【摘要】：背景： 1、制造小口徑組織工程血管是當(dāng)前的研究熱點(diǎn)，仿生應(yīng)力對(duì)小口徑組織工程血管的生長(zhǎng)和成熟非常重要，但目前的很多灌注培養(yǎng)式生物反應(yīng)器尚不能提供模擬生理血流的動(dòng)力學(xué)環(huán)境，而且未提供足夠的生物學(xué)數(shù)據(jù)來(lái)證明其優(yōu)越性； 2、小口徑組織工程血管的功能和生物力學(xué)特性都離不開(kāi)平滑肌細(xì)胞的良好增殖，但平滑肌細(xì)胞在接種到支架材料后的增殖情況并不令人滿意，從而影響小口徑組織工程血管的功能和生物力學(xué)強(qiáng)度。目的： 1、構(gòu)建小口徑組織工程血管，體外仿生搏動(dòng)流體應(yīng)力條件下進(jìn)行培養(yǎng)，觀察對(duì)小口徑組織工程血管的生物學(xué)作用； 2、構(gòu)建平滑肌細(xì)胞受仿生搏動(dòng)應(yīng)力模型，探討仿生搏動(dòng)應(yīng)力促平滑肌細(xì)胞增殖的轉(zhuǎn)錄后調(diào)控機(jī)制。 方法： 1、（1）酶消化法分離獲得內(nèi)皮細(xì)胞和平滑肌細(xì)胞，顯微鏡觀察細(xì)胞形態(tài)，VIII因子相關(guān)抗原免疫組化染色和α肌動(dòng)蛋白免疫熒光染色分別鑒定內(nèi)皮細(xì)胞和平滑肌細(xì)胞的表型； （2）用不使用胰酶的化學(xué)方法制備脫細(xì)胞兔主動(dòng)脈支架材料，觀察大體形態(tài)，行組織學(xué)觀察、掃描電鏡和透射電鏡觀察，CCK-8法評(píng)價(jià)生物相容性，計(jì)算接種細(xì)胞黏附率，評(píng)價(jià)親水性，接種內(nèi)皮細(xì)胞和平滑肌細(xì)胞后行組織學(xué)觀察、掃描電鏡和透射電鏡觀察； （3）內(nèi)皮細(xì)胞和平滑肌細(xì)胞接種到脫細(xì)胞兔主動(dòng)脈，構(gòu)建小口徑組織工程血管，置入我們自行研制的小口徑組織工程血管生物反應(yīng)器，分別給予仿生搏動(dòng)流體應(yīng)力和靜態(tài)培養(yǎng)后檢測(cè)：觀察大體形態(tài)，行HE染色、Masson染色和彈力纖維染色，掃描電鏡觀察，Real-time PCR檢測(cè)Collagen I、III、IV、堿性成纖維細(xì)胞生長(zhǎng)因子和內(nèi)皮素-1mRNA表達(dá)，免疫熒光檢測(cè)鈣調(diào)蛋白、α肌動(dòng)蛋白、VIII因子相關(guān)抗原和血管性假性血友病因子，NO和6-keto-PGF1a濃度檢測(cè)，血小板黏附實(shí)驗(yàn)，生物力學(xué)檢測(cè)。 2、（1）分組：①動(dòng)態(tài)培養(yǎng)組；②靜態(tài)培養(yǎng)組；③動(dòng)態(tài)培養(yǎng)+慢病毒組；④靜態(tài)培養(yǎng)+慢病毒組；⑤動(dòng)態(tài)培養(yǎng)+IGF-1R siRNA組；⑥靜態(tài)培養(yǎng)+IGF-1R siRNA組。檢測(cè)：細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡，Real-time PCR檢測(cè)IGF-1R、骨橋蛋白mRNA，Western Blot檢測(cè)IGF-1R、骨橋蛋白、pTyr-IGF-1R、p-AKT； （2）分組：①動(dòng)態(tài)培養(yǎng)組；②靜態(tài)培養(yǎng)組。檢測(cè)：microRNA芯片檢測(cè)，microRNA數(shù)據(jù)庫(kù)預(yù)測(cè)，Real-time PCR檢測(cè)差異表達(dá)的microRNA； （3）分組：①動(dòng)態(tài)培養(yǎng)組；②靜態(tài)培養(yǎng)組；③動(dòng)態(tài)培養(yǎng)+慢病毒組；④靜態(tài)培養(yǎng)+慢病毒組；⑤動(dòng)態(tài)培養(yǎng)+microRNA-223組；⑥靜態(tài)培養(yǎng)+microRNA-223組；⑦動(dòng)態(tài)培養(yǎng)+microRNA-153組；⑧靜態(tài)培養(yǎng)+microRNA-153組。檢測(cè)：靶基因驗(yàn)證實(shí)驗(yàn)、細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡，Real-time PCR檢測(cè)IGF-1R、骨橋蛋白mRNA，WesternBlot檢測(cè)IGF-1R、骨橋蛋白、pTyr-IGF-1R、p-AKT。 