本文選題：血管內(nèi)皮細(xì)胞 + 線粒體自噬�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景自噬是細(xì)胞將自身受損的細(xì)胞器或錯(cuò)誤折疊的蛋白質(zhì)包裹、吞噬,并運(yùn)輸?shù)饺苊阁w,降解,實(shí)現(xiàn)自身物質(zhì)代謝更新,能量再循環(huán)利用的生物學(xué)過(guò)程。自噬對(duì)維持血管內(nèi)環(huán)境的穩(wěn)態(tài)的十分重要,并且參與了血管內(nèi)各種細(xì)胞生長(zhǎng)和死亡的調(diào)控。在血管損傷動(dòng)物模型中,伴隨著血管內(nèi)皮細(xì)胞的首先死亡和血管平滑肌細(xì)胞的不斷增殖,這兩種細(xì)胞中都存在著不同程度的自噬反應(yīng)。血管內(nèi)皮細(xì)胞、血管平滑肌細(xì)胞是構(gòu)成血管組織最主要的兩種細(xì)胞類型,深入探討自噬對(duì)這兩種血管細(xì)胞的作用及調(diào)節(jié)機(jī)制意義重大。首先,血管內(nèi)皮細(xì)胞活躍地參與血管各功能活動(dòng),因此內(nèi)皮細(xì)胞能量代謝要迅速且準(zhǔn)確,而細(xì)胞內(nèi)的能量供應(yīng)站即線粒體,這個(gè)細(xì)胞器自身的穩(wěn)態(tài)和代謝更新也就更為重要。研究表明,在細(xì)胞中,通過(guò)自噬機(jī)制可以選擇性地清除那些受到損傷或者已經(jīng)老化的線粒體,也就是線粒體自噬,來(lái)維護(hù)線粒體結(jié)構(gòu)與功能的完整性。在代謝綜合征中,內(nèi)皮細(xì)胞中出現(xiàn)線粒體損傷、積聚,細(xì)胞內(nèi)ROS水平升高,而這些代謝應(yīng)激誘導(dǎo)的損傷線粒體又進(jìn)一步損傷了內(nèi)皮細(xì)胞的功能,加重了心血管疾病。PINK1/Parkin這條信號(hào)通路在控制線粒體自噬的過(guò)程中發(fā)揮了重要的作用。當(dāng)PINK1/Parkin分子突變后會(huì)引起神經(jīng)系統(tǒng)的帕金森疾病。因此研究PINK1/Parkin信號(hào)通路介導(dǎo)的線粒體自噬在代謝應(yīng)激中對(duì)內(nèi)皮細(xì)胞的作用機(jī)制,愈來(lái)愈重要。平滑肌細(xì)胞是血管中除內(nèi)皮細(xì)胞外另一重要細(xì)胞群,在血管損傷后的血管修復(fù)與重構(gòu)中,平滑肌細(xì)胞的增殖成為主要的細(xì)胞生命活動(dòng),因此研究自噬對(duì)血管損傷后平滑肌細(xì)胞的調(diào)控,也就顯得十分重要。但是近年來(lái)無(wú)論是高血壓,還是其它代謝性疾病等引起的血管疾病中,僅僅采用針對(duì)平滑肌細(xì)胞的藥物并沒(méi)有能夠有效的控制血管疾病病情的發(fā)展,更不用說(shuō)根治血管疾病。例如在嚴(yán)重的冠脈阻塞中,冠狀動(dòng)脈旁路移植術(shù)仍然是最有效的外科治療手段,但是,術(shù)后十年,50%的移植血管會(huì)發(fā)生再阻塞,這主要?dú)w因于血管平滑肌細(xì)胞的不斷增殖和血管新生內(nèi)膜的持續(xù)增生。接著人們采用直接或是間接的方法抑制血管平滑肌細(xì)胞的增殖,但是20年來(lái),冠狀動(dòng)脈旁路移植術(shù)術(shù)后十年再阻塞的概率并沒(méi)有得到很好的改善。因此針對(duì)抑制平滑肌細(xì)胞的治療就顯得讓人遲疑,但同時(shí)這也引發(fā)了人們新的思考,是否我們忽略了更為重要的治療耙點(diǎn)?已有證據(jù)顯示血管中存在著大量的血管干細(xì)胞,血管損傷后,這些干細(xì)胞會(huì)被激活。在小鼠靜脈移植這個(gè)模型中,徐清波教授已經(jīng)證明移植靜脈新生內(nèi)膜中,增殖的平滑肌細(xì)胞60%來(lái)源于供體靜脈血管。在我們的實(shí)驗(yàn)中發(fā)現(xiàn),移植術(shù)后3天,移植靜脈血管原有的成熟平滑肌細(xì)胞都已死亡,而血管外膜中Sca-1(干細(xì)胞抗原-1)陽(yáng)性的干細(xì)胞仍存活,并且在術(shù)后7-14天增殖達(dá)到高峰,這群干細(xì)胞無(wú)疑是構(gòu)成移植靜脈血管新生內(nèi)膜的主要來(lái)源。干細(xì)胞是一種長(zhǎng)壽命細(xì)胞,長(zhǎng)期處于靜息狀態(tài)。這種狀態(tài)的維持,需要嚴(yán)密的自我復(fù)制和分化潛能。自噬作為一種可誘導(dǎo)的分解代謝的細(xì)胞生命活動(dòng),對(duì)于細(xì)胞內(nèi)蛋白質(zhì)質(zhì)量控制及細(xì)胞器物質(zhì)代謝再利用起著重要作用。按照發(fā)育階段分類干細(xì)胞為：胚胎干細(xì)胞和成體干細(xì)胞。自噬在這些干細(xì)胞中的功能及作用已被不同的文獻(xiàn)報(bào)道：比如在胚胎形成過(guò)程中,自噬可以幫助清除死亡細(xì)胞,移除有缺陷的線粒體及某些泛素化的蛋白質(zhì),維持胚胎干細(xì)胞的增殖,降解在其分化過(guò)程中形成的中間體。在造血干細(xì)胞中作用,自噬主要表現(xiàn)為維持穩(wěn)定的增殖,有效移除有缺陷的線粒體,阻止DNA損傷,自我復(fù)制。自噬在間充質(zhì)干細(xì)胞中的作用則主要是細(xì)胞保護(hù)反應(yīng)和促進(jìn)分化。自噬在神經(jīng)干細(xì)胞中的作用則是促進(jìn)分化及營(yíng)養(yǎng)不足時(shí),維持存活。總之自噬對(duì)干細(xì)胞的生長(zhǎng)和分化起到了嚴(yán)密的質(zhì)量監(jiān)控,正因?yàn)樽允晒δ艿拇嬖?各種干細(xì)胞才能一方面維護(hù)自體干性狀態(tài),另一方面分化成成熟的不同的體細(xì)胞完成機(jī)體的各種修復(fù)過(guò)程。目前對(duì)血管干細(xì)胞和自噬相關(guān)的研究較少,但這無(wú)疑為治療血管損傷性疾病提供了新的方向。我們的研究則聚焦在尋找可以調(diào)控自噬的去泛素化酶分子CYLD(圓柱瘤基因編碼的蛋白)上面,CYLD可以通過(guò)激活mTOR信號(hào)通路,來(lái)控制自噬,進(jìn)而干預(yù)血管干細(xì)胞的激活、自我復(fù)制及分化潛能,維持血管內(nèi)環(huán)境穩(wěn)態(tài),達(dá)到治療血管病的最終目的。