本文關(guān)鍵詞：STIM1抗體封閉對(duì)fMLP誘導(dǎo)的人中性粒細(xì)胞極性及趨化性的影響　出處：《南方醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： STIM1 fMLP 中性粒細(xì)胞 極性 趨化性

【摘要】：研究背景 中性粒細(xì)胞是先天免疫系統(tǒng)的重要組成部分。當(dāng)機(jī)體遭受外來(lái)細(xì)菌和病原體入侵時(shí),充當(dāng)?shù)谝坏婪谰�(xiàn),它們的主要功能是滲出血管壁并遷移到炎癥灶,通過(guò)吞噬作用和釋放超氧化物和／或蛋白水解酶殺死細(xì)菌和入侵的微生物。這種外滲和遷移的機(jī)制是通過(guò)細(xì)胞的極性和趨化性來(lái)實(shí)現(xiàn)的。臨床上一些疾病例如敗血癥、哮喘、缺血／再灌注損傷和器官移植的排斥反應(yīng)、動(dòng)脈粥樣硬化、病毒性心肌炎、類(lèi)風(fēng)濕性關(guān)節(jié)炎、過(guò)敏反應(yīng)、炎癥性皮膚病甚至腫瘤的發(fā)生和轉(zhuǎn)移都與中性粒細(xì)胞極性功能的增強(qiáng)和減弱有關(guān)。因此深入探討中性粒細(xì)胞極性調(diào)節(jié)機(jī)制為抵抗中性粒細(xì)胞激活導(dǎo)致的機(jī)體組織損傷尋找到新的干預(yù)和治療靶點(diǎn)。 鈣信號(hào)調(diào)控細(xì)胞內(nèi)許多重要生理、病理過(guò)程,胞質(zhì)內(nèi)Ca2+濃度升高依賴(lài)于兩種途徑：內(nèi)質(zhì)網(wǎng)鈣庫(kù)釋放和細(xì)胞膜外鈣內(nèi)流。鈣池操縱的鈣流入(store-operated calcium entry, SOCE)是非興奮細(xì)胞的主要鈣流入形式。近年來(lái)隨著蛋白基質(zhì)交互分子(stromal interaction molecule,STIM)的發(fā)現(xiàn),將SOCE調(diào)控機(jī)制的研究向前推進(jìn)了一步。Liou, Roos等證實(shí)了STIM1而非STIM2調(diào)控著SOCE。STIMl作為SOCE的關(guān)鍵分子,主要存在于內(nèi)質(zhì)網(wǎng)(Endoplasmic Reticulum, ER)內(nèi),也有少量分布于質(zhì)膜(plasma membrane,PM)上,它包含著一個(gè)定位于ER內(nèi)的EF-hand基序,充當(dāng)著鈣離子感受器的角色。文獻(xiàn)報(bào)導(dǎo)SOCE在細(xì)胞極性及趨化過(guò)程中發(fā)揮著重要作用,但對(duì)于STIM1分子在其中的作用及作用機(jī)制缺乏研究。 細(xì)胞的極性機(jī)制研究中存在著依賴(lài)PI3K和非依賴(lài)PI3K兩條途徑。PI3K-Akt被認(rèn)為是經(jīng)典的極性信號(hào)通路,也有研究表明酪氨酸激酶Src、Rho小G蛋白參與極性形成、細(xì)胞的遷移、腫瘤的侵襲與轉(zhuǎn)移等。而對(duì)于fMLP誘導(dǎo)的人中性粒細(xì)胞極性及趨化過(guò)程中是否也存在這樣的信號(hào)傳導(dǎo)通路尚不清楚。 人中性粒細(xì)胞為終末血細(xì)胞,在體外存活時(shí)間短,難于進(jìn)行基因操作或RNA干擾,那么電轉(zhuǎn)抗體封閉技術(shù)對(duì)于這種短時(shí)程的研究不失為一種行之有效的手段。為了闡明SOCE在人中性粒細(xì)胞極性及趨化性的作用以及調(diào)控機(jī)制,本實(shí)驗(yàn)采用此技術(shù)封閉細(xì)胞內(nèi)STIM1蛋白分子,觀(guān)察其對(duì)中性粒細(xì)胞趨化率的影響,以及相關(guān)的信號(hào)蛋白分子Akt, Src, Rho小G蛋白的變化。 目的 本實(shí)驗(yàn)通過(guò)研究STIM1抗體封閉后對(duì)fMLP誘導(dǎo)的人中性粒細(xì)胞極性及趨化性的影響,探討SOCE在人中性粒細(xì)胞極性及趨化性中的作用及其調(diào)控機(jī)制。 方法 1.采用Dextran作用下紅細(xì)胞自然沉降,Ficoll-Hypaque密度梯度離心,低滲裂解紅細(xì)胞的方法急性分離人外周血中性粒細(xì)胞。 2.使用電轉(zhuǎn)儀將anti-STIM1抗體轉(zhuǎn)入人中性粒細(xì)胞內(nèi)以進(jìn)行后續(xù)的人中性粒細(xì)胞極性及趨化性的研究。 3.采用Immunoprecipitation和Western blotting檢測(cè)anti-STIM1抗體電轉(zhuǎn)效果。 4.使用Zigmond chamber觀(guān)察STIM1抗體封閉后fMLP誘導(dǎo)的人中性粒細(xì)胞趨化率的變化。 5.應(yīng)用GST pull-down and Western blotting方法檢測(cè)STIM1抗體封閉后fMLP誘導(dǎo)的人中性粒細(xì)胞極性及趨化過(guò)程中相關(guān)信號(hào)分子的變化。 結(jié)果 1. Immunoprecipitation和Western blotting檢測(cè)anti-STIM1抗體電轉(zhuǎn)效率顯著,電轉(zhuǎn)anti-STIM1抗體組檢測(cè)到的蛋白信號(hào)強(qiáng)于非電轉(zhuǎn)組(P0.001)。 2. Control組、IgG組、anti-STIM1三組間趨化率比較差異有統(tǒng)計(jì)學(xué)意義(P0.001),趨化率的大小為Control組IgG組anti-STIM1組。 3.免疫印跡結(jié)果顯示Control組加與不加fMLP刺激p-Akt檢測(cè)到的蛋白條帶信號(hào)很弱,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.505), Src磷酸化以及Racl,Rac2,Cdc42活化水平加fMLP刺激后高于不加fMLP(P0.001); IgG組有著相同趨勢(shì),p-Akt無(wú)變化(P=0.766), p-Src,GTP-Racl,GTP-Rac2,GTP-Cdc42在fMLP刺激后其含量高于不加fMLP(P0.001); anti-STIM1組p-Akt差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.476), p-Src (P=0.315), GTP-Racl (P=0.944), GTP-Rac2 (P=0.386), GTP-Cdc42 (P=0.386)加與不加fMLP刺激各組比較差異均無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論 1. STIM1抗體封閉后抑制了fMLP誘導(dǎo)的人中性粒細(xì)胞極性及趨化性,提示SOCE可能參與了fMLP誘導(dǎo)的人中性粒細(xì)胞極性和趨化性。 2. STIM1抗體封閉后下調(diào)Src和Rac/Cdc42的活性,說(shuō)明SOCE通過(guò)Src-Rac/Cdc42信號(hào)通路正性調(diào)節(jié)fMLP誘導(dǎo)的人中性粒細(xì)胞極性及趨化性。
[Abstract]:Background of the study Neutrophils are an important part of innate immune system . When the organism is invaded by foreign bacteria and pathogens , it acts as the first line of defense . Their main function is to exude the blood vessel wall and migrate to the inflammatory range . The mechanism of this extravasation and migration is achieved by the polarity and the convergence of the cells . The mechanism of this extravasation and migration is related to the enhancement and attenuation of the neutrophil function by phagocytosis and release of superoxide and / or