YAP及P-YAP在RAW264.7細(xì)胞膿毒癥模型中的表達(dá)*

左文 王海清 鐘武 胡迎春 陳睦虎

(西南醫(yī)科大學(xué)附屬醫(yī)院急診科,四川 瀘州 646000)

膿毒癥是機(jī)體對(duì)感染的過度免疫反應(yīng)，大量的炎癥介質(zhì)釋放到外周血引起全身炎癥反應(yīng)(systemic inflammatory response syndrome,SIRS)[1]。其發(fā)生率占住院患者的1%～2%，如果伴感染性休克和多器官功能障礙，其死亡率高達(dá)30%～60%[2]。膿毒癥的發(fā)生機(jī)制尚不明確，若能找到調(diào)控膿毒癥的關(guān)鍵信號(hào)通路及其關(guān)鍵基因，或許能為臨床治療膿毒癥提供新靶點(diǎn)。目前關(guān)于hippo信號(hào)通路在膿毒癥中的作用機(jī)制的報(bào)道較少。Yes相關(guān)蛋白(YAP)是hippo信號(hào)通路中重要的核心轉(zhuǎn)錄因子，參與細(xì)胞的增殖、分化及組織的穩(wěn)定[3]。本實(shí)驗(yàn)擬通過檢測(cè)YAP和P-YAP在細(xì)胞膿毒癥模型中的表達(dá)情況，以探討YAP和P-YAP與膿毒癥發(fā)生的相關(guān)性。

1 材料與方法

1.1 試劑及儀器 高糖DMEM培養(yǎng)基(Hyclone)，胎牛血清FBS(PAN),LPS(Sigma),YAP、P-YAP及IONS(abcom)一抗，β-actin一抗(碧云天)，山羊抗兔IgG二抗(博奧森)，綠色熒光山羊抗兔IgG(Protientech)，DAPI(索萊寶)，蛋白酶抑制劑和磷酸酶抑制劑(Roche)，蛋白裂解液(碧云天)。低溫離心機(jī)(Thermo),生物安全柜(Telstar)，熒光顯微鏡(olympus)，共聚焦顯微鏡(feica),細(xì)胞培養(yǎng)箱(Thermo)。

1.2 細(xì)胞株 小鼠巨噬細(xì)胞RAW264.7，購(gòu)自中國(guó)科學(xué)院上海細(xì)胞庫(kù)。

1.3 細(xì)胞培養(yǎng)及模型的建立 RAW264.7細(xì)胞用含10%FBS DMEM培養(yǎng)基在5%CO2,37℃條件下培養(yǎng)[4]。取對(duì)數(shù)期RAW264.7細(xì)胞，用100ng/ml的LPS刺激4小時(shí)，構(gòu)建膿毒癥模型[5]。取對(duì)數(shù)期raw264.7細(xì)胞在6孔板中培養(yǎng)，每孔接種5.0×106個(gè)/ml，分為正常組和實(shí)驗(yàn)組，實(shí)驗(yàn)組接種24小時(shí)后(即對(duì)數(shù)期)用100ng/ml的LPS刺激4小時(shí)建立膿毒癥模型，對(duì)照組加入相同體積的完全培養(yǎng)基。

1.4 免疫熒光對(duì)YAP及P-YAP進(jìn)行定位 取對(duì)數(shù)期的RAW264.7細(xì)胞在12孔板中爬片，每孔接種2.0×106個(gè)/ml，培養(yǎng)24小時(shí)后用LPS(100ng/ml)刺激RAW264.7細(xì)胞，培養(yǎng)4小時(shí)，PBS洗滌3次，每次3分鐘，4%多聚甲醛固定15分鐘，再用PBS洗滌3次，每次3分鐘，0.5%Triton-10打孔20分鐘，PBS洗滌3次，每次3分鐘，10%山羊血清封閉30分鐘，一抗過夜。第二天37℃復(fù)溫45分鐘，吸進(jìn)一抗后，PBS洗滌3次，每次3分鐘，37℃孵育二抗1小時(shí)，PBS洗滌3次，每次3分鐘，DAPI復(fù)染核5分鐘，PBS洗滌3次，每次5分鐘，加入適量的抗熒光淬滅劑。然后在熒光顯微鏡或共聚焦顯微鏡下觀察。

1.5 Weston blot檢測(cè)YAP及P-YAP的表達(dá)情況 用含有1%磷酸酶抑制劑和1%蛋白酶抑制劑的細(xì)胞裂解液提取蛋白,BCA檢測(cè)蛋白濃度，用10%的分離膠電泳，先用60V電壓電泳30分鐘，再用120V電壓電泳60分鐘，然后轉(zhuǎn)移到PVDF膜，用0.02A恒流轉(zhuǎn)60分鐘。用5%脫脂牛奶封閉1小時(shí)，PBST洗滌5分鐘，YAP(1:4000)，P-YAP(1:2000)一抗在4℃過夜。第二天，PBST洗滌3次，每次10分鐘，孵二抗(1:4000)一小時(shí)， PBST洗滌3次，每次10分鐘。用VisionCapt曝光。用Quantity One進(jìn)行灰度值檢測(cè)。

1.6 統(tǒng)計(jì)學(xué)分析 運(yùn)用SPSS16.0對(duì)YAP及P-YAP表達(dá)情況進(jìn)行分析，P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 RAW264.7細(xì)胞鑒定及細(xì)胞膿毒癥模型的建立 INOS為M1型巨噬細(xì)胞特異性分子[6],用100ng/ml LSP刺激RAW264.7細(xì)胞4h，用免疫熒光檢測(cè)模型的建立，100%細(xì)胞均有INOS表達(dá)，見圖1。說明RAW264.7濃毒血癥模型建立成功。