結(jié)果： 1、（1）平滑肌細(xì)胞成束平行的排列，呈典型的“峰與谷”樣生長(zhǎng)，α肌動(dòng)蛋白免疫熒光呈現(xiàn)強(qiáng)陽(yáng)性；內(nèi)皮細(xì)胞呈典型的“鵝卵石”樣生長(zhǎng)，排列密集，VIII因子相關(guān)抗原免疫組化染色為強(qiáng)陽(yáng)性； （2）①脫細(xì)胞兔主動(dòng)脈質(zhì)地與新鮮兔主動(dòng)脈相比無(wú)明顯變化；②HE染色：結(jié)構(gòu)疏松，質(zhì)地均勻，自身細(xì)胞基本完全脫除；Masson染色：結(jié)構(gòu)疏松，膠原纖維之間可見(jiàn)較多空隙；彈力纖維染色：結(jié)構(gòu)疏松，彈力纖維之間可見(jiàn)較多空隙；③掃描電鏡檢測(cè)：外表面毛糙，主要由粗大的膠原纖維交織而成，孔隙比內(nèi)表面多，內(nèi)表面光滑；④對(duì)平滑肌細(xì)胞和內(nèi)皮細(xì)胞的細(xì)胞毒性評(píng)價(jià)為0～1級(jí)；⑤平滑肌細(xì)胞和內(nèi)皮細(xì)胞的黏附率分別為64.32%±2.03%和52.77%±1.19%；⑥種子細(xì)胞-脫細(xì)胞兔主動(dòng)脈復(fù)合物的檢測(cè)：HE染色：平滑肌細(xì)胞生長(zhǎng)良好，并有部分遷移至管壁內(nèi)部，內(nèi)皮細(xì)胞附著于內(nèi)表面，生長(zhǎng)良好；掃描電鏡：外表面和內(nèi)表面均被平滑肌細(xì)胞和內(nèi)皮細(xì)胞附著，并且部分平滑肌細(xì)胞向支架表面的孔隙內(nèi)生長(zhǎng)；透射電鏡：平滑肌細(xì)胞胞漿內(nèi)富含肌絲，縱向平行排列，胞內(nèi)有密斑和密體，內(nèi)皮細(xì)胞間存在細(xì)胞連接，胞漿內(nèi)可見(jiàn)W-P小體；⑦親水性：浸泡15分鐘后吸水率227±21.2%，浸泡12小時(shí)后飽和(519%±23%)，而后維持在這一水平； （3）①仿生搏動(dòng)流體應(yīng)力培養(yǎng)的小口徑組織工程血管管腔通暢，色澤與天然血管接近；②HE染色：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管中平滑肌細(xì)胞和內(nèi)皮細(xì)胞增殖的數(shù)量較靜態(tài)培養(yǎng)的數(shù)量更多；③掃描電鏡：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管平滑肌細(xì)胞向鄰近的細(xì)胞伸出很多突起相連，靜態(tài)培養(yǎng)平滑肌細(xì)胞沿著支架形成紡錘形狀，細(xì)胞數(shù)量也非常少；仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管管腔內(nèi)面形成更完整的單層內(nèi)皮細(xì)胞層，并更廣泛的分布；④彈力纖維染色：天然血管和仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管中染色較為豐富；Masson染色：膠原表達(dá)沒(méi)有顯著差異；Real-time PCR檢測(cè)：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的平滑肌細(xì)胞Collagen I、III和IV基因表達(dá)水平明顯升高；⑤免疫熒光檢測(cè)：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管中，平滑肌細(xì)胞沿環(huán)形方向伸展排列，Calponin和a-actin免疫熒光強(qiáng)度更強(qiáng)，數(shù)量更多；仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管中，內(nèi)皮細(xì)胞幾乎鋪滿管腔內(nèi)壁，VIII因子相關(guān)抗原和血管性假性血友病因子免疫熒光強(qiáng)度更強(qiáng)，內(nèi)皮細(xì)胞數(shù)量更多；⑥Real-time PCR檢測(cè)：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管中堿性成纖維細(xì)胞生長(zhǎng)因子和內(nèi)皮素-1mRNA的表達(dá)較靜態(tài)培養(yǎng)明顯升高；⑦NO和6-keto-PGF1a檢測(cè)：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管的內(nèi)皮細(xì)胞能夠產(chǎn)生大量NO和6-keto-PGF1a，數(shù)量比靜態(tài)培養(yǎng)更接近天然血管；⑧血小板黏附實(shí)驗(yàn)：在仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管的管腔內(nèi)面粘附的血小板很少；⑨力學(xué)特性：仿生搏動(dòng)流體應(yīng)力培養(yǎng)的血管的拉伸彈性恢復(fù)率、斷裂伸長(zhǎng)率、抗拉強(qiáng)度明顯高于靜態(tài)培養(yǎng)的血管。 