發(fā)布時間：2018-04-27 17:50

  本文選題：哮喘 + 去整合素金屬蛋白酶��； 參考：《重慶大學》2012年博士論文

【摘要】：哮喘病是一種常見的呼吸道慢性炎癥疾病，是指支氣管在高反應(yīng)狀態(tài)下，由變應(yīng)原或其他因素引起的氣道狹窄的疾病，主要癥狀有胸悶、咳嗽、呼吸困難等。氣道平滑肌細胞Airway Smooth Muscle Cell，ASMC㖞是呼吸道的主要組成部分之一，具有重要的生理作用。當氣道平滑肌在受到外來因子的刺激時會發(fā)生氣道過度收縮反應(yīng)（Airway hyperrespinsiveness，AHR），臨床表現(xiàn)為患者氣道過度縮窄和發(fā)作性呼吸障礙，這是導致哮喘病成為高危疾病的最終原因所在。雖然在哮喘病的研究當中，對ASMC收縮特性的認識已經(jīng)有一個多世紀，但是關(guān)于氣道平滑肌病理變化的機制卻沒有研究透徹。氣道平滑肌是控制氣管管徑的效應(yīng)器，其病理性收縮成為氣道阻塞和呼吸困難的最終因素，它在決定哮喘病治療方法的有效性方面起到關(guān)鍵的作用，普遍認為其病理性收縮是哮喘難治的重要的病理基礎(chǔ)。去整合素堿金屬蛋白酶33（A disintegrin and metalloprotease33，ADAM33）基因已被證實是一個哮喘易感基因，且在大部分哮喘患者的氣道平滑肌和基底膜上其表達水平較健康受試者要高，說明ADAM33可能參與ASMC力學環(huán)境的調(diào)控，進而與哮喘病人ASMC力學特性異常及氣道重構(gòu)有關(guān)。然而，至今還沒有直接的證據(jù)證明ASMC生物力學行為與其ADAM33表達之間的關(guān)系。因此，本文采用分子生物學方法調(diào)控ASMC的ADAM33表達，同時采用細胞生物力學方法測量超表達和沉默ADAM33的ASMC的力學行為，以揭示二者之間的直接關(guān)系。本文的主要研究內(nèi)容和研究結(jié)果如下： ①ADAM33的表達水平隨著SD大鼠霧化周數(shù)的增加而顯著性升高。首先，利用氫氧化鋁作為佐劑，卵清蛋白（OVA）致敏并激發(fā)哮喘，建立SD(SpragueDawley)大鼠哮喘模型。每周隔天OVA霧化3次，每次30分鐘，共霧化12周，每2周取大鼠肺組織。通過對肺功能（Penh）的測定及氣道組織HE(Hematoxylin-Eosin staining)染色觀察結(jié)果表明我們已成功構(gòu)建大鼠哮喘模型。接著，利用定量RT-PCR和Western blot技術(shù)檢測ADAM33在OVA霧化不同周數(shù)的SD大鼠哮喘平滑肌細胞中的表達，結(jié)果顯示ADAM33的表達水平隨著OVA霧化周數(shù)的增加而顯著性升高，且定量RT-PCR與Western blot的實驗結(jié)果基本一致。從而說明ADAM33的表達水平與哮喘的嚴重程度呈正相關(guān)，該結(jié)果與Lee等在哮喘病人中的研究發(fā)現(xiàn)相符合，更近一步證明我們實驗中所構(gòu)建的SD大鼠哮喘模型能真實地模擬哮喘病。 ②獲得ADAM33超表達和沉默的ASMC。首先，構(gòu)建ADAM33基因超表達和干擾慢病毒重組載體，然后進行慢病毒轉(zhuǎn)染，以GFP空載及未轉(zhuǎn)染細胞作為對照。在熒光顯微鏡下觀察到重組慢病毒轉(zhuǎn)染正常SD大鼠ASMC，24h后GFP表達逐漸增強，至培養(yǎng)72h時GFP表達最強到高峰，并發(fā)現(xiàn)當MOI(Multiplicity ofInfection)=100時，慢病毒感染ASMC的效率為95%。定量RT-PCR檢測結(jié)果顯示，超表達組ADAM33基因表達水平是正常對照組的8倍（P＜0.01），沉默組中pLVT453沉默效率最高達到75%（P＜0.01）；同時，Western blot測定各組細胞的ADAM33蛋白表達水平結(jié)果表明，超表達組細胞內(nèi)ADAM33蛋白表達較兩組對照明顯增高（P＜0.01），而沉默組表達明顯降低（P＜0.01），ADAM33蛋白在兩組對照組中的表達量無明顯差別。本實驗成功獲得了穩(wěn)定的ADAM33超表達和沉默ASMC，且重組慢病毒轉(zhuǎn)染明顯上調(diào)或抑制了ASMC中ADAM33基因mRNA和蛋白的表達。 ③ADAM33基因表達水平的變化改變了ASMC的遷移能力和增殖能力。采用MTT法觀察不同4組細胞增殖情況，發(fā)現(xiàn)超表達組pLV-ADAM33的細胞增殖能力與空白對照組相比沒有顯著性差異；而沉默組pMa-ADAM33相對于對空白照組細胞增殖能力明顯降低（P＜0.05）。GFP對照組的細胞增殖能力顯著低于未轉(zhuǎn)染對照組（P＜0.01），，由此我們可以看出慢病毒轉(zhuǎn)染對細胞增殖能力有抑制作用，雖然上調(diào)ADAM33對細胞增殖能力對于未轉(zhuǎn)染對照組來說無差異，但是相對GFP對照組的細胞增殖能力顯著增強（P＜0.01）。劃痕法(Wound healing)檢測細胞橫向遷移能力的結(jié)果顯示，在12h時，4組細胞遷移無明顯差異。但是在24h時，超表達組pLV-ADAM33的橫向遷移能力與兩組對照相比明顯增強（P＜0.01）（P＜0.05）；而沉默組pMa-ADAM33的細胞顯著降低（P＜0.01），該結(jié)果表明通過特異性改變ADAM33基因的表達，能夠影響ASMC橫向遷移能力。 ④ADAM33基因能調(diào)控ASMC的細胞基礎(chǔ)剛度、收縮力及牽張力等細胞力學特性。運用光學捕捉（Optical trapping）與磁力扭轉(zhuǎn)細胞測量術(shù)（Magnetic TwistingCytometry）和傅里葉變換牽引力顯微術(shù)（Fourier Transform Traction Microscopy，F(xiàn)TTM）檢測由慢病毒轉(zhuǎn)染所獲得的穩(wěn)定超表達和干擾ADAM33的ASMC的生物力學特性。研究結(jié)果顯示：1)超表達ADAM33的ASMC組pLV-ADAM33的細胞基礎(chǔ)剛度、KCL激動劑處理后的收縮力、牽張力顯著高于對照組，而ADAM33沉默的細胞組其上述參數(shù)明顯低于對照。2)共聚焦免疫熒光結(jié)果顯示ADAM33和黏著斑蛋白vinculin存在共定位關(guān)系，并且還有部分ADAM33有沿著應(yīng)力纖維分布的情況，說明ADAM33可能調(diào)控細胞骨架結(jié)構(gòu)和粘著斑的形成。3)通過定量RT-PCR檢測結(jié)果發(fā)現(xiàn)，ADMA33基因表達水平的改變能調(diào)控細胞力學相關(guān)基因Calponin和Integrin-β1的表達。 從以上實驗結(jié)果，我們推測：調(diào)控ADAM33的表達直接影響到ASMCs的生物力學行為，提示ASMCs的生物力學行為與其ADAM33的表達存在直接的聯(lián)系。因此，哮喘患者中ADAM33基因表達及修復(fù)的異常有可能在氣道重塑和氣道收縮高反應(yīng)性等方面扮演決定性的角色，這對于深入理解哮喘的發(fā)病機制，特別是氣道功能變換的內(nèi)在調(diào)控機制，從而探尋有效的哮喘治療靶點和藥物具有深遠的意義。
[Abstract]:Asthma is a common chronic inflammatory disease of the respiratory tract. It refers to the bronchial stenosis caused by allergen or other factors in the high response state. The main symptoms are chest tightness, cough, and dyspnea. Airway smooth muscle cells? Airway Smooth Muscle Cell, ASMC? Is one of the main parts of the respiratory tract. When the airway smooth muscle is stimulated by foreign factors, the airway overcontraction (Airway hyperrespinsiveness, AHR) may occur. The clinical manifestation is the patient's airway overcontraction and paroxysmal breathing disorder, which is the ultimate cause of asthma to become