研究目的第一部分研究線粒體自噬對(duì)游離脂肪酸代謝應(yīng)激誘導(dǎo)的內(nèi)皮細(xì)胞損傷的保護(hù)機(jī)制：1研究線粒體自噬對(duì)維持內(nèi)皮細(xì)胞線粒體結(jié)構(gòu)和功能完整性的作用。2研究PINK1/Parkin介導(dǎo)的線粒體自噬對(duì)游離脂肪酸(FFA)誘導(dǎo)的內(nèi)皮細(xì)胞損傷的保護(hù)機(jī)制。第二部分研究CYLD是否通過(guò)控制自噬,調(diào)節(jié)血管干細(xì)胞的功能,進(jìn)一步影響血管損傷后的重構(gòu)過(guò)程：1在同種異體小鼠頸外靜脈向頸總動(dòng)脈移植模型中,解析血管干細(xì)胞在移植靜脈新生內(nèi)膜形成過(guò)程中的巨大貢獻(xiàn)。2在CYLD基因敲除小鼠頸外靜脈向野生型小鼠頸總動(dòng)脈移植模型中,分析移植靜脈新生內(nèi)膜形成情況。3 CYLD是否可以調(diào)控自噬,干預(yù)血管干細(xì)胞的自我更新和向前體細(xì)胞分化功能,最終影響血管重構(gòu)。研究方法第一部分1動(dòng)物模型1.1Ⅰ型糖尿病小鼠模型：C57BL/6J小鼠,50 mg/kg STZ連續(xù)注射五天,10天后檢測(cè)小鼠血糖水平、體重及4周后進(jìn)行胸主動(dòng)脈的取材固定。1.2高脂喂養(yǎng)小鼠模型：用高脂飼料對(duì)C57BL/6J小鼠進(jìn)行持續(xù)喂養(yǎng),三個(gè)月后檢測(cè)小鼠的血脂水平及進(jìn)行胸主動(dòng)脈的取材固定。2細(xì)胞培養(yǎng)和轉(zhuǎn)染：2.1用含有5%的胎牛血清(FBS)、內(nèi)皮細(xì)胞生長(zhǎng)因子、雙抗等物質(zhì)的內(nèi)皮細(xì)胞培養(yǎng)基培養(yǎng)人主動(dòng)脈弓內(nèi)皮細(xì)胞(HAECs)。2.2用lipofectamine 2000對(duì)PINK1/Parkin的小干擾RNA及過(guò)表達(dá)質(zhì)粒進(jìn)行內(nèi)皮細(xì)胞的轉(zhuǎn)染。3棕櫚酸(PA)的配制：將PA溶入200 mmol/L的甲醇后,加入10%牛血清白蛋白(BSA), PH=7.5,-20℃凍存。4 Western blot實(shí)驗(yàn)分析：使用Western細(xì)胞裂解液裂解不同刺激條件處理的細(xì)胞,檢測(cè)蛋白濃度,使用10% SDS-PAGE進(jìn)行電泳,免疫印跡法檢測(cè)不同目的蛋白的表達(dá)情況。5細(xì)胞免疫熒光染色：將不同刺激條件處理后的細(xì)胞接種在細(xì)胞爬片上面,固定后,一抗二抗孵育,封片,用Ultra VIEW VOX共聚焦顯微鏡觀察,拍照,最后用Volocigy軟件分析輸出高質(zhì)量圖片。6線粒體檢測(cè)：使用Mito Tracker Deep Red染色試劑盒進(jìn)行內(nèi)皮細(xì)胞線粒體染色。7線粒體自噬檢測(cè)：使用Mito Tracker Deep Red進(jìn)行內(nèi)皮細(xì)胞線粒體染色的同時(shí),進(jìn)行自噬小體標(biāo)志物L(fēng)C3或溶酶體標(biāo)志物L(fēng)amp1的共定位染色。8透射電鏡檢測(cè)：將不同刺激條件處理后的細(xì)胞用戊二醛固定過(guò)夜,第二天送往濟(jì)南微亞生物技術(shù)公司,由該公司進(jìn)行透射電鏡的檢測(cè)JEOL-1200EX透射電鏡觀察,MORADA-G2記錄結(jié)果。9線粒體膜電位的檢測(cè)：羰基氰-3-氯苯腙(CCCP)是一種強(qiáng)效的線粒體氧化磷酸化解偶聯(lián)劑,用它或PA處理內(nèi)皮細(xì)胞后進(jìn)行四甲基羅丹明甲酯(TMRM)和Mito Tracker Deep Red線粒體共定位雙染色,用Ultra VIEW VOX共聚焦顯微鏡進(jìn)行活細(xì)胞觀察,拍照,最后用Volocigy軟件分析輸出高質(zhì)量圖片。10 ROS水平的檢測(cè)：將不同刺激條件處理后的內(nèi)皮細(xì)胞用ROS檢測(cè)試劑盒進(jìn)行檢測(cè)。11 ATP水平的檢測(cè)：將不同刺激條件處理后的內(nèi)皮細(xì)胞用ATP檢測(cè)試劑盒進(jìn)行檢測(cè)。12原位末端標(biāo)記法(TUNEL):使用羅氏TUNEL檢測(cè)試劑盒檢測(cè)不同刺激條件處理后的內(nèi)皮細(xì)胞凋亡情況。13數(shù)據(jù)分析：使用Student's t檢驗(yàn)或單因素方差分析進(jìn)行統(tǒng)計(jì)學(xué)分析。當(dāng)P0.05認(rèn)為有統(tǒng)計(jì)學(xué)差異意義。第二部分1靜脈移植模型：用帶有柄和體的CUFF管進(jìn)行頸外靜脈的移植,移植后,觀察靜脈血管中有動(dòng)脈血液流通,及血管搏動(dòng),說(shuō)明手術(shù)成功。2用藥：2.1局部用藥：辛伐他汀(30umol/L)/SenexinA (3 umol/L)用20% F-127膠包裹在移植血管周圍。2.2全身系統(tǒng)用藥：辛伐他汀(1.6mg/kg)/SenexinB (20 mg/kg)分別采用灌胃和腹腔內(nèi)注射的方法。用藥間為：術(shù)前三天至術(shù)后28天連續(xù)用藥或術(shù)后三天至術(shù)后28天連續(xù)用藥。3組織免疫熒光染色法：分別在術(shù)后0、1、3、7、14、21、42天后取材,固定,近心端開(kāi)始切片,切片厚度為5μm,連續(xù)切片,每隔501μm或是500μm各取一張切片。進(jìn)行HE染色和組織免疫熒光染色。4細(xì)胞培養(yǎng)：取8周大C57BL/6J小鼠一只,取出胸主動(dòng)脈及根部,顯微鏡下分離出外膜,剪碎,并用膠原酶Ⅱ進(jìn)行適度消化后,過(guò)濾血管碎片,用干細(xì)胞培養(yǎng)基培養(yǎng)數(shù)天,當(dāng)細(xì)胞長(zhǎng)到足夠數(shù)量后,用Sca-1磁珠釣取Sca-1陽(yáng)性細(xì)胞,再進(jìn)行培養(yǎng)。5統(tǒng)計(jì)分析：使用Student's t檢驗(yàn)或單因素方差分析進(jìn)行統(tǒng)計(jì)學(xué)分析。當(dāng)P0.05認(rèn)為有統(tǒng)計(jì)學(xué)差異意義。研究結(jié)果第一部分1 PA處理后內(nèi)皮細(xì)胞自噬和線粒體自噬均增強(qiáng)。