2、（1）①動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞，與靜態(tài)培養(yǎng)靜脈平滑肌細(xì)胞相比較，細(xì)胞增殖明顯增強(qiáng)，凋亡明顯減弱；動(dòng)態(tài)培養(yǎng)干擾IGF-1R mRNA翻譯后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)的靜脈平滑肌細(xì)胞相比較，細(xì)胞增殖明顯減弱，凋亡明顯增強(qiáng)；②Real-timePCR檢測(cè)：動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞，與靜態(tài)培養(yǎng)靜脈平滑肌細(xì)胞相比較，IGF-1R mRNA的表達(dá)明顯上調(diào)；動(dòng)態(tài)培養(yǎng)干擾IGF-1R mRNA翻譯后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)的靜脈平滑肌細(xì)胞相比較，IGF-1R mRNA的表達(dá)明顯下調(diào)；③Western Blot檢測(cè)：動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞，與靜態(tài)培養(yǎng)靜脈平滑肌細(xì)胞相比較，，IGF-1R、pTyr-IGF-1R、p-AKT的表達(dá)明顯上調(diào)；動(dòng)態(tài)培養(yǎng)干擾IGF-1R mRNA翻譯后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)的靜脈平滑肌細(xì)胞相比較，IGF-1R、pTyr-IGF-1R、p-AKT的表達(dá)明顯下調(diào)； （2）①動(dòng)態(tài)培養(yǎng)的靜脈平滑肌細(xì)胞組中篩選出2個(gè)表達(dá)上調(diào)2倍以上的microRNA，6個(gè)表達(dá)下調(diào)2倍以上的microRNA；②Real-time PCR對(duì)芯片結(jié)果進(jìn)行驗(yàn)證,microRNA-153的表達(dá)在動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞中約為靜態(tài)培養(yǎng)靜脈平滑肌細(xì)胞中的1/2，microRNA-223的表達(dá)在動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞中約為靜態(tài)培養(yǎng)靜脈平滑肌細(xì)胞中的1/4；③Real-time PCR檢測(cè)發(fā)現(xiàn)隨著時(shí)間的延長(zhǎng)，microRNA-153和microRNA-223的表達(dá)呈下降趨勢(shì)，并均在4小時(shí)達(dá)到最低，此后microRNA-153和microRNA-223的表達(dá)一直維持在最低水平； （3）①EGFP/RFP報(bào)告系統(tǒng)結(jié)果顯示：IGF-1R是microRNA-153和microRNA-223作用的靶基因，并且microRNA-223對(duì)靶基因IGF-1R的調(diào)控作用強(qiáng)于microRNA-153；②Real-time PCR檢測(cè)：靜態(tài)培養(yǎng)microRNA-153和microRNA-223分別轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與靜態(tài)培養(yǎng)的靜脈平滑肌細(xì)胞相比較，microRNA-153和microRNA-223的表達(dá)明顯上調(diào)，上調(diào)約4倍左右；動(dòng)態(tài)培養(yǎng)microRNA-153和microRNA-223分別轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與靜態(tài)培養(yǎng)microRNA-153和microRNA-223分別轉(zhuǎn)染后的靜脈平滑肌細(xì)胞相比較，microRNA-153和microRNA-223的表達(dá)明顯下調(diào)；③動(dòng)態(tài)培養(yǎng)microRNA-153和microRNA-223分別轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞相比較，細(xì)胞增殖明顯減弱，凋亡明顯增強(qiáng)；動(dòng)態(tài)培養(yǎng)microRNA-223轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)microRNA-153轉(zhuǎn)染后的靜脈平滑肌細(xì)胞相比較，細(xì)胞增殖減弱更加明顯，凋亡增強(qiáng)更加明顯；④Real-time