a high-risk disease. Although in asthma studies, A The understanding of the contractile characteristics of SMC has been over a century, but the mechanism of the pathological changes of the airway smooth muscle is not thoroughly studied. The airway smooth muscle is an effector that controls the tracheal tube, and its pathological contraction is the ultimate factor in airway obstruction and dyspnea. It plays a key role in determining the effectiveness of asthma treatment. It is generally believed that the pathological contraction is an important pathological basis for the refractory asthma. The A disintegrin and metalloprotease33 (ADAM33) gene has been proved to be an asthma susceptible gene, and the expression level of the airway smooth muscle and basement membrane in most of the asthmatic patients is more than that of the healthy subjects. High, indicating that ADAM33 may be involved in the regulation of the mechanical environment of ASMC, which is related to the abnormal mechanical properties of ASMC and airway remodeling in asthmatic patients. However, there is no direct evidence to prove the relationship between the biomechanical behavior of ASMC and the expression of ADAM33. Therefore, the molecular biology method is used to regulate the ADAM33 expression of ASMC and to adopt a fine method. Cell biomechanical methods were used to measure the mechanical behavior of overexpression and silent ADAM33 ASMC, in order to reveal the direct relationship between the two. The main contents and results of this paper are as follows:
(1) the expression level of ADAM33 was significantly increased with the increase of the number of nebulization weeks in SD rats. First, using aluminum hydroxide as an adjuvant, ovalbumin (OVA) sensitized and stimulated asthma and established a model of asthma in SD (SpragueDawley) rats. OVA atomization 3 times a week, 12 weeks in a total of 12 weeks, and the lung function (P) was taken every 2 weeks. The determination of enh) and the observation of airway tissue HE (Hematoxylin-Eosin staining) staining showed that we had successfully constructed the rat model of asthma. Then, the quantitative RT-PCR and Western blot techniques were used to detect the expression of ADAM33 in the asthmatic smooth muscle cells of SD rats with different weeks of OVA atomization. The results showed that the ADAM33 expression level was with OVA atomization. The number of weeks increased significantly, and the quantitative RT-PCR was basically consistent with the experimental results of Western blot, indicating that the expression level of ADAM33 was positively correlated with the severity of asthma. This result was consistent with the research findings of Lee in the patients with asthma, and a closer step proved that the asthma model of the SD rat constructed in our experiment could be true. To simulate asthma.
(2) to obtain ADAM33 overexpression and silence ASMC. first, construct ADAM33 gene overexpression and interference with lentivirus recombinant vector, then transfection of lentivirus, with GFP empty and untransfected cells as control. The recombinant lentivirus transfected to normal SD rats ASMC, 24h after 24h, and GFP expression gradually increased, to GFP expression when 72h was cultured. When MOI (Multiplicity ofInfection) =100 was found, the efficiency of lentivirus infection ASMC was 95%. quantitative RT-PCR detection results. The expression of ADAM33 gene expression level in overexpression group was 8 times (P < 0.01), and pLVT453 silencing efficiency was up to 75% (P < 0.01) in silent group. The expression of ADAM33 protein showed that the expression of ADAM33 protein in the cells of the overexpression group was significantly higher than that in the two groups (P < 0.01), while the expression of the silent group decreased significantly (P < 0.01). The expression of ADAM33 protein in the two control groups was not significantly different. The stable ADAM33 overexpression and the silent ASMC were successfully obtained and the recombinant lentivirus was reorganized in this experiment. Transfection obviously increased or inhibited the expression of ADAM33 gene mRNA and protein in ASMC.