2 PINK1/Parkin信號(hào)通路參與了PA誘導(dǎo)內(nèi)皮細(xì)胞的線粒體自噬：3 PINK1/Parkin信號(hào)通路保護(hù)了PA誘導(dǎo)的內(nèi)皮細(xì)胞中損傷的線粒體的膜電位：4 PINK1/Parkin信號(hào)通路阻止了PA誘導(dǎo)內(nèi)皮細(xì)胞中線粒體失功能。5 PINK1/Parkin信號(hào)通路阻止了PA誘導(dǎo)內(nèi)皮細(xì)胞死亡。6在肥胖小鼠和Ⅰ型糖尿病小鼠模型中,PINK1/Parkin信號(hào)通路在小鼠血管中被激活。第二部分：1在同種異體小鼠頸外靜脈向頸總動(dòng)脈移植模型中,抑制血管干細(xì)胞后,移植靜脈的新生內(nèi)膜也得到抑制。1.1在靜脈移植早期,供體血管平滑肌細(xì)胞死亡,同時(shí)伴有血管干細(xì)胞的增殖,分化最終參于移植靜脈新生內(nèi)膜的形成。1.2建立了一個(gè)更全面的可以定量測(cè)量和分析移植靜脈新生內(nèi)膜中平滑肌細(xì)胞的方法。1.3在移植術(shù)后前三天,成熟平滑肌細(xì)胞死亡而血管干細(xì)胞存活并增殖最終參于血管新生內(nèi)膜的形成。1.4辛伐他汀可能主要通過(guò)抑制早期血管干細(xì)胞的激活來(lái)抑制移植術(shù)后血管新生內(nèi)膜的形成與增生。1.5 Senexin A/B, CDK8抑制物,可以通過(guò)抑制移植術(shù)后血管干細(xì)胞的激活而非抑制血管平滑肌細(xì)胞來(lái)抑制血管新生內(nèi)膜的形成與增生。2在CYLD基因敲除小鼠頸外靜脈向野生型小鼠頸總動(dòng)脈移植模型中,CYLD基因的敲除促進(jìn)了血管新生內(nèi)膜形成。3 CYLD通過(guò)激活mTOR信號(hào)通路,抑制自噬。研究結(jié)論1 PINK1/Parkin信號(hào)通路通過(guò)線粒體自噬對(duì)內(nèi)皮細(xì)胞維持線粒體功能完整性發(fā)揮了重要作用,可以清除代謝應(yīng)激中受損線粒體,從而保護(hù)了內(nèi)皮細(xì)胞的功能。2血管干細(xì)胞參于血管損傷后新生內(nèi)膜形成過(guò)程并發(fā)揮了重要作用,CYLD通過(guò)調(diào)控mTOR信號(hào)通路來(lái)調(diào)控自噬,進(jìn)而干預(yù)血管干細(xì)胞的自我更新,向前體細(xì)胞的分化,增殖,最終影響血管重構(gòu)。
[Abstract]:Background autophagy is an autophagy that is encapsulated by a cell or a wrongly folded protein, phagocytic, and transported to the lysosome, degrading, realizing the metabolic renewal of its own material, and the biological process of energy recirculation. Autophagy is important to maintain the homeostasis of the intravascular environment and participates in the growth and death of various cells in the blood vessels. In the animal model of vascular injury, with the first death of vascular endothelial cells and the continuous proliferation of vascular smooth muscle cells, there are different degrees of autophagy in these two cells. Vascular endothelial cells, vascular smooth muscle cells are the two most important types of vascular tissue, and the autophagy is discussed in depth. The role and regulation mechanism of these two kinds of vascular cells are of great significance. First, vascular endothelial cells actively participate in various functional activities of blood vessels, so the energy metabolism of endothelial cells should be quickly and accurately, and the energy supply station in the cell is mitochondria, and the homeostasis and metabolic renewal of this organelle itself is more important. In the metabolic syndrome, mitochondrial damage, accumulation, elevated levels of ROS in cells, and these metabolic stress induced damage lines, are possible to maintain mitochondrial structure and function integrity in the metabolic syndrome. The body further damages the function of endothelial cells and aggravates the cardiovascular disease.PINK1/Parkin signaling pathway plays an important role in controlling mitochondrial autophagy. When the PINK1/Parkin molecule mutates, it will cause the Parkinson disease of the nervous system. Therefore, the study of the mitochondrial autophagy mediated by the PINK1/ Parkin signaling pathway Smooth