PCR檢測(cè)：動(dòng)態(tài)培養(yǎng)microRNA-153和microRNA-223分別轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞相比較，IGF-1R mRNA的表達(dá)無(wú)明顯差異；動(dòng)態(tài)培養(yǎng)microRNA-223轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)microRNA-153轉(zhuǎn)染后的靜脈平滑肌細(xì)胞相比較，IGF-1R mRNA的表達(dá)無(wú)明顯差異；⑤Western Blot檢測(cè)：動(dòng)態(tài)培養(yǎng)microRNA-153和microRNA-223分別轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)靜脈平滑肌細(xì)胞相比較，IGF-1R的表達(dá)明顯下調(diào)；動(dòng)態(tài)培養(yǎng)microRNA-223轉(zhuǎn)染后的靜脈平滑肌細(xì)胞，與動(dòng)態(tài)培養(yǎng)microRNA-153轉(zhuǎn)染后的靜脈平滑肌細(xì)胞相比較，IGF-1R、pTyr-IGF-1R、p-AKT的表達(dá)下調(diào)更加明顯。結(jié)論： 1、酶消化法分離內(nèi)皮細(xì)胞和平滑肌細(xì)胞方法簡(jiǎn)單可行，細(xì)胞獲取率高，獲得細(xì)胞的細(xì)胞形態(tài)和免疫表型符合內(nèi)皮細(xì)胞和平滑肌細(xì)胞的特征； 2、用不使用胰酶的化學(xué)方法制備脫細(xì)胞兔主動(dòng)脈支架材料，方法簡(jiǎn)便可行，制備的脫細(xì)胞兔主動(dòng)脈支架材料的親水性、細(xì)胞黏附性、細(xì)胞相容性和組織學(xué)等特性均適合作為小口徑組織工程血管的支架材料； 3、仿生搏動(dòng)流體應(yīng)力可以有效促進(jìn)平滑肌細(xì)胞和內(nèi)皮細(xì)胞增殖并引導(dǎo)平滑肌細(xì)胞的同向排列和內(nèi)皮細(xì)胞的均勻分布，可能會(huì)引導(dǎo)小口徑組織工程血管形成接近于天然血管的組織結(jié)構(gòu)；仿生搏動(dòng)流體應(yīng)力可以有效促進(jìn)膠原和彈力纖維的分泌，可能會(huì)引導(dǎo)小口徑組織工程血管具備接近于天然血管的生物力學(xué)性能；仿生搏動(dòng)流體應(yīng)力可以有效促進(jìn)平滑肌細(xì)胞和內(nèi)皮細(xì)胞成熟表型的表達(dá)和細(xì)胞因子的分泌，可能會(huì)引導(dǎo)小口徑組織工程血管向天然血管的方向成熟分化；仿生搏動(dòng)流體應(yīng)力可以有效促進(jìn)內(nèi)皮細(xì)胞舒張血管和抗血栓形成功能的發(fā)揮，可能會(huì)引導(dǎo)小口徑組織工程血管擁有接近于天然血管的舒張血管和抗血栓形成能力； 4、IGF-1R的表達(dá)上調(diào)、活性及其下游信號(hào)通路激活在仿生搏動(dòng)應(yīng)力促進(jìn)靜脈平滑肌細(xì)胞的增殖和減少其凋亡中起重要作用； 5、仿生搏動(dòng)應(yīng)力可以促進(jìn)靜脈平滑肌細(xì)胞IGF-1R相關(guān)的microRNA-223和microRNA-153表達(dá)下調(diào)；仿生搏動(dòng)應(yīng)力作用4小時(shí)，microRNA-223和microRNA-153下調(diào)幅度最大，此后維持在這一表達(dá)水平； 6、microRNA-223和microRNA-153的表達(dá)下調(diào)在仿生搏動(dòng)應(yīng)力促進(jìn)靜脈平滑肌細(xì)胞的增殖和減少其凋亡中，通過(guò)上調(diào)IGF-1R的表達(dá)、激活其活性及其下游信號(hào)通路起重要作用。
[Abstract]:Background:
1. Fabrication of small-caliber tissue-engineered blood vessels is a hot research topic at present. Bionic stress is very important to the growth and maturation of small-caliber tissue-engineered blood vessels. However, many perfusion-culture bioreactors can not provide a dynamic environment to simulate physiological blood flow, and do not provide enough biological data to prove their superiority.