(3) the change of ADAM33 gene expression level changed the migration ability and proliferation ability of ASMC. The proliferation of different 4 groups was observed by MTT, and the proliferation ability of pLV-ADAM33 in the overexpression group was not significantly different from that in the blank control group, while the silent group pMa-ADAM33 was significantly lower than the cell proliferation ability of the blank group. The cell proliferation ability of the low (P < 0.05).GFP control group was significantly lower than that in the untransfected control group (P < 0.01). Therefore, we can see that the lentivirus transfection has a inhibitory effect on the cell proliferation ability. Although up regulation of ADAM33 has no difference in the cell proliferation ability to the untransfected control group, the cell proliferation ability of the GFP control group is significantly increased. Strong (P < 0.01). The results of detection of lateral migration of cells by scratch method (Wound healing) showed that there was no significant difference in the migration of 4 groups at 12h, but at 24h, the transversal migration of pLV-ADAM33 in the overexpression group was significantly increased (P < 0.01) (P < 0.05) in the two group (P < 0.05), while the cells in the silent group decreased significantly (P < 0.01). The results showed that the lateral migration of ASMC could be affected by specific changes in the expression of ADAM33 gene.
(4) ADAM33 gene can regulate the cellular mechanical properties of ASMC, such as cell base stiffness, contractile force and traction tension, etc. using optical capture (Optical trapping) and magnetic force torsional cell measurement (Magnetic TwistingCytometry) and Fu Liye transform traction microscopy (Fourier Transform Traction Microscopy, FTTM) detection by lentivirus transfection The biomechanical properties of the stable overexpression and interference of ADAM33 ASMC. The results showed: 1) the basal stiffness of pLV-ADAM33 in the ASMC group of ADAM33 overexpressed, the contractile force after the KCL agonist was significantly higher than that of the control group, while the above parameters in the ADAM33 silent cell group were significantly lower than that of the control.2) confocal immunofluorescence. The results show that there is a co localization relationship between ADAM33 and macula vinculin, and some ADAM33 has a distribution along the stress fiber, indicating that ADAM33 may regulate the cytoskeleton structure and the formation of.3 in the plaque. The changes in the expression level of the ADMA33 gene can regulate the cell mechanics related gene Calpon by the quantitative RT-PCR detection results. Expression of in and Integrin- beta 1.
From the above results, we speculate that the regulation of the expression of ADAM33 directly affects the biomechanical behavior of ASMCs, suggesting that the biomechanical behavior of ASMCs has a direct connection with the expression of ADAM33. Therefore, the abnormal expression of ADAM33 gene and the abnormality of the repair of the ADAM33 in the asthmatic patients may be played in the air channel remodeling and the hyperresponsiveness of the airway contraction. The decisive role is of profound significance in understanding the pathogenesis of asthma, especially the internal regulation mechanism of the airway function transformation, so as to explore effective targets and drugs for asthma treatment.

