本文選題：內(nèi)皮細(xì)胞 + 乙醇��； 參考：《河北醫(yī)科大學(xué)》2014年博士論文

【摘要】：我國酒文化源遠(yuǎn)流長，但隨著飲酒在社會(huì)交往活動(dòng)中更加頻繁，飲酒所帶來的社會(huì)問題也日益凸顯。近年來飲酒后傷害案件屢見不鮮，加之案情復(fù)雜，給檢案人員造成很大壓力。早在1963年有國外法醫(yī)提出外傷性蛛網(wǎng)膜下腔出血（traumatic subarachnoid hemorrhage，tSAH）與酗酒有關(guān)，實(shí)際檢案過程發(fā)現(xiàn)酒后受到輕微外力作用，顱內(nèi)出血的發(fā)病率和死亡率明顯增高。雖然已知飲酒可以調(diào)節(jié)心率和局部血流，改變血管通透性，影響血管舒縮功能，破壞凝血、抗凝血平衡，但這些改變尚不足以解釋飲酒后外力作用易于引發(fā)顱內(nèi)出血的原因。此外，長期困擾法醫(yī)工作者的難題是：如何界定飲酒和外力各自的參與度，進(jìn)而判定案件性質(zhì)。所以更深入地研究飲酒易于造成腦血管破裂出血機(jī)制，有助于指導(dǎo)法醫(yī)實(shí)踐、改進(jìn)檢案思路和明確判定結(jié)果。 乙醇（ethanol，EtOH）又名酒精，是酒類飲品中的主要成分。飲酒后乙醇可以以原型形式進(jìn)入血液，血乙醇濃度（blood alcohol concentration,BAC）快速升高，乙醇隨血流遍布全身。內(nèi)皮細(xì)胞（endotheilial cell，EC）位于血管腔面最內(nèi)側(cè)，直接接觸血液中乙醇及其代謝產(chǎn)物；另一方面內(nèi)皮細(xì)胞生物功能活躍，擁有乙醇代謝相關(guān)酶體參與乙醇代謝，這就決定了內(nèi)皮細(xì)胞可能遭受乙醇的直接和間接毒性作用，成為乙醇首當(dāng)其沖的損傷靶點(diǎn)。 由于血管內(nèi)皮承擔(dān)著重要的血管調(diào)節(jié)功能，在物質(zhì)交換、血管張力調(diào)控、凝血、血管發(fā)生、白細(xì)胞游走等多種生物過程中發(fā)揮不可替代的作用；并可以“感知”血流切應(yīng)力、周期應(yīng)力等各種機(jī)械應(yīng)力及各種血液成分改變，協(xié)調(diào)其他細(xì)胞共同作出相應(yīng)反應(yīng)，所以乙醇引起的內(nèi)皮細(xì)胞損傷會(huì)危及到整個(gè)血管系統(tǒng)。 其中腦血管具有其他部位血管不可比擬的自主調(diào)控能力以保持穩(wěn)定的腦灌流量，對(duì)內(nèi)皮細(xì)胞損傷愈發(fā)敏感。本課題以內(nèi)皮細(xì)胞作為主要研究對(duì)象，探索乙醇的血管毒性作用，為進(jìn)一步研究飲酒后輕微外力引發(fā)蛛網(wǎng)膜下腔出血（subarachnoid hemorrhage，SAH）的機(jī)制做好鋪墊。 血管內(nèi)皮以單層細(xì)胞形式分布，其結(jié)構(gòu)、功能完整性特別依賴于與細(xì)胞外基質(zhì)（extracellular matrix，ECM）的連接、交流。黏著斑復(fù)合體是細(xì)胞與ECM連接處的復(fù)合結(jié)構(gòu)，實(shí)現(xiàn)了細(xì)胞骨架與ECM的連接。一方面它可以將細(xì)胞外的信號(hào)傳入胞內(nèi)，通過“引導(dǎo)”應(yīng)力纖維分布實(shí)現(xiàn)對(duì)細(xì)胞形態(tài)、功能的調(diào)整；另一方面將細(xì)胞生命活動(dòng)產(chǎn)生的細(xì)胞內(nèi)牽引力（tractionforce）傳遞到ECM，進(jìn)而轉(zhuǎn)化為生物信號(hào)傳遞給周圍細(xì)胞，使機(jī)體能作出及時(shí)的宏觀調(diào)整。 黏著斑激酶（focal adhesion kinase，F(xiàn)AK）是黏著斑復(fù)合體重要的組成成分。