本文選題：自發(fā)性高血壓大鼠 + 腸系膜�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：第一部分自發(fā)性高血壓大鼠腸系膜微循環(huán)的改變目的：動(dòng)態(tài)監(jiān)測(cè)并定量分析不同周齡自發(fā)性高血壓大鼠(spontaneously hypertensive rats, SHR)腸系膜微靜脈內(nèi)白細(xì)胞流變特性、真毛細(xì)血管內(nèi)紅細(xì)胞流動(dòng)速度、肥大細(xì)胞脫顆粒及微血管迂曲度的變化。方法：取3、8和13周齡的雄性SHR口Wistar大鼠,使用活體顯微鏡動(dòng)態(tài)監(jiān)測(cè)腸系膜微靜脈中白細(xì)胞流變特性、真毛細(xì)血管內(nèi)紅細(xì)胞流速、肥大細(xì)胞脫顆粒及微血管迂曲度的變化。運(yùn)用VasTrack自動(dòng)測(cè)量系統(tǒng)對(duì)動(dòng)態(tài)視頻錄像進(jìn)行定量和分析。結(jié)果：(1)白細(xì)胞流變特性的改變：3周齡的SHR與Wist ar大鼠的微靜脈內(nèi)白細(xì)胞滾動(dòng)速度、滾動(dòng)的白細(xì)胞數(shù)及微靜脈內(nèi)白細(xì)胞-內(nèi)皮接觸時(shí)間均無顯著差異。然而,與同周齡的8周齡和13周齡的Wistar大鼠相比,SHR的微靜脈內(nèi)白細(xì)胞滾動(dòng)速度明顯加快(8周齡：72.73±36.12μm/s vs 36.09±23.39μm/s, P0.01:13周齡：8133±24.37μm/s vs 35.89±11.37μm/s, P0.01),滾動(dòng)的白細(xì)胞數(shù)明顯減少(8周齡：41.3±21.79 cells/min vs 60.83±28.87 cells/min, P0.01; 13周齡 38.73±22.79 cells/min vs 56.33±29.54 cells/min,P0.01),白細(xì)胞-內(nèi)皮接觸時(shí)間明顯延長(8周齡：142.67±27.51 s/min·100μm vs 116.24±17.33 s/min·100μm, P0.01;13周齡：327.29±56.44 s/min·100μm vs 150.34±35.81 s/min·100μm, P0.01).而且,隨著SHR周齡的增大,微靜脈內(nèi)滾動(dòng)的白細(xì)胞數(shù)逐漸增多、白細(xì)胞-內(nèi)皮接觸時(shí)間逐漸延長。(2)真毛細(xì)血管內(nèi)紅細(xì)胞流速：3周齡的SHR和Wistar大鼠的真毛細(xì)血管內(nèi)紅細(xì)胞流速?zèng)]有顯著的統(tǒng)計(jì)學(xué)差異。8周齡時(shí),SHR的流速明顯高于同周齡的Wistar大鼠(1402±684μm/s vs 982±560μm/s, P0.01)。然而,13周齡時(shí),SHR的流速又低于同周齡的Wistar大鼠(874±335μm/s vs 1153±617μm/s,P0.05)。(3)肥大細(xì)胞脫顆粒：3周齡的Wistar大鼠與SHR的腸系膜內(nèi),肥大細(xì)胞計(jì)數(shù)無統(tǒng)計(jì)學(xué)差異。對(duì)于8周齡和13周齡的大鼠,SHR的肥大細(xì)胞計(jì)數(shù)明顯多于Wistar大鼠(8周齡：10.51±3.8vs8.4±2.2,P0.01；13周齡：15.6±3.8vs10.13±3.3,P0.01)。而且,8周齡和13周齡的SHR腸系膜內(nèi)的肥大細(xì)胞明顯要比3周的多。與同周齡的Wistar大鼠比,SHR腸系膜內(nèi)脫顆粒肥大細(xì)胞的百分比都升高(3周齡：75%±14%vs25%±18%,P0.01；8周齡：83%±9%vs33%±8%,P0.01；13周齡：90%±4%vs 46%±20%,P0.01)。而且隨著周齡增大,血壓增高,SHR腸系膜內(nèi)脫顆粒的肥大細(xì)胞比例也在增加。(4)微血管迂曲度：與同周齡的Wistar大鼠相比,3、8、13周齡的SHR腸系膜內(nèi)微動(dòng)脈迂曲度,均無統(tǒng)計(jì)學(xué)差異。而對(duì)于微靜脈迂曲度,僅3周齡SHR比同周齡的Wistar大鼠迂曲度大(1.83±0.49vs1.29±0.16,P0.01),其余周齡均無明顯統(tǒng)計(jì)學(xué)差異。