2. The function and biomechanical properties of small-caliber tissue-engineered blood vessels can not be separated from the good proliferation of smooth muscle cells. However, the proliferation of smooth muscle cells after implantation of scaffolds is not satisfactory, which affects the function and biomechanical strength of small-caliber tissue-engineered blood vessels.
1. To construct small-diameter tissue-engineered blood vessels and culture them in vitro under the condition of bionic pulsating fluid stress.
2. To construct a bionic pulsatile stress model of smooth muscle cells and explore the post-transcriptional regulation mechanism of the proliferation of smooth muscle cells induced by bionic pulsatile stress.
Method:
1. (1) Endothelial cells and smooth muscle cells were isolated by enzymatic digestion. Cell morphology was observed under microscope. The phenotypes of endothelial cells and smooth muscle cells were identified by factor VIII associated antigen immunohistochemical staining and alpha actin immunofluorescence staining respectively.
(2) The acellular rabbit aorta scaffolds were prepared by chemical method without trypsin. The morphology of the scaffolds was observed by histological observation, scanning electron microscopy and transmission electron microscopy. The biocompatibility was evaluated by CCK-8 method. The adhesion rate of the inoculated cells was calculated and the hydrophilicity was evaluated. Observation by electron microscope.
(3) Endothelial cells and smooth muscle cells were inoculated into the aorta of acellular rabbits to construct small-caliber tissue-engineered blood vessels, and then implanted into a small-caliber tissue-engineered blood vessel bioreactor developed by ourselves. Bionic pulsatile fluid stress and static culture were performed respectively. The gross morphology was observed, HE staining, Masson staining and elastic fiber staining were performed. The expression of Collagen I, III, IV, basic fibroblast growth factor and endothelin-1 mRNA, calmodulin, alpha actin, factor VIII related antigen and von Willebrand factor, NO and 6-keto-PGF1a, platelet adhesion test and biomechanical test were detected by Real-time PCR and immunofluorescence.