一方面FAK受到integrin聚集、生長因子受體連接、切應(yīng)力等多種因素調(diào)節(jié)活化；另一方面FAK可以連接并激活其他黏著斑結(jié)構(gòu)蛋白，參與黏著斑復(fù)合體形成與成熟，調(diào)節(jié)細(xì)胞粘附、遷移等基本功能，還可以招募多種功能蛋白，參加到不同的信號(hào)通路調(diào)節(jié)中去�，F(xiàn)已證實(shí)FAK在內(nèi)皮細(xì)胞中發(fā)揮著舉足輕重的作用。用特異失活內(nèi)皮細(xì)胞內(nèi)FAK的方法研究胚胎發(fā)育，發(fā)現(xiàn)FAK失活的胚胎雖然早期發(fā)育正常，但是在胚胎發(fā)育晚期出現(xiàn)胚胎、卵黃囊和胎盤的血管發(fā)育異常，內(nèi)皮細(xì)胞死亡增多，血管破裂進(jìn)而出現(xiàn)出血、水腫。多種疾病中也發(fā)現(xiàn)內(nèi)皮細(xì)胞FAK表達(dá)、活化異常。對(duì)于過量乙醇刺激對(duì)內(nèi)皮細(xì)胞FAK的影響及相應(yīng)的生物學(xué)意義尚缺乏研究。本研究將著眼于過量乙醇對(duì)內(nèi)皮細(xì)胞FAK的影響，探討乙醇血管毒性的相關(guān)機(jī)制。 第一部分：乙醇對(duì)內(nèi)皮細(xì)胞的毒性作用 目的：本部分將從動(dòng)物整體水平和體外細(xì)胞水平觀察乙醇對(duì)血管的毒性作用，全面研究乙醇對(duì)內(nèi)皮細(xì)胞結(jié)構(gòu)和功能的影響。 方法： 1采用灌胃器給酒制備大鼠飲酒模型，檢測(cè)飲酒后BAC變化。運(yùn)用Masson三色染色和透射電子顯微鏡（transmission electron microscope，TEM）技術(shù)觀察不同飲酒時(shí)間大鼠腦實(shí)質(zhì)毛細(xì)血管和基底動(dòng)脈形態(tài)改變； 2給予人臍靜脈內(nèi)皮細(xì)胞（human umbilical vein endothelial cell，HUVEC）不同濃度、不同時(shí)間的乙醇刺激，利用MTT檢測(cè)HUVEC存活率，反映乙醇的細(xì)胞毒性； 3利用流式細(xì)胞術(shù)觀測(cè)乙醇誘導(dǎo)的HUVCE凋亡； 4用光鏡、掃描電子顯微鏡（scanning electron microscope，SEM）和TEM觀察乙醇引起的內(nèi)皮細(xì)胞形態(tài)結(jié)構(gòu)改變； 5待乙醇干預(yù)結(jié)束后用胰酶替代物消化HUVEC，重新接種于纖維連接蛋白（fibronectin，F(xiàn)N）包被的培養(yǎng)板中，測(cè)量1-2h內(nèi)的粘附率和粘附過程中細(xì)胞鋪展的面積以評(píng)價(jià)內(nèi)皮細(xì)胞粘附能力；利用特異阻斷抗體預(yù)處理細(xì)胞實(shí)現(xiàn)整合素（integrin）阻斷后，進(jìn)行粘附實(shí)驗(yàn)，觀察integrin在其中作用； 6給予乙醇干預(yù)后，利用劃痕實(shí)驗(yàn)檢驗(yàn)乙醇對(duì)HUVEC遷移能力的影響。 結(jié)果： 1短期飲酒大鼠腦內(nèi)既可見明顯血管源性水腫，毛細(xì)血管周圍間隙明顯增大，內(nèi)皮細(xì)胞微絨毛減少，胞質(zhì)內(nèi)線粒體空泡化； 2長期飲酒大鼠基底動(dòng)脈中膜平滑肌成分與外膜膠原成分增多，壁/腔比明顯增大（p0.05），并可見內(nèi)皮細(xì)胞脫落； 3乙醇使內(nèi)皮細(xì)胞生存率呈劑量、時(shí)間依賴性下降，與對(duì)照組相比出現(xiàn)較多早期凋亡細(xì)胞（p0.05），這些凋亡細(xì)胞在TEM下呈現(xiàn)出細(xì)胞核形態(tài)不規(guī)則，核染色質(zhì)邊集呈半月狀且電子密度增高，細(xì)胞胞漿減少等征象； 4光鏡、SEM、TEM觀察可見乙醇刺激使HUVEC失去正常形態(tài)，胞內(nèi)細(xì)胞器表現(xiàn)出空泡化等損傷征象； 5乙醇明顯降低HUVEC粘附率（p0.05）和平均細(xì)胞鋪展面積（p0.05）。特異integrin β1抗體預(yù)處理后可以降低正常組細(xì)胞粘附率，，但對(duì)乙醇組影響不明顯； 6乙醇明顯降低HUVEC單細(xì)胞層劃痕的愈合速度（p0.05）。 