范麗娟,李慧,張慧敏,李含含,黃鳳,張子健,戴周彤,項(xiàng)園,姚奧,李佳蓬,廖興華
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信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3通過趨化因子CX3C配體1促進(jìn)血管內(nèi)皮細(xì)胞增殖遷移
范麗娟,李慧,張慧敏,李含含,黃鳳,張子健,戴周彤,項(xiàng)園,姚奧,李佳蓬,廖興華
武漢科技大學(xué) 生命科學(xué)與健康學(xué)院,湖北 武漢 430000
范麗娟, 李慧, 張慧敏, 等. 信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3通過趨化因子CX3C配體1促進(jìn)血管內(nèi)皮細(xì)胞增殖遷移. 生物工程學(xué)報(bào), 2019, 35(4): 677–686.Fan LJ, Li H, Zhang HM, et al. Signal transducer and activator of transcription 3 promotes vascular endothelial cell proliferation and migration by fractalkine. Chin J Biotech, 2019, 35(4): 677–686.
信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3 (STAT3) 與趨化因子CX3C配體1 (Fractalkine/CX3CL1) 在血管炎癥和損傷中起重要作用,為了探討STAT3是否通過CX3CL1促進(jìn)血管內(nèi)皮細(xì)胞增殖和遷移,在血管內(nèi)皮細(xì)胞 (HUVEC) 中過表達(dá)或敲降STAT3,通過quantitative real-time PCR、Western blotting實(shí)驗(yàn)確定STAT3對(duì)CX3CL1表達(dá)的影響。構(gòu)建含有STAT3結(jié)合位點(diǎn)及突變STAT3結(jié)合位點(diǎn)的CX3CL1啟動(dòng)子熒光素酶報(bào)告基因質(zhì)粒,利用熒光素酶活性分析實(shí)驗(yàn)研究STAT3對(duì)CX3CL1啟動(dòng)子轉(zhuǎn)錄活性的作用。利用MTT實(shí)驗(yàn)檢測(cè)過表達(dá)或敲降STAT3或CX3CL1對(duì)血管內(nèi)皮細(xì)胞增殖率的影響。利用劃痕實(shí)驗(yàn)檢測(cè)過表達(dá)或敲降STAT3或CX3CL1對(duì)血管內(nèi)皮細(xì)胞遷移率的影響。結(jié)果顯示,過表達(dá)STAT3可以促進(jìn)CX3CL1表達(dá),敲降STAT3可以使CX3CL1表達(dá)下調(diào)。STAT3可以直接結(jié)合到CX3CL1的啟動(dòng)子促進(jìn)其轉(zhuǎn)錄激活,其促進(jìn)作用依賴于CX3CL1啟動(dòng)子上的GAS位點(diǎn)。敲降STAT3可以抑制血管內(nèi)皮細(xì)胞的遷移,過表達(dá)CX3CL1拮抗該抑制作用??偨Y(jié)得出,STAT3通過結(jié)合到CXCL1啟動(dòng)子促進(jìn)CX3CL1轉(zhuǎn)錄與表達(dá)進(jìn)而促進(jìn)血管內(nèi)皮的增殖與遷移。
人臍靜脈血管內(nèi)皮細(xì)胞,CX3CL1,STAT3,增殖,遷移
動(dòng)脈粥樣硬化 (Atherosclerosis,AS) 是許多心血管病形成的主要病理基礎(chǔ),由多因素共同作用引起,發(fā)病機(jī)制復(fù)雜,目前尚未完全闡明。炎癥學(xué)說認(rèn)為AS是一種慢性炎性反應(yīng),長(zhǎng)期反復(fù)的內(nèi)皮細(xì)胞 (Endothelial cells,EC) 損傷是其發(fā)生的病理基礎(chǔ)。在高脂血癥、高血壓、糖尿病或吸煙等危險(xiǎn)因素作用下,EC發(fā)生形態(tài)改變及功能障礙,導(dǎo)致血管損傷[1]。關(guān)于信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3 (Signal transducer and activator of transcription 3,STAT3) 在參與動(dòng)脈粥樣硬化炎癥機(jī)制方面的研究是目前的熱點(diǎn)。各種炎癥因子可能通過STAT3轉(zhuǎn)導(dǎo)的信號(hào)通路在血管炎癥中發(fā)揮作用。