【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R562.25

【共引文獻】

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1 周治國;劉志文;范哲意;;微懸臂梁矩陣細胞牽引力測量傳感器研制[J];傳感技術(shù)學報;2009年05期

2 周歷;馬德順;楊毓;高淑云;;Pinch-3蛋白對細胞形態(tài)及牽引力的影響[J];沈陽大學學報(自然科學版);2012年01期

3 黃建永;鄧昊;彭小玲;李姍姍;熊春陽;方競;;細胞牽引力顯微鏡反演方法研究進展[J];實驗力學;2011年05期

4 馬德順;高淑云;路春霞;易正鑫;;前交叉韌帶干細胞分化后其牽引力的變化[J];中國細胞生物學學報;2010年01期

5 周治國;劉志文;范哲意;;基于BioMEMS微柱矩陣的細胞牽引力測量(英文)[J];醫(yī)用生物力學;2009年02期

6 李姍姍;黃建永;鄧昊;龐明姝;彭小玲;熊春陽;方競;;高分辨率細胞牽引力頻域反演技術(shù)[J];醫(yī)用生物力學;2011年03期

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1 劉凱;原媛;龐明姝;鄧昊;熊春陽;方競;;基于微圖案技術(shù)的高通量細胞牽引力顯微鏡[A];第十屆全國生物力學學術(shù)會議暨第十二屆全國生物流變學學術(shù)會議論文摘要匯編[C];2012年

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1 陳誠;短暫機械拉伸導致的人體膀胱平滑肌細胞的液化及其再固化[D];重慶大學;2011年

2 宋愛晶;大氣污染物二氧化硫?qū)Υ笫笙∧Ｐ蜌獾榔交∩锪W行為影響的研究[D];重慶大學;2011年

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4 劉彬;大花旋覆花內(nèi)酯及其衍生物抑制細胞增殖的作用機制[D];河北醫(yī)科大學;2010年

5 閆策;表面微納米圖案化技術(shù)及細胞黏附臨界面積等問題的研究[D];復(fù)旦大學;2012年

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1 高淑云;小鼠干細胞牽引力在分化過程中的變化研究[D];沈陽大學;2011年

2 周歷;小鼠髕腱干細胞分化過程中的力學特性研究[D];沈陽大學;2012年

3 李中港;過敏反應(yīng)動物模型的建立及在雙黃連注射劑過敏反應(yīng)檢測中的應(yīng)用[D];山東大學;2010年

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