muscle cells are another important cell group except endothelial cells in the blood vessels. The proliferation of smooth muscle cells is the main cell life activity in vascular repair and reconstruction after vascular injury. Therefore, the regulation of self macrophages on vascular smooth muscle cells after vascular injury is also studied. It is important, but in recent years, in vascular diseases caused by hypertension and other metabolic diseases, only the use of drugs aimed at smooth muscle cells has not been able to effectively control the development of vascular disease, let alone to cure vascular diseases. For example, coronary artery bypass graft in severe coronary occlusion. Surgery is still the most effective surgical treatment. However, ten years after the operation, 50% of the transplanted vessels will be re blocked, which is attributed to the continuous proliferation of vascular smooth muscle cells and the continuous proliferation of neointima in the vascularization. Then, the proliferation of vascular smooth muscle cells is suppressed by direct or indirect methods, but for 20 years, coronary movement has been performed. The probability of re blocking ten years after the vein bypass grafting has not been well improved. Therefore, the treatment of the suppression of smooth muscle cells appears to be hesitant, but it also causes new thinking. Do we ignore the more important treatment rake? There is evidence that there are a large number of vascular stem cells in the blood vessels, blood. In the mouse vein transplant model, Professor Xu Qingbo has proved that 60% of the proliferating smooth muscle cells are derived from the donor vein. In our experiment, we found that the original smooth muscle cells of the vein graft were dead at 3 days after the transplantation. The stem cells of the Sca-1 (stem cell antigen -1) positive in the epicardium still survive and proliferate at the peak 7-14 days after the operation. This group of stem cells is undoubtedly the main source of the neointima of the vein graft. The stem cells are a long life life cell and remain resting for a long time. Autophagy. Autophagy, as an induced metabolic cell life activity, plays an important role in intracellular protein quality control and organelle metabolism reutilization. Stem cells are classified as stem cells and adult stem cells according to the developmental stages. The function and function of autophagy in these stem cells has been different In the process of embryogenesis, autophagy, for example, helps to remove dead cells, remove defective mitochondria and some ubiquitin proteins, maintain the proliferation of embryonic stem cells, and degrade intermediates formed during their differentiation. In hematopoietic stem cells, autophagy is mainly shown to maintain stable proliferation and be effectively removed. The function of autophagy in mesenchymal stem cells is mainly cell