細(xì)胞因子和生長(zhǎng)因子識(shí)別表面受體后,受體偶聯(lián)的JAK激酶與STAT3相互作用發(fā)生磷酸化,形成同源或異源二聚體,并作為轉(zhuǎn)錄激活因子轉(zhuǎn)運(yùn)到細(xì)胞核參與調(diào)控[2-3]。近年來JAK-STAT信號(hào)通路被發(fā)現(xiàn)與細(xì)胞因子刺激而形成的信號(hào)轉(zhuǎn)導(dǎo)過程密切相關(guān),并通過這一形式參與許多重要生物學(xué)過程,該通路的異常會(huì)導(dǎo)致各種疾病的形成。炎癥因子IL-6就被報(bào)道通過激活JAK/STAT3通路參與了動(dòng)脈粥樣硬化的病變[4]。
趨化因子CX3C的配體1 (Chemokine CX3C ligand 1,F(xiàn)ractalkine /CX3CL1) 是一種獨(dú)特的膜結(jié)合分子,在內(nèi)皮細(xì)胞 (ECs) 中表達(dá),分為可溶性 (游離型) 和膜結(jié)合性兩種形式。它是一種長(zhǎng)蛋白質(zhì),具有擴(kuò)展的粘蛋白樣莖和頂部的趨化因子結(jié)構(gòu)域。粘蛋白樣莖可以使其與某些細(xì)胞表面結(jié)合??扇苄訡X3CL1能有效地化學(xué)誘導(dǎo)T細(xì)胞和單核細(xì)胞[5],相對(duì)可溶性CX3CL1的趨化性而言,細(xì)胞結(jié)合趨化因子促進(jìn)白細(xì)胞與活化的內(nèi)皮細(xì)胞強(qiáng)烈粘附[6-7]。CX3C趨化因子受體1 (CX3C chemokine receptor 1,CX3CR1) 是CX3CL1的特異性受體,主要在某些白細(xì)胞群上表達(dá)[8-9],也被發(fā)現(xiàn)來源于血管壁損傷后平滑肌細(xì)胞分化的物質(zhì)[10]。有研究報(bào)道CX3CL1和CX3CR1在血管炎癥和損傷中起重要作用,促進(jìn)動(dòng)脈粥樣硬化的形成[11]。
雖然由ECs表達(dá),但CX3CL1和STAT3對(duì)內(nèi)皮細(xì)胞的增殖遷移的影響并不清楚。根據(jù)生物信息學(xué)發(fā)現(xiàn)CX3CL1基因序列上有STAT3的作用位點(diǎn),STAT3是否也同時(shí)與CX3CL1相互作用共同調(diào)節(jié)內(nèi)皮細(xì)胞的增殖遷移情況也并不明確。這些問題的解決可能為動(dòng)脈粥樣硬化不穩(wěn)定斑塊里的血管新生機(jī)制提供依據(jù)。又根據(jù)其他研究表明,STAT3能夠上調(diào)平滑肌細(xì)胞中CX3CL1的表達(dá)[12]。因此本文創(chuàng)新性研究STAT3介導(dǎo)的CX3CL1對(duì)血管內(nèi)皮細(xì)胞本身增殖能力的影響,為動(dòng)脈粥樣硬化血管新生的研究奠定基礎(chǔ)。
1.1.1 細(xì)胞
采用的人臍靜脈內(nèi)皮細(xì)胞株來源于中國(guó)上海細(xì)胞庫(kù)。所用細(xì)胞系為EA.HY926,置于?80 ℃保存。在進(jìn)行動(dòng)脈血管內(nèi)皮細(xì)胞實(shí)驗(yàn)時(shí),通常選用的細(xì)胞模型為人臍靜脈內(nèi)皮細(xì)胞 (Human umbilical vein endothelial cells,簡(jiǎn)稱HUVECs),因?yàn)槟氺o脈內(nèi)皮細(xì)胞具有干細(xì)胞的潛能,其特性與動(dòng)脈血管內(nèi)皮細(xì)胞相似,它們常被用作研究?jī)?nèi)皮細(xì)胞功能和病理學(xué) (例如血管生成)[13]。
1.1.2 試劑
DMEM培養(yǎng)基 (Hyclone,美國(guó));胰蛋白酶 (Biosharp,中國(guó));胎牛血清FBS (Gibco,美國(guó));雙抗/青鏈霉素合劑 (Gibco,美國(guó))。培養(yǎng)細(xì)胞使用的完全培養(yǎng)基含10% FBS 1%的雙抗。PBS液、TBST (自配);RIPA裂解液;磷酸酶抑制劑 (Roche,瑞士);SDS-PAGE蛋白上樣緩沖液 (碧云天,中國(guó));Trizol (Invitrogen,美國(guó));熒光定量試劑盒 (TaKaRa,日本);人源STAT3抗體 (鼠抗人,STAT3 (124H6) (mAbs,CST),二抗使用羊抗鼠抗體;人源CX3CL1抗體 (兔抗人,(CX3CL1 (ab25088) (mAbs,Abcam)),二抗使用羊抗兔抗體;PVDF膜 (Bio-Rad,美國(guó));SIRNA-STAT3及對(duì)照、SIRNA-CX3CL1及對(duì)照 (RIBOBIO,廣州銳博);一步法克隆試劑盒 (上海翊圣,中國(guó));Western Bright ECL (Advansta,美國(guó));熒光素酶報(bào)告基因檢測(cè)試劑盒 (Promega,美國(guó))。
