房艷宇　羅曉光　蘇　陽　謝　欣　田旭聰　范麗婷　任　艷

(齊齊哈爾醫(yī)學(xué)院附屬第三醫(yī)院神經(jīng)內(nèi)科，齊齊哈爾161000)

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探討B(tài)V2細(xì)胞活化中是否存在p38MAPK及JAK2-STAT通路的磷酸化激活①

房艷宇羅曉光蘇陽謝欣田旭聰范麗婷任艷

(齊齊哈爾醫(yī)學(xué)院附屬第三醫(yī)院神經(jīng)內(nèi)科，齊齊哈爾161000)

[摘要]目的：探索CD200R在LPS誘導(dǎo)的小膠質(zhì)細(xì)胞炎癥模型中的作用以及是否有pho-p38MAPK及phoJAK2-STAT通路的激活。方法：采用小膠質(zhì)細(xì)胞進(jìn)行研究，將細(xì)胞用CD200R抗體及LPS處理，ELISA檢測(cè)TNF-α及IL-1β的分泌。Western blot檢驗(yàn)p38MAPK、JAK2、pho-p38MAPK及pho-JAK2-STAT表達(dá)。為證實(shí)這兩條通路是炎癥的下游通路,分別應(yīng)用兩者的阻斷劑處理細(xì)胞后再次檢測(cè)炎癥因子的分泌。結(jié)果：在小膠質(zhì)細(xì)胞表面有CD200R的表達(dá)；應(yīng)用CD200R的阻斷性抗體后，TNF-α及IL-1β分泌均增多,較對(duì)照組及LPS組比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)，并且有pho-p38MAPK及pho-JAK2-STAT的激活；阻斷劑SB203580及AG490均能抑制TNF-α及IL-1β的分泌。結(jié)論：pho-p38MAPK及pho-JAK2-STAT兩條通路是LPS及CD200R抗體誘導(dǎo)的小膠質(zhì)細(xì)胞炎癥反應(yīng)的下游通路。

[關(guān)鍵詞]CD200R；BV2 細(xì)胞；TNF-α；IL-1β；pho-p38MAPK；pho-JAK2-STAT

帕金森病(PD)是中樞神經(jīng)系統(tǒng)常見的變性病，其發(fā)病機(jī)制尚不完全清楚，目前認(rèn)為：遺傳因素、環(huán)境因素、免疫/炎癥反應(yīng)等參與了其病理過程[1,2]。眾所周知，小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)內(nèi)的常駐免疫細(xì)胞，在中樞神經(jīng)系統(tǒng)固有免疫中發(fā)揮重要作用[3]，然而，小膠質(zhì)細(xì)胞的慢性活化可以導(dǎo)致TNF-α、IL-1β及IL-6等炎性因子的異常分泌，進(jìn)而引發(fā)神經(jīng)炎癥及神經(jīng)變性疾病[4]。近年來的研究表明，CD200-CD200R通路是中樞神經(jīng)系統(tǒng)內(nèi)下調(diào)小膠質(zhì)細(xì)胞活化的重要機(jī)制[5,6]。CD200主要表達(dá)在包括神經(jīng)元在內(nèi)的多種細(xì)胞中，而其受體CD200R則特異表達(dá)在包括小膠質(zhì)細(xì)胞在內(nèi)的髓系細(xì)胞(Myeloid)中，兩者結(jié)構(gòu)相似,均屬于跨膜糖蛋白[7-10]。動(dòng)物炎癥模型中，阻斷兩者中任何一方的表達(dá)均可以使小膠質(zhì)細(xì)胞激活，并且在細(xì)胞活化過程中伴有炎癥相關(guān)通路的激活，而MAPK(Mitogen-activated protein kinase，絲裂原活化蛋白激酶)及JAK-STAT(Janus kinase-signal transducer and activator of transcription，酪氨酸激酶-信號(hào)轉(zhuǎn)導(dǎo)子與轉(zhuǎn)錄激活子)[11]是與炎癥密切相關(guān)的兩條通路。本實(shí)驗(yàn)應(yīng)用BV2細(xì)胞代替小膠質(zhì)細(xì)胞，應(yīng)用CD200R抗體及LPS處理細(xì)胞誘導(dǎo)其活化，發(fā)現(xiàn)了其下游有pho-p38MAPK及pho-JAK2-STAT通路的激活。

1材料與方法

1.1細(xì)胞培養(yǎng)用含10% FBS的DMEM高糖培養(yǎng)基(購(gòu)自Hyclone，美國(guó))培養(yǎng)BV2細(xì)胞(購(gòu)自北京協(xié)和細(xì)胞庫)每2～3 d傳代一次，待細(xì)胞生長(zhǎng)良好貼壁達(dá)到90%左右時(shí)用于實(shí)驗(yàn)。

