAA的WT小鼠血管中KCNQ4蛋白表達(dá)水平明顯高于未形成AAA的WT小鼠(P0.05)。4 KCNQ4缺陷可抑制Ang II誘導(dǎo)血管炎癥引發(fā)AAA的形成。采用免疫組織化學(xué)染色和免疫組織熒光染色技術(shù)檢測CD68、IL-6、VCAM-1、ICAM-1、MMP2、MMP9蛋白表達(dá)情況。結(jié)果顯示:灌注Ang II后,形成動脈瘤的WT組小鼠血管組織中CD68、IL-6、VCAM-1、ICAM-1、MMP2、MMP9蛋白表達(dá)均明顯高于KCNQ4~(-/-)小鼠(P0.05)。結(jié)論:1 KCNQ4敲除可引起小鼠血壓升高,并且給予KCNQ4通道開放劑能夠降低血壓。2 KCNQ4敲除可降低Ang II誘發(fā)AAA的發(fā)生率。3 KCNQ4敲除可抑制血管炎性因子和粘附分子的表達(dá),減輕血管炎性反應(yīng)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R543.16

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本文編號：1982258