1.1.3 儀器
電泳儀 (BIO-RAD,美國(guó)),核酸蛋白凝膠圖像系統(tǒng) (BIO-RAD,美國(guó);酶標(biāo)儀 (Leica,德國(guó));熒光定量PCR儀 (BIO-RAD);冷凍超速離心機(jī) (Beckman,美國(guó));氣套式二氧化碳恒溫培養(yǎng)箱 (Astec,日本);倒置顯微鏡 (Olympus,日本)。
1.2.1 質(zhì)粒構(gòu)建
在CX3CL1啟動(dòng)子上發(fā)現(xiàn)有STAT3的結(jié)合元件GAS區(qū),以人臍靜脈內(nèi)皮細(xì)胞基因組為模板構(gòu)建CX3CL1啟動(dòng)子的熒光素酶報(bào)告基因質(zhì)粒 pGL3-CX3CL1-WT,并構(gòu)建其突變質(zhì)粒pGL3- CX3CL1-MUT,空載為pGL3。同時(shí)以人臍靜脈內(nèi)皮細(xì)胞的cDNA為模板構(gòu)建STAT3、CX3CL1的表達(dá)質(zhì)粒pCDNA3.1-STAT3、pCDNA3.1-CX3CL1,空載為pCDNA3.1。質(zhì)粒構(gòu)建引物設(shè)計(jì)采用上海翊圣一步法克隆試劑盒所提供的方法,原理為末端同源重組,引物序列見表1。實(shí)時(shí)熒光定量核酸擴(kuò)增檢測(cè)所用的引物來源于PrimerBank,引物序列見表2。
1.2.2 細(xì)胞培養(yǎng)及轉(zhuǎn)染
為研究過表達(dá)和干擾CX3CL1和STAT3的表達(dá)對(duì)HUVEC細(xì)胞的增殖遷移作用,構(gòu)建表達(dá)質(zhì)粒和合成相應(yīng)的SIRNA,由廣州銳博公司設(shè)計(jì)合成。合成的SIRNA要在無(wú)RNA酶環(huán)境下進(jìn)行溶解分裝并置于?20 ℃保存。實(shí)驗(yàn)分組:轉(zhuǎn)染STAT3過表達(dá)質(zhì)粒 (STAT3組) 及其對(duì)照質(zhì)粒pCDNA3.1 (NC組);轉(zhuǎn)染SIRNA-STAT3 (SI-STAT3組)干擾STAT3的表達(dá)及其對(duì)照SIRNA-STAT3-Control (SI-NC1組)。轉(zhuǎn)染CX3CL1過表達(dá)質(zhì)粒 (CX3CL1組) 及其對(duì)照組pCDNA3.1 (NC組);轉(zhuǎn)染SIRNA-CX3CL1 (SI-CX3CL1組) 干擾CX3CL1的表達(dá)及其對(duì)照組SIRNA-CX3CL1-Control (SI-NC2組)。轉(zhuǎn)染前保證培養(yǎng)細(xì)胞處于對(duì)數(shù)生長(zhǎng)期,此時(shí)轉(zhuǎn)染細(xì)胞狀態(tài)最好。應(yīng)用FPF-g-PEI (Folate-poly (Ethylene glycol)-folate-grafted-polyethylenimine,PEI) 化合物作為轉(zhuǎn)染試劑進(jìn)行轉(zhuǎn)染。根據(jù)6孔板1×106/2 mL、12孔板5×105/mL濃度種植細(xì)胞,用完全培養(yǎng)基培養(yǎng)細(xì)胞6 h后完全貼壁,準(zhǔn)備轉(zhuǎn)染液配制。配制前提前半小時(shí)將細(xì)胞培養(yǎng)更換為無(wú)血清低糖DMEM培養(yǎng)基,然后按質(zhì)?!肞EI化合物=1∶3比例分別用無(wú)血清低糖DMEM培養(yǎng)基配置細(xì)胞轉(zhuǎn)染液200 μL,每100 μL中分別加轉(zhuǎn)染質(zhì)粒和對(duì)應(yīng)的轉(zhuǎn)染試劑,然后在室溫下放置5–10 min后混勻;混勻后37 ℃放置30 min。最后根據(jù)每個(gè)孔總培養(yǎng)基的量加入轉(zhuǎn)染液并補(bǔ)足無(wú)血清培養(yǎng)基,作好標(biāo)記放置于37 ℃、5% CO2培養(yǎng)箱內(nèi)培養(yǎng)8 h后棄掉轉(zhuǎn)染液更換為完全培養(yǎng)基繼續(xù)培養(yǎng)至48 h。