小結(jié)：在體和細(xì)胞實(shí)驗(yàn)均證明過量乙醇具有明顯的內(nèi)皮毒性作用，影響內(nèi)皮細(xì)胞形態(tài)、降低生存率、抑制其與ECM粘附及粘附后鋪展，以及降低內(nèi)皮細(xì)胞遷移能力；乙醇作用后HUVEC粘附力下降可能與integrin信號(hào)通路有關(guān)。 第二部分：過量乙醇對(duì)內(nèi)皮細(xì)胞黏著斑激酶的影響及其產(chǎn)生機(jī)制 目的：本部分將系統(tǒng)研究過量乙醇對(duì)內(nèi)皮細(xì)胞中FAK分布、蛋白表達(dá)及磷酸化的影響，并探討相關(guān)機(jī)制。 方法： 1用細(xì)胞免疫熒光標(biāo)記細(xì)胞粘附、鋪展和遷移過程中的磷酸化的FAK酪氨酸397位點(diǎn)（phosphorylated FAK at Tyr397，pFAK Y397），觀察黏著斑復(fù)合體分布變化； 2利用western blot檢測(cè)乙醇作用對(duì)FAK總蛋白水平和磷酸化水平的影響； 3給予HUVEC特異乙醇脫氫酶（alcohol dehydrogenase，ADH）抑制劑4-甲基吡唑（4-methyle-pyrazole，4MP）預(yù)孵或給予乙醇主要的代謝產(chǎn)物乙醛（acetaldehyde，Ach）刺激，研究乙醇代謝在FAK變化中的作用； 4利用活性氧簇（reactive oxygen species，ROS）特異探針DCFH-DA標(biāo)記ROS觀察乙醇、乙醛引起的氧化應(yīng)激變化； 5給予外源性過氧化氫（H2O2）或非特異性內(nèi)源性ROS清除劑N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine，NAC），研究氧化應(yīng)激在FAK變化中的作用。 結(jié)果： 1乙醇并未影響integrin β1的表達(dá)和分布； 2在鋪展過程中的對(duì)照組細(xì)胞內(nèi)pFAK Y397形成較大的片狀分布，尤以細(xì)胞邊緣明顯，遷移過程中細(xì)胞遷移方向的前緣亦可形成大片狀pFAK Y397表達(dá)；而過量乙醇干預(yù)過的HUVEC中形成的片狀結(jié)構(gòu)小而少； 3乙醇增加FAK磷酸化，且呈劑量、時(shí)間依賴性（p0.05，p0.01），但并不影響FAK總蛋白水平； 4給予4MP可以削弱過量乙醇引起的FAK磷酸化增高（p0.05）；給予乙醛增加pFAK Y397（p0.01）； 5乙醇、乙醛刺激均可激發(fā)內(nèi)皮細(xì)胞中ROS產(chǎn)生； 6給予外源性H2O2可以激發(fā)FAK磷酸化（p0.01）；給予NAC可部分降低乙醇、乙醛引起的FAK磷酸化水平（p0.05）。 小結(jié)：過量乙醇一方面干擾了FAK參與的內(nèi)皮細(xì)胞黏著斑成熟，不能有效“引導(dǎo)”F-actin骨架，導(dǎo)致內(nèi)皮細(xì)胞生存、粘附、遷移功能受損；另一方面增加pFAK水平，且乙醇和（或）其代謝產(chǎn)生的乙醛、氧化應(yīng)激是引起FAK酪氨酸磷酸化的重要因素之一；過量乙醇并不影響FAK總蛋白水平。 第三部分：黏著斑激酶磷酸化在過量乙醇引起的內(nèi)皮細(xì)胞毒性中的作用 目的：本部分實(shí)驗(yàn)將以PI3K/AKT信號(hào)通路著手研究過量乙醇干預(yù)后FAK磷酸化增高在內(nèi)皮細(xì)胞一氧化氮（nitric oxide，NO）內(nèi)穩(wěn)態(tài)破壞中的作用。 方法： 1利用NO特異探針DAF-FM檢測(cè)內(nèi)皮細(xì)胞NO產(chǎn)生，并利用eNOS特異抑制劑N-硝基-L-精氨酸甲酯鹽酸鹽（ω-nitro-L-argine methyl ester，L-NAME）、 iNOS特異抑制劑L-canavanine和FAK自主磷酸化抑制劑PF573228（PF228）研究NO來源； 2在給予HUVEC乙醇刺激前給予PI3K/AKT通路抑制劑wortmannin和PF228預(yù)處理研究eNOS活化的相關(guān)通路； 3用western blot檢測(cè)硝基酪氨酸（nitrotyrosine，NT）產(chǎn)生量。 