圖1 LPS(100ng/ml)刺激RAW264.7細(xì)胞后檢測(cè)INOSFigure 1 LPS stimulated RAW 264.7 cells，INOS were used to evaluate the model of sepsis

2.2 免疫熒光對(duì)RAW264.7細(xì)胞濃度血癥模型中YAP及P-YAP進(jìn)行表達(dá)定位 在RAW264.7細(xì)胞膿毒癥模型中，P-YAP表達(dá)于胞質(zhì)中，在實(shí)驗(yàn)組中熒光亮度減弱，見圖2。YAP蛋白表達(dá)于胞核中，在實(shí)驗(yàn)組中熒光強(qiáng)度增強(qiáng)，見圖3。

2.3 用Weston blot檢測(cè)正常組及模型組YAP蛋白及P-YAP蛋白的情況 實(shí)驗(yàn)組YAP蛋白表達(dá)明顯高于正常組(P<0.5),見圖4；實(shí)驗(yàn)組P-YAP蛋白表達(dá)明顯低于正常組(P<0.5)，見圖5。

3 討論

膿毒癥是嚴(yán)重的全身炎癥反應(yīng)，嚴(yán)重威脅患者生命[7]。內(nèi)毒素是革蘭陰性桿菌的重要成分，是導(dǎo)致膿毒癥的主要原因[8]。當(dāng)膿毒癥發(fā)生時(shí)，血液中微生物的引起了一種先天性免疫反應(yīng)，其特征是單核細(xì)胞的刺激和促炎細(xì)胞因子的釋放以及各種免疫途徑的激活。目前，Toll樣受體(LPS-TLR4-MD-2)在最初的免疫激活中發(fā)揮關(guān)鍵作用，作為天然免疫系統(tǒng)的傳感器通過高度保守的成分，參與各種微生物的識(shí)別[9]。 但是，阻斷TLR4通路并不能完全抑制炎癥介質(zhì)的釋放[10]，推測(cè)有其他通路參與其中。

巨噬細(xì)胞在膿毒癥的發(fā)生中有著重要的作用。巨噬細(xì)胞可以分化成M1、M2型巨噬細(xì)胞。M1型巨噬細(xì)胞產(chǎn)生TNF-α，IL-1和一氧化氮合酶,促進(jìn)細(xì)菌的去除和損壞的細(xì)胞,已被證明具有抗腫瘤性質(zhì)，而M2巨噬細(xì)胞產(chǎn)生精氨酸酶,促進(jìn)組織修復(fù)和腫瘤增殖[11-12]。在膿毒癥時(shí)，巨噬細(xì)胞可分化為M1型[13]，大量釋放炎癥介質(zhì)進(jìn)入外周血，引發(fā)廣泛炎癥，引起多器官功能障礙(MODS)的發(fā)生，引起嚴(yán)重感染和感染性休克[14]。故本實(shí)驗(yàn)通過LPS刺激RAW264.7細(xì)胞構(gòu)建細(xì)胞膿毒癥模型。

圖2 P-YAP表達(dá)于細(xì)胞質(zhì)Figure 2 In the sepsis model of RAW264.7 cells, the YAP protein was expressed in the nucleus

圖3 YAP表達(dá)于細(xì)胞核Figure 3 In the sepsis model of RAW264.7 cells,,the P-YAP was expressed in the cytoplasm

圖4 YAP在膿毒血癥模型中高表達(dá)Figure 4 In the model of sepsis, the expression of YAP protein was significantly higher than that in the normal group

圖5 P-YAP在膿毒血癥模型中表達(dá)減少Figure 5 The expression of P-YAP protein in the sepsis model was lower than that in the normal group

Hippo信號(hào)通路下游的效應(yīng)分子YAP/TAZ復(fù)合物在器官的大小的調(diào)節(jié)，組織的穩(wěn)態(tài)，腫瘤的發(fā)生中有著重要的影響，YAP/TAZ復(fù)合物的活性主要同過磷酸化來實(shí)現(xiàn)。去磷酸化的YAP/TAZ在細(xì)胞核中與TEAD結(jié)合，引發(fā)下游基因的表達(dá)[15]。YAP在心肌的增殖過程中有重要作用，缺乏YAP的小鼠在圍產(chǎn)期就發(fā)生死亡[16]。研究發(fā)現(xiàn)在結(jié)腸癌細(xì)胞中β-catenin能調(diào)節(jié)YAP的表達(dá)在炎癥性腸病和結(jié)腸癌中[17]，通過YAP和NOTCH通路，腸上皮gp130激活能增強(qiáng)腸黏膜抵抗性[18]。腫瘤的發(fā)生與炎癥反應(yīng)有著密切的關(guān)系，腫瘤組織中癌細(xì)胞的浸潤(rùn)，炎癥因子的釋放[19]，而YAP在腫瘤的發(fā)生中有重要影響。研究發(fā)現(xiàn)YAP與CD8+T細(xì)胞表達(dá)Blimp-1有關(guān)[20]。猜想YAP參與膿毒癥的發(fā)生；下一步深入研究YAP對(duì)膿毒癥時(shí)炎癥因子表達(dá)的情況，探討與其他通路的關(guān)系。

4 結(jié)論

本研究通過構(gòu)建細(xì)胞膿毒癥模型檢測(cè)YAP的表達(dá)情況，發(fā)現(xiàn)YAP高表達(dá)于M1型巨噬細(xì)胞，而P-YAP低表達(dá)。因此推測(cè)去磷酸化的YAP參與膿毒癥的發(fā)生，可作為新的治療靶標(biāo)。

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