結(jié)論：隨著高血壓進(jìn)展,白細(xì)胞滾動(dòng)速度加快、滾動(dòng)的白細(xì)胞數(shù)量減少、白細(xì)胞-內(nèi)皮接觸時(shí)間延長；真毛細(xì)血管的紅細(xì)胞流速先代償性增高、后失代償而降低；微血管周圍肥大細(xì)胞數(shù)量增加、脫顆粒細(xì)胞百分比增大；微血管的迂曲度增高主要發(fā)生于高血壓早期的微靜脈。第二部分內(nèi)皮細(xì)胞對(duì)自發(fā)性高血壓大鼠主動(dòng)脈平滑肌細(xì)胞鈣化的促進(jìn)作用及其機(jī)制的研究目的：探討內(nèi)皮細(xì)胞(endothelial cells, ECs)在自發(fā)性高血壓大鼠(SHR)主動(dòng)脈平滑肌細(xì)胞(smooth muscle cells, SMCs)鈣化中的促進(jìn)作用及其機(jī)制。方法：原代分離和培養(yǎng)8周齡的Wistar大鼠和SHR主動(dòng)脈SMCs和ECs,并以形態(tài)學(xué)特征以及a-SMA和vWF免疫熒光鑒定。將每一種屬的SMCs和ECs分為三組,分別用于共培養(yǎng)SMCs和ECs、以ECs的培養(yǎng)基條件培養(yǎng)SMCs、以及未進(jìn)行任何處理的對(duì)照培養(yǎng)。6d后,以von Kossa染色和鈣定量分析SMCs的鈣沉積程度,以堿性磷酸酶(alkaline phosphatase, ALP)活力分析SMCs成骨樣細(xì)胞表型轉(zhuǎn)化程度,以、Vestern blot分析BMP2、Runx2、Msx2、 MMP-2、MMP-9、 OPN、MGP在SMCs的表達(dá)水平變化,以及BMP2、MMP-2、MMP-9、OPN、MGP在ECs的表達(dá)水平變化情況。結(jié)果：(1)成功分離培養(yǎng)Wistar大鼠和SHR主動(dòng)脈的SMCs (rat aortic smooth muscle cells, RASMCs)和ECs (rat aortic endothelial cells, RAECs),經(jīng)形態(tài)學(xué)和免疫熒光鑒定后,細(xì)胞無混雜,無其它細(xì)胞污染。(2)鈣沉積改變：當(dāng)RASMCs與RAECs共培養(yǎng)6 d后,與對(duì)照組相比,SHR RASMCs的細(xì)胞外鈣沉積明顯增加(鈣含量：58.38±7.95 μg/mg protein vs 24.30±3.22μg/mg protein, P 0.01),用ECs培養(yǎng)基條件培養(yǎng)SMCs時(shí),SHR RASMCs的鈣化程度依然要比對(duì)照組高(鈣含量：30.15±5.42μg/mg protein vs 24.30±3.22μg/mg protein, P 0.01),而Wistar RASMCs在共培養(yǎng)和條件培養(yǎng)時(shí)均無統(tǒng)計(jì)學(xué)意義的變化。(3)ALP活力的改變：當(dāng)RAECs與RASMCs共培養(yǎng)6 d后,SHR RASMCs的ALP活力明顯高于對(duì)照組(106.28±16.63 unit/mg protein vs 75.87±6.06 unit/mg protein,P0.01),而且當(dāng)RASMCs用RAECs的培養(yǎng)基條件培養(yǎng)時(shí),SHR RASMCs的ALP活力(86.73±9.15unit/mg protein)依然比對(duì)照組高(P0.05)。然而,共培養(yǎng)和條件培養(yǎng)的WistarRASMCs的ALP活力改變卻無統(tǒng)計(jì)學(xué)意義。(4)BMP2、Runx2、Msx2的表達(dá)水平變化：共培養(yǎng)6 d后,SHR RASMCs中BMP2、Runx2和Msx2的表達(dá)水平(相對(duì)于對(duì)照表達(dá)水平的倍數(shù))顯著高于VVistar RASMCs (BMP2:2.88±0.12 fold vs 1.43±0.21 fold, P0.01; Runx2:1.76±0.09 fold vs 1.26±0.08 fold, P0.01; Msx2: 3.10±0.45 fold vs 1.53±0.48 fold, P0.01)。而且當(dāng)SHR RASMCs 以SHR RAECs培養(yǎng)基進(jìn)行條件培養(yǎng)時(shí),這些蛋白的表達(dá)水平依然要比Wistar RASMCs高(BMP2:1.84±0.37 fold vs 1.22±0.22 fold, P0.05; Runx2:1.39±0.02 fold vs 1.07±0.13 fold, P0.05; Msx2:1.94±0.34 fold vs 1.26±0.19 fold, P0.01)。