結(jié)果： 1乙醇引起NO明顯增高（p0.05）；L-NAME或PF228可以減弱乙醇引起的NO增加（p0.05），而L-canavanine作用不明顯； 2給予wortmannin和PF228均可明顯減弱乙醇引起AKT和eNOS磷酸化（p0.05）； 3200mM乙醇干預(yù)24h后明顯增加內(nèi)皮細(xì)胞NT生成量（p0.05）。 小結(jié)：過量乙醇刺激引起的FAK磷酸化通過PI3K/AKT通路促進(jìn)的eNOS活化，產(chǎn)生過量NO，生成具有細(xì)胞毒性的NT，造成內(nèi)皮細(xì)胞損傷。 結(jié)論： 過量乙醇可以引起腦血管內(nèi)皮細(xì)胞損傷，其機(jī)制主要表現(xiàn)在兩個(gè)方面： 1乙醇干擾黏著斑復(fù)合體成熟，影響內(nèi)皮細(xì)胞功能； 2乙醇及其代謝產(chǎn)生的乙醛、氧化應(yīng)激通過增加FAK磷酸化水平，激活PI3K/AKT通路，上調(diào)eNOS/NO系統(tǒng)，使NT過量生成，造成內(nèi)皮細(xì)胞損傷。
[Abstract]:The wine culture in China has a long history, but with the more frequent drinking in social communication activities, the social problems brought about by drinking are also becoming increasingly prominent. In recent years, the injury cases are often seen after drinking, and the cases are complicated, causing great pressure to the prosecutors. In 1963, there were foreign forensic doctors to put forward traumatic subarachnoid hemorrhage (traumatic Su). Barachnoid hemorrhage, tSAH) was associated with alcohol abuse. The actual case detection process found a slight external force, the incidence and mortality of intracranial hemorrhage were significantly higher. Although it is known that drinking can regulate heart rate and local blood flow, change vascular permeability, affect vasomotor energy, destroy clotting and anticoagulant balance, but these changes are still insufficient. To explain the causes of intracranial hemorrhage caused by the effect of external force after drinking. In addition, the problem that puzzles forensic workers for a long time is: how to define the participation of alcohol and external force, and then determine the nature of the case. Therefore, it is helpful to guide forensic practice and improve the thinking of forensic medicine by studying the mechanism of drinking alcohol which is easy to cause cerebral vascular rupture and bleeding. Road and clear decision results.