2. (1) Grouping: dynamic culture group; static culture group; dynamic culture + lentivirus group; static culture + lentivirus group; _dynamic culture + IGF-1R siRNA group; _static culture + IGF-1R siRNA group. Detection: cell proliferation, cell cycle, apoptosis, Real-time PCR detection of IGF-1R, osteopontin mRNA, Western Blot detection of IGF-1R Osteopontin, pTyr-IGF-1R, p-AKT;
(2) Grouping: dynamic culture group; static culture group; detection: microRNA chip detection, microRNA database prediction, Real-time PCR detection of differentially expressed microRNA;
(3) Grouping: dynamic culture group; static culture group; dynamic culture + lentiviral group; static culture + lentiviral group; static culture + microRNA-223 group; static culture + microRNA-223 group; dynamic culture + microRNA-153 group; static culture + microRNA-153 group; detection: target gene validation experiment, cell proliferation, fine Cell cycle, apoptosis, Real-time PCR for IGF-1R, osteopontin mRNA, Western Blot for IGF-1R, osteopontin, pTyr-IGF-1R, p-AKT.
Result:
1. (1) Smooth muscle cells were arranged in bundles parallel to each other, showing a typical "peak and valley" like growth, and alpha actin immunofluorescence showed a strong positive; endothelial cells showed a typical "cobblestone" like growth, dense arrangement, and factor VIII related antigen immunohistochemical staining showed a strong positive;
(2) The texture of acellular rabbit aorta had no obvious change compared with fresh rabbit aorta; HE staining: the structure was loose, the texture was uniform, and the cells were completely removed; Masson staining: the structure was loose, and there were more gaps between collagen fibers; elastic fiber staining: the structure was loose, there were more gaps between elastic fibers; Electron microscopic examination showed that the outer surface was rough, mainly composed of thick collagen fibers, with more pores than the inner surface and smooth inner surface. The cytotoxicity of smooth muscle cells and endothelial cells was evaluated as 0-1 grade. _The adhesion rates of smooth muscle cells and endothelial cells were 64.32% + 2.03% and 52.77% + 1.19% respectively. HE staining: smooth muscle cells grew well, and some of them migrated to the inner wall, endothelial cells adhered to the inner surface and grew well; scanning electron microscopy: smooth muscle cells and endothelial cells adhered to the outer surface and inner surface, and some of the smooth muscle cells grew into the pores of the scaffold surface; The cytoplasm of smooth muscle cells was rich in myofilaments, arranged in parallel and longitudinal directions, and there were spots and dense bodies in the cells. There were junctions between endothelial cells and W-P bodies in the cytoplasm.
(3) Bionic pulsatile fluid stress culture of small-caliber tissue engineering vascular lumen unobstructed, color and color close to natural vessels; HE staining: bionic pulsatile fluid stress culture of vascular smooth muscle cells and endothelial cells proliferation more than static culture; Scanning electron microscopy: bionic pulsatile fluid stress culture of blood The smooth muscle cells of the tubules extend many processes to adjacent cells and connect with each other. Static cultured smooth muscle cells form a spindle shape along the scaffold, and the number of cells is very small. A more complete monolayer of endothelial cells is formed in the lumen of the vascular lumen cultured by bionic pulsing fluid stress, and more widely distributed. 4 Elastic fiber staining: natural blood vessels. Masson staining showed no significant difference in collagen expression; Real-time PCR showed that the expression levels of Collagen I, III and IV genes in cultured smooth muscle cells were significantly increased; _Immunofluorescence detection: blood vessels cultured under bionic pulsatile fluid stress. In the bionic pulsatile fluid stress cultured vessels, endothelial cells almost covered the lumen wall, factor VIII associated antigen and von Willebrand factor immunofluorescence intensity was stronger, the number of endothelial cells was more; The expression of basic fibroblast growth factor (bfgf) and endothelin-1 (et-1) mRNA in blood vessels cultured under pulsatile fluid stress was significantly higher than that in static culture. _NO and 6-keto-PGF1a detection: endothelial cells cultured under pulsatile fluid stress could produce large amounts of NO and 6-keto-PGF1a, which were closer to those cultured in static culture. Natural blood vessels; _Platelet adhesion test: There were few platelets adhered to the lumen of blood vessels cultured by bionic pulsating fluid stress; _Mechanical properties: The elastic recovery rate, breaking elongation rate and tensile strength of blood vessels cultured by bionic pulsing fluid stress were significantly higher than those cultured by static fluid stress.