表1 質(zhì)粒構(gòu)建引物設(shè)計(jì)
表2 實(shí)時(shí)熒光定量核酸擴(kuò)增檢測(cè)引物序列
1.2.3 熒光素酶報(bào)告基因活性的測(cè)定 (Luciferase)
熒光素酶報(bào)告基因是指以熒光素 (Luciferin) 為底物來檢測(cè)螢火蟲熒光素酶 (Firefly luciferase) 活性的一種報(bào)告系統(tǒng)。熒光素酶可以催化luciferin氧化成oxyluciferin,在luciferin氧化的過程中,會(huì)發(fā)出生物熒光 (Bioluminescence)。先按pCDNA3.1+CX3CL1-WT組 (共轉(zhuǎn)pCDNA3.1和pGL3-CX3CL1-WT)、STAT3+CX3CL1-WT組 (共轉(zhuǎn)pCDNA3.1-STAT3和pGL3-CX3CL1-WT)、pCDNA3.1+CX3CL1-MUT組 (共轉(zhuǎn)pCDNA3.1和pGL3-CX3CL1-MUT)、STAT3+CX3CL1-MUT組(共轉(zhuǎn)pCDNA3.1-STAT3和pGL3-CX3CL1-MUT) 轉(zhuǎn)染細(xì)胞并進(jìn)行培養(yǎng)。然后從孵育箱中取出經(jīng)過處理好并培養(yǎng)48 h的細(xì)胞,棄去24孔板培養(yǎng)基,用PBS洗3次,動(dòng)作輕柔避免細(xì)胞漂浮。后按150 μL/孔加入細(xì)胞裂解液,輕輕搖動(dòng)讓裂解液完全覆蓋細(xì)胞后放冰上裂解30 min。小心刮下細(xì)胞,吸走裂解液轉(zhuǎn)到EP管,在冷凍超速離心機(jī)中于4 ℃、12 000 r/min離心20 min。離心后每個(gè)樣取上清和檢測(cè)底物,測(cè)20 s內(nèi)的發(fā)光值。
1.2.4 MTT細(xì)胞增殖檢測(cè)
將對(duì)數(shù)期細(xì)胞均勻鋪在96孔板中,細(xì)胞濃度為1×104個(gè)/mL,每孔加100 μL。按照實(shí)驗(yàn)分組,設(shè)6個(gè)復(fù)孔,37 ℃、5% CO2培養(yǎng)至24 h后,每孔加MTT溶液 (5 mg/mL,用pH 7.4的PBS緩沖液配制) 20 μL。繼續(xù)孵育4 h,終止培養(yǎng),小心棄去孔內(nèi)培養(yǎng)上清液,對(duì)于懸浮細(xì)胞,需要離心后再吸棄孔內(nèi)培養(yǎng)上清液。每孔加150 μL DMSO,振蕩10 min,使結(jié)晶物充分溶解。后選擇490 nm波長(zhǎng),在酶聯(lián)免疫監(jiān)測(cè)儀上測(cè)各組光密度 (Optical density,) 值。記錄測(cè)定結(jié)果。
1.2.5 細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力
將處理好的對(duì)數(shù)期細(xì)胞均勻鋪在12孔板中,待細(xì)胞貼壁并找到合適的密度開始劃線,劃線采用20 μL移液槍頭筆直地劃4條線,然后用PBS將漂浮的細(xì)胞洗滌3次。加入培養(yǎng)基放入37 ℃、5% CO2培養(yǎng)箱培養(yǎng),針對(duì)同一視野選取不同的時(shí)間來拍攝記錄細(xì)胞增殖遷移情況。
1.2.6 實(shí)時(shí)熒光定量核酸擴(kuò)增檢測(cè) (qPCR)
將細(xì)胞培養(yǎng)并進(jìn)行轉(zhuǎn)染處理后培養(yǎng)48 h,分別收集各組細(xì)胞至EP管中,離心后用Trizol裂解細(xì)胞快速提取RNA,提取時(shí)應(yīng)防止污染導(dǎo)致RNA降解。然后進(jìn)行逆轉(zhuǎn)錄合成cDNA。最后用熒光定量PCR試劑盒檢測(cè)基因的表達(dá),獲取數(shù)據(jù)進(jìn)行處理。
1.2.7 蛋白質(zhì)印跡法檢測(cè) (Western blotting)