Ethanol (EtOH), also known as alcohol, is the main ingredient in alcoholic beverages. After drinking alcohol, ethanol can enter the blood in a prototype form. The concentration of blood alcohol concentration (BAC) increases rapidly and the ethanol is all over the body with the blood flow. The endothelial cells (endotheilial cell, EC) are located at the most inside of the vascular cavity and directly contact the blood ethanol and the blood. On the other hand, the endothelial cells are active in biological function and have ethanol metabolism related enzymes involved in ethanol metabolism, which determines that endothelial cells may suffer direct and indirect toxic effects of ethanol and become the first damage target of ethanol.
The vascular endothelium plays an important role in vascular regulation, which plays an irreplaceable role in a variety of biological processes, such as material exchange, vascular tension regulation, coagulation, angiogenesis, and leukocyte migration, and can "perceive" the shear stress, periodic stress and various kinds of mechanical stress and various blood components to coordinate other cells. Accordingly, the injury of endothelial cells caused by ethanol can endanger the whole blood vessel system.
In this study, endothelium is used as the main research object to explore the toxic effect of ethanol on the vascular toxicity of ethanol, and to further study the subarachnoid hemorrhage after drinking mild external force (subarachnoid The mechanism of hemorrhage, SAH) is well paved.
The vascular endothelium is distributed in the form of single layer cells. Its structure, function integrity depends particularly on the connection with the extracellular matrix (ECM), and communication. The adhesion complex is a complex structure between the cell and the ECM junction, realizing the connection between the cytoskeleton and ECM. On the one hand, it can pass the extracellular signal into the cell and pass through the cell. The distribution of the stress fiber is guided to adjust the cell morphology and function; on the other hand, the intracellular tractive force (tractionforce) produced by cell life activities is transferred to ECM, and then transformed into the biological signal to the surrounding cells, so that the body can make a timely macro adjustment.
Focal adhesion kinase (FAK) is an important component of the macula complex. On the one hand, FAK is activated by a variety of factors such as integrin aggregation, growth factor receptor connection, shear stress and so on. On the other hand, FAK can connect and activate other plaque structural proteins and participate in the formation and maturation of the plaque complex and regulate the cells. The basic functions such as adhesion and migration can also recruit functional proteins and participate in the regulation of different signaling pathways. It has been proved that FAK plays an important role in endothelial cells. The development of embryo in the specific inactivated endothelium FAK method is studied. Although the embryo of FAK inactivation is normal in the early stage, it is found in the embryo. There is an embryo in the late stage of breeding. The vascular development of the yolk sac and placenta is abnormal, the death of endothelial cells increases, blood vessels rupture and bleeding and edema. The FAK expression of endothelial cells is also found in a variety of diseases. The effect of excessive ethanol stimulation on the endothelial cell FAK and the corresponding biological significance are still not studied. This study will focus on the study. The effects of excessive ethanol on FAK of endothelial cells were investigated.
Part one: toxicity of ethanol on endothelial cells
Objective: To observe the toxic effects of ethanol on blood vessels from the overall level of animal and in vitro cell level, and to study the effect of ethanol on the structure and function of endothelial cells.
Method錛

本文編號(hào)：1972895