protection and promotion of differentiation. Autophagy plays a role in promoting differentiation and insufficient nutrition in neural stem cells. In short, autophagy is closely monitored for the growth and differentiation of stem cells. Due to the existence of autophagy, all kinds of stem cells can maintain autologous dry state on the one hand and differentiate into mature different somatic cells to complete various repair processes. There are few studies on vascular stem cells and autophagy, but this will undoubtedly provide a new direction for the treatment of vascular injury. It is focused on the search for autophagy, which regulates the autophagy molecule CYLD (the protein encoded by the cylindric gene). CYLD can control autophagy by activating the mTOR signaling pathway, and then interferes with the activation of vascular stem cells, self replication and differentiation potential, and maintains the homeostasis of vascular intravascular environment to achieve the ultimate goal of treating vascular diseases. The first part study the protective mechanism of mitochondrial autophagy induced endothelial cell damage induced by free fatty acid metabolism stress: 1 study the role of mitochondrial autophagy to maintain mitochondrial structure and functional integrity of endothelial cells.2 study the protection of mitochondrial autophagy mediated endothelial cell injury induced by free fatty acid (FFA) induced by PINK1/Parkin Mechanism. The second part studies whether CYLD regulates the function of vascular stem cells by controlling autophagy and regulates the function of vascular stem cells and further affects the reconstruction process after vascular injury. 1 in the allograft mouse model of external jugular vein to common carotid artery transplantation, the great contribution of.2 to CYLD gene knockout in the neointima formation process of the transplanted vein In the model of common carotid artery transplantation in mice from the external jugular vein to the wild type mouse, the analysis of the formation of neointima of the transplanted vein.3 CYLD can regulate autophagy, interfere with the self renewal of the vascular stem cells and the function of the differentiation of the forward body cells, and ultimately affect the vascular remodeling. The first part of the 1 animal model of the animal model of type 1.1 diabetic mice C57BL/6J mice, 50 mg/kg STZ continuous injection for five days, 10 days after the test of blood glucose level, body weight and 4 weeks after the thoracic aorta were used to fix the.1.2 high fat feeding mice model: high fat feed for continuous feeding of C57BL/6J mice, three months after the detection of blood lipid level in mice and the thoracic aorta material fixed.2 cell culture. Cultured and transfected: 2.1 the human aortic arch endothelial cells (HAECs).2.2 with 5% fetal bovine serum (FBS), endothelial cell growth factor, double resistance