與SMCs共培養(yǎng)6 d后,SHR RAECs中BMP2的表達(dá)水平也明顯高于Wistar RAECs (3.03±0.52 fold vs 1.19±0.29 fold, P0.01)。(5) MMP-2和MMP-9表達(dá)水平的變化：無論共培養(yǎng)還是條件培養(yǎng),MMP-2和MMP-9在SHR RASMCs中的表達(dá)水平與Wistar RASMCs相比,差異無統(tǒng)計(jì)學(xué)意義。但在SHR RAECs的表達(dá)水平卻明顯高于Vistar RAECs (MMP-2:2.18±0.48 fold vs 1.38±0.38 fold, P0.01; MMP-9:1.84±0.40 fold vs 1.16±0.21 fold,P0.01)。(6) OPN和MGP表達(dá)水平的變化：當(dāng)共培養(yǎng)6 d以后,OPN和MGP在SHR RASMCs的表達(dá)水平明顯高于Wistar RASMCs (MGP:3.13±0.51 fold vs 1.51±0.37 fold,P0.01; OPN:1.91±0.16 fold vs 1.56±0.23 fold, P0.05)。而且它們?cè)赟HR RAECs的表達(dá)水平也明顯高于Wistar RAECs (MGP:2.33±0.34 fold vs 1.26±0.48 fold, P0.01; OPN:1.74±0.09 fold vs 1.28±0.32 fold, P0.05)。結(jié)論：本研究首次證實(shí)SHR RAECs具有促進(jìn)SHR RASMCs鈣化的能力。其促進(jìn)作用是通過增強(qiáng)BMP2-Runx2和BMP2-Msx2信號(hào)通路中蛋白的表達(dá)來實(shí)現(xiàn)的。SHR RAECs分泌的MMP-2和MMP-9也參與了這一促進(jìn)鈣化的過程。而且代償性增高的MGP和OPN不能阻斷SHR RAECs 對(duì)SHR RASMCs鈣化的促進(jìn)作用。
[Abstract]:The first part of the mesenteric microcirculation in spontaneously hypertensive rats. Objective: to change the different week old spontaneously hypertensive rats (spontaneously dynamic monitoring and quantitative analysis of hypertensive, rats, SHR) white blood cell rheological characteristics of mesenteric veins, capillary red blood cell flow velocity, the change of mast cell degranulation and microvessel tortuosity. Methods: male SHR Wistar rats were 3,8 and 13 weeks of age, white blood cell rheological properties using the dynamic monitoring of mesenteric venules in intravital microscopy, really red cell velocity in the capillaries, changes of mast cell degranulation and microvessel tortuosity. Using the VasTrack automatic measurement system for the quantitative and dynamic analysis of the video. Results: (1) white blood cell rheology change: Micro vein 3 week old SHR and Wist ar rats in leukocyte rolling speed, the number of leukocyte rolling and micro vein in white 緇嗚優(yōu)-鍐呯毊鎺ヨЕ鏃墮棿鍧囨棤鏄捐憲宸紓.鐒惰，

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