proteolytic enzymes . There are many important physiological and pathological processes in calcium signal regulation cells . The increase of intracellular Ca 2 + concentration depends on two ways : calcium influx in endoplasmic reticulum and intracellular calcium influx . In recent years , the study of SOCE control mechanism has been advanced by one step . In recent years , as the key molecule of SOCE , it contains an EF - hand motif located in ER , which acts as a role of calcium ion receptor . The literature reports that SOCE plays an important role in the process of cell polarity and chemotropism . In the study of the polarity mechanism of the cells , there are two pathways , which are dependent on the K 3 + and the non - dependent kinase , which are considered to be classical polar signal pathways . It is also found that there is a clear signal transduction pathway in the process of human neutrophil polarity and chemotropism induced by fmlp . In order to clarify the effect of SOCE on human neutrophil polarity and chemotropism and the mechanism of regulation and regulation , this experiment used this technique to close the molecule of STIM1 protein in the cell and observe its effect on neutrophil chemoattractant , as well as the changes of the related signal protein molecules such as signal protein kinase , protein kinase and protein . Purpose The effects of SOCE on the polarity and chemokine of human neutrophil in human neutrophil and its mechanism were investigated by studying the effect of STIM1 antibody on the polarity and chemoattractant of human neutrophil in human neutrophils . method 1 . Human peripheral blood neutrophils were isolated from human peripheral blood by the method of natural sedimentation of red blood cells , Ficoll - Hyacinth density gradient centrifugation and hypotonic lysis of red blood cells . 2 . The anti - STIM1 antibody was transferred to human neutrophils using an electrometer to conduct subsequent studies on the polarity and chemokine of human neutrophils . 3 . The effect of anti - STIM1 antibody was detected by Immunoppressing and Western blotting . 4 . Observe the change of human neutrophil chemoattractant induced by the STIM1 antibody after closure of the STIM1 antibody . 5 . GST pull - down and Western blotting were used to detect the changes of related signal molecules in the human neutrophil polarity and chemoattractant induced by STIM1 antibody . Results 1 . Immunoperoxidase and Western blotting showed that the anti - STIM1 antibody was significantly more efficient , and the protein signal detected by anti - STIM1 antibody group was stronger than that of non - electric rotating group ( P0.001 ) . 2 . There was significant difference between the three groups of control group , IgG group and anti - STIM1 group ( P0.001 ) , and the size of Chemotactic rate was control group IgG group anti - STIM1 group . 3 . There was no statistical significance ( P = 0.505 ) , there was no significant difference ( P = 0.505 ) , no change ( P = 0.766 ) , p - src ( P = 0.315 ) , GTP - Racl ( P = 0.944 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.396 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.386 ) , and no statistical significance . P=0.4 1 . After the blocking of the STIM1 antibody , the human neutrophil polarity and the chemoattractant induced by fmlp were inhibited , suggesting that SOCE might be involved in the human neutrophil polarity and chemokine induced by fmlp . 2 . After the STIM1 antibody was closed down , the activity of c / c / cdc42 was downregulated , indicating that SOC EON was positive to regulate the polarity and chemokine of human neutrophils induced by fL - Rac / Cdc42 signal pathway .

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363

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