在6孔板中種板細(xì)胞,待細(xì)胞進(jìn)入對(duì)數(shù)生長(zhǎng)期后,進(jìn)行實(shí)驗(yàn)分組并對(duì)細(xì)胞進(jìn)行轉(zhuǎn)染。培養(yǎng)至48 h后分別收集細(xì)胞約5×106個(gè),離心去上清,用裂解液裂解細(xì)胞,提取總蛋白進(jìn)行定量,根據(jù)相應(yīng)的實(shí)驗(yàn)分組進(jìn)行SDS-PAGE、300 mA轉(zhuǎn)膜 1 h。用5%奶牛封閉1 h,一抗 (一抗STAT3孵育濃度為1∶1 000,一抗CX3CL1孵育濃度為1∶1 500) 4 ℃孵育過夜,TBST洗膜5次, 5 min/次。用二抗37 ℃孵育2 h,洗膜后加化學(xué)底物發(fā)光試劑ECL,用核酸蛋白凝膠圖像系統(tǒng)顯影。用Origin 8軟件分析。
1.2.8 統(tǒng)計(jì)學(xué)方法
數(shù)據(jù)采用SSPS 13.0統(tǒng)計(jì)軟件單因素方差分析,以±表示,組間比較用檢驗(yàn)或非參數(shù)檢驗(yàn),以<0.05為差異顯著,以α=0.05為顯著檢驗(yàn)水準(zhǔn)。
圖1A顯示PCR擴(kuò)增出帶有CX3CL1啟動(dòng)子片段、表達(dá)CX3CL1基因片段、表達(dá)STAT3基因片段以及構(gòu)建成功的CX3CL1啟動(dòng)子熒光素酶質(zhì)粒和突變質(zhì)粒、CX3CL1表達(dá)質(zhì)粒和STAT3表達(dá)質(zhì)粒。圖1B中紅色是轉(zhuǎn)染CY3后在熒光顯微鏡下觀察到的結(jié)果,代表SIRNA的轉(zhuǎn)染效率。綠色是轉(zhuǎn)染GFP后在熒光顯微鏡下觀察到的結(jié)果,代表質(zhì)粒的轉(zhuǎn)染效率。CY3的轉(zhuǎn)染效率為70%左右,GFP的轉(zhuǎn)染效率為30%左右。
Western blotting、QPCR檢測(cè)結(jié)果顯示,過表達(dá)STAT3組STAT3、CX3CL1水平比對(duì)照組高,SI-STAT3組STAT3、CX3CL1水平有所降低 (圖2A、2B)。
CX3CL1啟動(dòng)子上TGCTGGGAA序列為GAS位點(diǎn),為檢測(cè)STAT3與CX3CL1啟動(dòng)子的相互作用,故將該位點(diǎn)序列突變?yōu)榉荊AS位點(diǎn) (圖3A)。進(jìn)行熒光素酶活性分析時(shí),實(shí)驗(yàn)組為共轉(zhuǎn)STAT3和野生型或者突變型CX3CL1啟動(dòng)子熒光素酶質(zhì)粒,共轉(zhuǎn)pCDNA3.1和CX3CL1啟動(dòng)子熒光素酶質(zhì)粒作為對(duì)照組。結(jié)果顯示,共轉(zhuǎn)STAT3和野生型CX3CL1啟動(dòng)子熒光素酶質(zhì)粒的實(shí)驗(yàn)組熒光素酶活性明顯高于對(duì)照組 (<0.05),而共轉(zhuǎn)STAT3和突變型CX3CL1啟動(dòng)子熒光素酶質(zhì)粒的熒光素酶活性相比對(duì)照組變化不顯著 (圖3B)。
圖1 質(zhì)粒構(gòu)建和轉(zhuǎn)染效率
MTT檢測(cè)結(jié)果顯示,轉(zhuǎn)染STAT3、CX3CL1組HUVEC細(xì)胞在490 nm處測(cè)得的值與對(duì)照組相比明顯增高,而分別用SI-STAT3、SI-CX3CL1處理后的實(shí)驗(yàn)組值明顯下降 (<0.05) (圖4A、4B)。
采用細(xì)胞劃痕試驗(yàn)檢測(cè)各組細(xì)胞遷移能力。48 h后,觀察到STAT3、CX3CL1組相對(duì)于對(duì)照組細(xì)胞增殖遷移能力明顯增強(qiáng),SI-STAT3和SI-CX3CL1組細(xì)胞遷移能力下降 (圖5A、5B)。表明STAT3和CX3CL1促進(jìn)HUVECs遷移,而干擾STAT3以后,可能下調(diào)CX3CL1的水平從而抑制細(xì)胞遷移。
為進(jìn)一步驗(yàn)證STAT3對(duì)HUVEC的遷移能力的影響是通過作用于CX3CL1來實(shí)現(xiàn)的,在敲降CX3CL1水平的同時(shí)過表達(dá)STAT3,通過圖6A可觀察到并沒有明顯地促進(jìn)細(xì)胞遷移,而圖6B中敲低STAT3時(shí)過表達(dá)CX3CL1仍然能促進(jìn)細(xì)胞遷移。這說明,CX3CL1可能作為STAT3的下游靶基因,對(duì)HUVEC的遷移有著更直接的影響。