2. (1) The proliferation and apoptosis of VSMCs cultured dynamically were significantly enhanced and weakened compared with those cultured statically. The proliferation and apoptosis of VSMCs cultured dynamically interfered with the translation of IGF-1R mRNA were significantly weakened compared with those cultured dynamically. The expression of IGF-1R mRNA was up-regulated in the cultured VSMCs compared with the static VSMCs, while the expression of IGF-1R mRNA was down-regulated in the VSMCs after the translation of IGF-1R mRNA. Measurement: The expression of IGF-1R, pTyr-IGF-1R and p-AKT was significantly up-regulated in cultured VSMCs, and the expression of IGF-1R, pTyr-IGF-1R and p-AKT was significantly down-regulated in cultured VSMCs after interfering with the translation of IGF-1R mRNA.
(2) Two microRNAs with up-regulation of more than two times and six microRNAs with down-regulation of more than two times were screened out in the dynamic cultured VSMC group; and (2) Real-time PCR was used to verify the results of the microRNA-153 chip. The expression of microRNA-153 in the dynamic cultured VSMC was about 1/2 of that in the static cultured VSMC and microRNA-22. The expression of microRNA-153 and microRNA-223 in VSMCs was about 1/4 of that in VSMCs cultured in static state. Flat;
(3) The results of EGFP/RFP reporting system showed that IGF-1R was the target gene of microRNA-153 and microRNA-223, and the regulatory effect of microRNA-223 on target gene IGF-1R was stronger than that of microRNA-153. Real-time PCR detection: static culture of microRNA-153 and microRNA-223 were transfected into venous smooth muscle cells, which were flat with static culture of venous smooth muscle cells. The expression of microRNA-153 and microRNA-223 was up-regulated by about 4 times in SMCs, and the expression of microRNA-153 and microRNA-223 in VSMCs transfected with microRNA-153 and microRNA-223 was up-regulated by microRNA-153 and microRNA-223 respectively. (3) The proliferation and apoptosis of VSMCs transfected with microRNA-153 and microRNA-223 were significantly decreased compared with those of VSMCs transfected with microRNA-153, and the VSMCs transfected with microRNA-223 were cultured dynamically to smooth veins after microRNA-153 transfection. The expression of IGF-1R mRNA in the vein smooth muscle cells transfected with microRNA-153 and microRNA-223 was not significantly different from that in the vein smooth muscle cells transfected with microRNA-223. The expression of IGF-1R mRNA in VSMCs transfected with microRNA-153 was not significantly different from that in VSMCs transfected with microRNA-153. _Western Blot assay: The expression of IGF-1R in VSMCs transfected with microRNA-153 and microRNA-223 was detected by dynamic culture. The expression of IGF-1R, pTyr-IGF-1R and p-AKT in the vein smooth muscle cells transfected with microRNA-223 was significantly down-regulated compared with the vein smooth muscle cells transfected with microRNA-153.
1. Enzymatic digestion is a simple and feasible method for isolation of endothelial cells and smooth muscle cells. The cell morphology and immunophenotype of the obtained cells conform to the characteristics of endothelial cells and smooth muscle cells.
2. Acellular rabbit aorta scaffolds were prepared by chemical method without trypsin. The method was simple and feasible. The acellular rabbit aorta scaffolds were suitable for scaffolds with small diameter for tissue engineering.
3. Bionic pulsatile fluid stress can effectively promote the proliferation of smooth muscle cells and endothelial cells and induce the co-arrangement of smooth muscle cells and the uniform distribution of endothelial cells, which may lead to the formation of small-diameter tissue-engineered blood vessels close to the structure of natural blood vessels; bionic pulsatile fluid stress can effectively promote collagen and elastic fibers. Biomimetic pulsatile fluid stress can effectively promote the expression of mature phenotype of smooth muscle cells and endothelial cells.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.11


本文編號(hào)：2190398