本文關鍵詞： 高血壓 微循環(huán) 微淋巴管 自律運動 自發(fā)性高血壓 血管鈣化 血管平滑肌細胞 基質金屬蛋白酶-2 凋亡　出處：《北京協(xié)和醫(yī)學院》2015年博士論文　論文類型：學位論文

【摘要】：第一部分 自發(fā)性高血壓大鼠腸系膜微淋巴管自律運動的改變目的動態(tài)監(jiān)測并定量分析不同周齡自發(fā)性高血壓大鼠(SHR)腸系膜微淋巴管自律運動的特點。方法取3周、8周和13周的雄性Wistar大鼠和SHR,麻醉、固定并暴露腸系膜后,在活體顯微鏡下動態(tài)監(jiān)測腸系膜微淋巴管的自律運動并錄像,運用VasTrack自動測量系統(tǒng)對錄像進行定量和分析。分析的指標包括微淋巴管自律運動的相對振幅、頻率、收縮期及舒張期的幅度、時長和速度,并根據(jù)頻率、最大舒張管徑和最大收縮管徑計算出腸系膜微淋巴管收縮功能的兩個指標：收縮分數(shù)和總收縮活性指數(shù)。結果(1)SHR與Wistar大鼠腸系膜微淋巴管自律運動在不同周齡的差別：3周齡SHR與3周齡Wistar大鼠微淋巴管自律運動在各個指標方面均無顯著差異(均為P0.05)。到8周齡時,SHR微淋巴管自律運動的相對振幅、收縮幅度、收縮時長、收縮速度、舒張幅度、舒張速度及總收縮活性指數(shù)均顯著低于Wistar大鼠(分別為P＜0.001,P＜0.01, P＜0.05,P0.01, P＜0.001,P0.01,P0.05), SHR微淋巴管自律運動的頻率、舒張時長和收縮分數(shù)與Wistar大鼠無顯著差異(均為P0.05)。到13周齡時,SHR微淋巴管自律運動的相對振幅、收縮幅度、收縮時長、舒張幅度、舒張時長和收縮分數(shù)顯著低于Wistar大鼠(均為P0.001),頻率和舒張速度顯著高于Wistar大鼠(均為P0.05),收縮速度和總收縮活性指數(shù)與Wistar大鼠無顯著差異(均為P0.05)。(2)SHR腸系膜微淋巴管自律運動在不同周齡的差別：與3周齡SHR相比,8周齡SHR淋巴管自律運動的相對振幅、頻率、舒張幅度、舒張速度和總收縮活性指數(shù)顯著降低(分別為P＜0.05, P0.05, P0.05, P0.01,P＜0.01)。8周齡SHR的收縮幅度、收縮時長、收縮速度、舒張時長和收縮分數(shù)與3周齡SHR相比,均無顯著差異(均為P0.05)。13周齡SHR的收縮頻率、收縮速度、舒張速度和總收縮活性指數(shù)均顯著高于8周齡SHR(分別為P0.05,P0.05,P0.01,P0.01)；13周齡SHR的收縮時長、舒張時長和收縮分數(shù)均顯著低于8周齡SHR(分別為P0.05,P0.01,P0.05)；13周齡SHR的相對振幅、收縮幅度和舒張幅度與8周齡SHR相比,均無顯著差異(均為P0.05)。結論(1)SHR與Wistar大鼠的腸系膜微淋巴管均存在自律運動。(2)高血壓可能是引起SHR腸系膜微淋巴管自律運動障礙的一個重要因素。(3)高血壓發(fā)生后,SHR腸系膜微淋巴管自律運動的舒張期活動減弱,收縮功能顯著下降。(4)高血壓進展過程中,SHR微淋巴管自律運動的紊亂體現(xiàn)在收縮功能增強,而收縮功能增強更有利于機體內環(huán)境的穩(wěn)態(tài)。第二部分 基質金屬蛋白酶抑制劑通過下調BMP-2抑制大鼠主動脈血管平滑肌細胞鈣化目的探討基質金屬蛋白酶(MMPs)在血管鈣化中的作用及其機制。方法取8周齡雄性VVistar大鼠,貼塊法培養(yǎng)主動脈血管平滑肌細胞(VSMCs),形態(tài)學和免疫熒光法鑒定VSMCs。對照組的VSMCs采用DMEM培養(yǎng)基,鈣化組用p-甘油磷酸(p-GP)誘導VSMCs發(fā)生鈣化,MMPs抑制劑組用β-GP+MMPs抑制劑多西環(huán)素(doxycycline, Doxy)。培養(yǎng)12天后,對VSMCs進行von kossa染色和鈣含量分析,以檢測VSMCs鈣化。用試劑盒檢測堿性磷酸酶(ALP)活性,用、Westernblot檢測a-SMA, Desmin及ALP的蛋白表達,以觀察VSMCs表型改變。、Western blot檢測MMP-2、MMP-9蛋白表達水平,明膠酶譜法檢測MMP-2、MMP-9的酶活性,以觀察MMPs抑制劑多西環(huán)素的抑制效果。Western blot檢測骨形態(tài)發(fā)生蛋白-2(BMP-2)及其受體、RUNX2. Msx-2的表達水平,以探討MMPs印制后BMP-2通路的變化。用TUNEL法檢測VSMCs凋亡狀況,Western blot檢測凋亡相關蛋白caspase-3、cleaved-caspase-3、Bax和Bcl-2的表達,caspase-3活性試劑盒檢測caspase-3活性。結果(1)培養(yǎng)的細胞鏡下呈梭形、紡錘形或菱形,細胞有多個突起,胞漿豐富,12天后,細胞出現(xiàn)“峰和谷”樣的生長模式。平滑肌a-actin (smooth muscle a-actin, SM a-actin)抗體免疫熒光染色后,可見胞漿呈綠色熒光,胞漿內見大量與細胞長軸平行的纖維細絲,表明培養(yǎng)的細胞為VSMCs.(2)鈣化組的鈣含量和鈣化面積顯著高于對照組(P＜0.001,P＜0.001); MMPs抑制劑組的鈣含量和鈣化面積顯著低于鈣化組(P0.001,P0.001)。鈣化組可見VSMCs表型改變,而MMPs抑制導致VSMCs表型改變減輕。(3)Western blot檢測結果發(fā)現(xiàn),與鈣化組相比,MMPs抑制導致MMP-2和MMP-9蛋白表達降低(P0.01,P0.01)；明膠酶譜檢測結果表明,與鈣化組相比,MMPs抑制導致MMP-2和MMP-9酶活性降低(P0.01,P＜0.01)。(4) MMPs抑制導致BMP-2、RUNX2和Msx-2蛋白表達降低(P＜0.01,P＜0.001, P0.01)。 (5)BMP-2可促進VSMCs表達RUNX2和Msx-2(P0.01, P0.01),而noggin可減少兩者的表達(P0.05, P0.05)。(6) TUNEL檢測結果表明,鈣化組的凋亡細胞比例顯著高于對照組(P0.001)；與鈣化組相比,MMPs抑制劑組的凋亡細胞比例顯著降低(P0.001)。鈣化組的caspase-3活性及cleaved-caspase-3蛋白表達均顯著關于對照組(P0.001,P0.001)。與對照組相比,鈣化組的Bax表達顯著升高、Bcl-2表達顯著降低(P0.01,P0.05)。結論(1)成功培養(yǎng)了大鼠VSMC并誘導鈣化。(2)抑制MMPs可減輕VSMCs鈣化,并減輕鈣化過程中VSMCs的表型改變。(3)抑制MMPs可通過下調BMP-2.進而降低RUNX2口Msx-2的表達,減輕血管鈣化。(4)抑制MMPs可能通過減輕VSMCs凋亡來減輕血管鈣化。
[Abstract]:Different week old spontaneously hypertensive rats to dynamic monitoring and quantitative analysis of changes in the first part of spontaneously hypertensive rat mesentery micro lymphatic vasomotion (SHR) characteristics of mesentery micro lymphatic vasomotion. Methods 3 weeks, 8 weeks and 13 weeks of male Wistar rats and SHR, anesthesia, fixed and mesentery after exposure in vivo microscopy, dynamic monitoring of mesentery micro lymphatic self motion and video, using the VasTrack automatic measurement system for quantitative analysis of video analysis. And the indexes including relative amplitude, microlymphatics vasomotion frequency, systolic and diastolic amplitude, duration and speed, and according to the frequency, maximum diastolic diameter and maximum the contraction diameter to calculate two indicators of mesentery micro lymphatic systolic function: the systolic fraction and total systolic activity index. Results (1) SHR and Wistar in rat mesentery micro lymphatic self-discipline The difference in motion in different age: 3 week old SHR and Wistar rats aged 3 weeks microlymphatics vasomotion were no significant differences in all indexes (P0.05). By 8 weeks of age, relative amplitude, SHR microlymphatics vasomotion contraction amplitude, contraction velocity, shrinkage, diastolic amplitude, diastolic velocity and total systolic activity index were significantly lower than that of Wistar rats (P < 0.001, P < 0.01, P < 0.05, P0.01 < 0.001, P, P0.01, P0.05, SHR) microlymphatics vasomotion frequency, duration and diastolic systolic fraction and Wistar rats no significant the difference (P0.05). By 13 weeks of age, relative amplitude, SHR microlymphatics vasomotion contraction, contraction duration, diastolic amplitude, duration and diastolic systolic fraction was significantly lower than that of Wistar rats (P0.001), diastolic velocity and frequency was significantly higher than that of Wistar rats (P0.05) the systolic velocity and total shrinkage, active index 鏁頒笌Wistar澶ч紶鏃犳樉钁楀樊寮，

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