信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子STAT3參與機(jī)體內(nèi)多種生物學(xué)過程,包括細(xì)胞存活、增殖、纖維化、氧化應(yīng)激、免疫功能改變及炎性反應(yīng)等[14-15]。STAT3基因中的功能獲得性突變會(huì)引起多種疾病的產(chǎn)生。在與血管有關(guān)的疾病中,STAT3作為重要轉(zhuǎn)錄因子參與血管平滑肌細(xì)胞增殖過程[16-17]。JAK/STAT信號(hào)通路與CX3CL1密切相關(guān),其調(diào)節(jié)異??纱偈笴X3CL1的表達(dá)[18]。CX3CL1是一種結(jié)構(gòu)獨(dú)特的趨化因子,膜結(jié)合性CX3CL1被 2個(gè)肽酶ADAM10和ADAM17裂解后能形成可溶性CX3CL1[19]。CX3CL1-CX3CR1軸活性在不穩(wěn)定性心絞痛和斑塊破裂或心血管疾病患者中表現(xiàn)增強(qiáng)[20-21]。據(jù)報(bào)道,CX3CL1和CX3CR1在動(dòng)脈粥樣硬化中起重要作用,最有可能是通過促進(jìn)血管壁炎癥[22]。在動(dòng)脈粥樣硬化晚期的人動(dòng)脈中觀察到高水平的CX3CL1 mRNA,在載脂蛋白基因apoE缺陷 (apoE-/-) 小鼠的動(dòng)脈粥樣硬化病變中CX3CL1上調(diào)[23]。CX3CL1在正常的ECs中表達(dá)水平較低,由促炎因子如脂多糖、腫瘤壞死因子-α、白細(xì)胞介素-1和干擾素-α激活的EC中表達(dá)顯著增加[24]。腫瘤壞死因子-α和白細(xì)胞介素-1通過核因子kβ信號(hào)通路誘導(dǎo)CX3CL1的表達(dá),而干擾素-β通過JAK/STAT1誘導(dǎo)CX3CL1的表達(dá)[25-26]。
圖4 MTT測(cè)STAT3、CX3CL1對(duì)HUVEC細(xì)胞增殖的影響
圖5 STAT3和CX3CL1對(duì)HUVEC的遷移能力的影響
圖6 敲低CX3CL1時(shí)過表達(dá)STAT3對(duì)HUVEC的遷移能力的影響
為研究STAT3參與細(xì)胞的增殖過程是否通過調(diào)控CX3CL1來實(shí)現(xiàn),以及探討在STAT3轉(zhuǎn)錄因子介導(dǎo)下CX3CL1對(duì)血管內(nèi)皮細(xì)胞本身增殖能力的影響,本文針對(duì)STAT3對(duì)CX3CL1作用方式、STAT3對(duì)CX3CL1表達(dá)的調(diào)控以及其相互作用對(duì)內(nèi)皮細(xì)胞增殖和遷移的影響也進(jìn)行了初步的探索。結(jié)果表明STAT3可能通過作用于CX3CL1啟動(dòng)子上的GAS區(qū)來調(diào)控CX3CL1水平,并且結(jié)果也顯示CX3CL1可以促進(jìn)細(xì)胞增殖和遷移,STAT3能誘導(dǎo)血管內(nèi)皮細(xì)胞CX3CL1表達(dá)上調(diào)。以上說明STAT3可能是通過直接作用于CX3CL1的啟動(dòng)子上調(diào)CX3CL1的水平,從而促進(jìn)細(xì)胞增殖和遷移。而干擾血管內(nèi)皮細(xì)胞中STAT3的表達(dá),則能下調(diào)CX3CL1的水平并能抑制血管內(nèi)皮細(xì)胞增殖和遷移。這些結(jié)論對(duì)動(dòng)脈粥樣硬化的機(jī)制研究具有重大意義,并且炎癥因子CX3CL1成為動(dòng)脈粥樣硬化的治療靶點(diǎn)對(duì)尋找動(dòng)脈粥樣硬化的治療藥物具有潛在的應(yīng)用價(jià)值。
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Signal transducer and activator of transcription 3 promotes vascular endothelial cell proliferation and migration by fractalkine
Lijuan Fan, Hui Li, Huimin Zhang, Hanhan Li, Feng Huang, Zijian Zhang, Zhoutong Dai, Yuan Xiang, Ao Yao, Jiapeng Li, and Xinghua Liao
College of Life Sciences and Health, Wuhan University of Science and Technology, Wuhan 430000, Hubei, China