and other substances,.2.2 with Lipofectamine 2000 on PINK1/Parkin small interference RNA and overexpressed plasmids for.3 palmitic acid (PA) preparation: PA into 200 MMO After l/L methanol, adding 10% bovine serum albumin (BSA), PH=7.5, -20 C,.4 Western blot experimental analysis: using Western cell lysate to crack the cells treated with different stimulation conditions, detecting protein concentration, using 10% SDS-PAGE for electrophoresis, and detecting the expression of different target proteins by Western blot,.5 cell immunofluorescence staining After the cells treated with different stimulation conditions were inoculated on the cell climbing tablets, after immobilization, a anti two anti incubation and sealing film was observed and photographed with Ultra VIEW VOX confocal microscope. Finally, the Volocigy software was used to analyze the high quality images of.6 mitochondria: Mito Tracker Deep Red staining kit was used to carry out mitochondrial infection of endothelial cells. Autophagy detection of mitochondrial.7 mitochondria: using Mito Tracker Deep Red for mitochondrial staining of endothelial cells, simultaneous localization of autophagic marker LC3 or lysosome marker Lamp1 is carried out by transmission electron microscopy (TEM) detection of.8: the cells treated with different stimuli were fixed with glutaraldehyde for the night and sent to Ji'nan micro biotechnology for second days. The company, the company carries out transmission electron microscope examination JEOL-1200EX transmission electron microscope observation, MORADA-G2 record results.9 mitochondrial membrane potential detection: carbonyl cyanogen -3- chlorobenzene hydrazone (CCCP) is a powerful mitochondrial oxidative phosphorylation uncoupling agent, using it or PA treatment of endothelial cells after four methyl Luo Danming methyl Luo Danming methyl ester (TMRM) and Mito Tracker Deep Red mitochondrial co localization double staining, using Ultra VIEW VOX confocal microscope for living cell observation, photographing, and finally using Volocigy software to analyze the level of.10 ROS output of high quality pictures: the detection of.11 ATP level by ROS detection kit after different stimulation conditions: the different stimulus conditions will be detected. ATP detection kit was used to detect.12 in situ terminal labeling (TUNEL):.13 data analysis was used to detect the apoptosis of endothelial cells treated with different stimuli using Roche TUNEL Kit: Student's t test or single factor analysis of variance analysis. When P0.05 believed there was a statistical difference. Second part 1 vein transplantation model: transplantation of the external jugular vein with the CUFF tube with a handle and body. After transplantation, we observe the circulation of blood in the veins of the veins in the veins of the veins and the pulsation of the blood vessels. It shows that the operation is