Signal transducer and activator of transcription 3 (STAT3) and Chemokine CX3C ligand 1 (Fractalkine/CX3CL1) play important roles in vascular inflammation and injury. To study if STAT3 promotes vascular endothelial cell proliferation and migration through fractalkine, we overexpressed or knocked down STAT3 in vascular endothelial cells, and used quantitative real-time PCR and Western blotting to determine the effect of STAT3 on fractalkine expression. The wild type and STAT3 binding site mutantfractalkine promoter luciferase reporter plasmids were constructed, and luciferase activity assays were used to explore the effect of STAT3 on the transcriptional activity of the fractalkine promoter. MTT assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on the proliferation rate of vascular endothelial cells. Scratch assays were used to detect the effect of overexpression or knockdown of STAT3 orfractalkine on vascular endothelial cell migration. There results showed that overexpression of STAT3 could promote fractalkine expression, and knockdown of STAT3 could down-regulate fractalkine expression. STAT3 could directly bind to the promoter of fractalkine to promote its transcriptional activity via binding the GAS site of the fractalkine promoter. Knockdown of STAT3 could inhibit the migration of vascular endothelial cell, and overexpression of fractalkine antagonized this inhibition. Our data concluded that STAT3 promotes the proliferation and migration of vascular endothelial cell by binding the GAS site of the fractalkine promoter to promote fractalkine transcriptional activity and expression.
human umbilical vein endothelial cells, CX3CL1, STAT3, proliferation, migration
10.13345/j.cjb.180355
September 3, 2018;
December 20, 2018
National Natural Science Foundation of China (No. 31501149),Hubei Science and Technology Program Natural Science Fund General Project (No. 2017CFB537).
Xinghua Liao. Tel: +86-27-68893396; E-mail: xinghualiao@hotmail.com
國(guó)家自然科學(xué)基金 (No. 31501149),湖北省科技計(jì)劃自然科學(xué)基金 (No. 2017CFB537) 資助。
2019-02-19
http://kns.cnki.net/kcms/detail/11.1998.q.20190218.0956.002.html
(本文責(zé)編 陳宏宇)