successful for.2 medication: 2.1 local medication: simvastatin (30umol/L) / SenexinA (3 umol/L) wrapped around the transplanted vascular.2 with 20% F-127 glue .2 systemic medication: simvastatin (1.6mg/kg) /SenexinB (20 mg/kg) were treated with intraperitoneal injection and intraperitoneal injection respectively. The use of the medication was continuous medication from three days before operation to 28 days after operation or three days after operation to 28 days after operation:.3 tissue immunofluorescence staining, respectively, after 0,1,3,7,14,21,42 after operation, fixed, near the end of the heart. The slice thickness was 5 mu m, sliced continuously, each 501 m or 500 micron m were sectioned. HE staining and tissue immunofluorescence staining.4 cell culture were taken to remove the thoracic aorta and the root of a large C57BL/6J mouse, the outer membrane was removed under microscope, and the collagenase II was used to digest the blood vessel fragments. After a few days in the stem cell culture medium, when the cells grew enough, the Sca-1 positive cells were caught with Sca-1 magnetic beads and then cultured for.5 statistical analysis: Student's t test or single factor analysis of variance analysis. When P0.05 believed that there was statistical difference. The results of the first part of the study were 1 PA after the endothelium autophagy. And mitochondrial autophagy enhanced the.2 PINK1/Parkin signaling pathway involved in the mitochondrial autophagy of PA induced endothelial cells: the 3 PINK1/Parkin signaling pathway protects the mitochondrial membrane potential damaged by PA induced endothelial cells: the 4 PINK1/Parkin signaling pathway prevents PA induced mitochondrial dysfunction.5 PINK1/Parkin signaling in the endothelial cells. The road prevented PA induced endothelial cell death.6 in the obese mice and the model of type I diabetic mice, the PINK1/Parkin signaling pathway was activated in the mouse blood vessels. Second: 1 in the allograft mouse model of the external jugular vein to the common carotid artery, the neointima of the vein graft is also suppressed after the suppression of the vascular stem cells. The vascular smooth muscle cells died at the early stage of vein transplantation, accompanied by the proliferation of vascular stem cells, and differentiation finally involved the formation of the neointima of the transplanted vein..1.2 established a more comprehensive method for quantitative measurement and analysis of the smooth muscle cells in the neointima of the transplanted vein..1.3 was mature smooth muscle cells three days after the transplantation. Death and vascular stem cells survive and proliferate and eventually participate in neointimal formation..1.4 simvastatin may mainly inhibit early vascular stem.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R363
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本文編號(hào)：2067729