1.2方法

1.2.1具體分組A：CD200R抗體(Abcam公司，美國(guó))及LPS(Sigma公司，美國(guó))處理組；B：LPS處理組；C：對(duì)照組；收集各組中的細(xì)胞及上清液。

1.2.2免疫熒光檢測(cè)CD200R的表達(dá)96孔板培養(yǎng)BV2細(xì)胞，4%多聚甲醛固定15 min后，0.3%Triton打孔15 min，5%BSA封閉0.5 h，加入1∶1000稀釋的CD200R過夜，熒光二抗孵育1.5 h，每步驟之間均用PBS洗3次，每次5 min，熒光顯微鏡下觀察。

1.2.3ELISA檢測(cè)TNF-α及IL-1β的分泌以非處理組上清做對(duì)照，每孔均設(shè)立2個(gè)復(fù)孔，實(shí)驗(yàn)共重復(fù)3次，分別按照TNF-α(R&D)及IL-1β(R&D)的使用說明書,在抗體包被的96孔板中分別加入標(biāo)準(zhǔn)樣品及待測(cè)樣本、生物標(biāo)記二抗及酶標(biāo)試劑，37℃，反應(yīng)1 h，洗板5次，加入顯色劑A、B，37℃顯色10 min，加終止液，以酶標(biāo)儀檢測(cè)波長(zhǎng)為450nm的光密度值。

1.2.4Western blot檢測(cè)p38MAPK、pho-p38MAPK及pho-JAK2-STAT的濃度分別收集A、B、C三組細(xì)胞，提取蛋白，BCA蛋白定量法用PAGE蛋白上樣緩沖液配制等體積等濃度的待測(cè)蛋白，配膠、上樣電泳，冰上轉(zhuǎn)膜，5%BSA封閉1 h，敷一抗p38MAPK(1∶1 000稀釋，cellsignal)、pho-p38MAPK(1∶1 000稀釋，cellsignal)，pho-JAK2-STAT(1∶1 000稀釋，Abcam)，α-tubulin(1∶1 000稀釋，CST)，4℃過夜，TBST洗膜后，二抗孵育2 h，ECL發(fā)光儀發(fā)光。

2結(jié)果

2.1免疫熒光結(jié)果熒光顯微鏡(20倍下)觀察BV2細(xì)胞為單層貼壁細(xì)胞，胞體細(xì)長(zhǎng)呈梭形，發(fā)出較多分支，見圖1。結(jié)果表明：CD200R可表達(dá)于BV2細(xì)胞表面，這為我們接下來使用CD200R阻斷性抗體阻斷BV2細(xì)胞表面CD200R的表達(dá)使細(xì)胞更大程度上活化，進(jìn)而研究細(xì)胞活化過程中相關(guān)激活的炎癥通路奠定了基礎(chǔ)。

2.2ELISA檢測(cè)炎癥因子表達(dá)結(jié)果在不加任何處理因素的情況下TNF-α及IL-1β均有基礎(chǔ)分泌，單加LPS后細(xì)胞活化，兩者分泌增多，而用CD200R抗體及LPS處理后較單獨(dú)的LPS處理而言，BV2細(xì)胞活化更加明顯，見圖2、3，兩種炎癥因子分泌繼續(xù)增多，各組比較差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)。

圖1　BV2細(xì)胞表面CD200R的表達(dá)情況Fig.1　Expression of CD200R on surface of BV2 cellsNote: A.CD200R;B.Hoechst；C.Merge.

圖2　TNF-α分泌(ng/L)Fig.2　 Expression of TNF-α(ng/L)Note: A.The group of CD200R and LPS handle the microglia;B.The group of LPS handle the microglia;C.Control group.*.P<0.05.

圖3　IL-1β分泌(ng/L)Fig.3　Expression of IL-1β(ng/L)Note: A.The group of CD200R and LPS handle the microglia;B.The group of LPS handle the microglia;C.Control group.*.P<0.05.

圖4　pho-p38MAPK及p38MAPK的表達(dá)Fig.4　Expression of pho-p38MAPK and p38MAPKNote: A.The group of CD200R and LPS handle the microglia;B.The group of LPS handle the microglia;C.Control group.*.P<0.05.

圖5　pho-p38MAPK與p38MAPK比值Fig.5　Ratio of pho-p38MAPK and p38MAPKNote: A.The group of CD200R and LPS handle the microglia;B.The group of LPS handle the microglia;C.Control group.*.P<0.05.

圖6　pho-JAK2-STAT 表達(dá)Fig.6　Expression of pho-JAK2-STATNote: A.The group of CD200R and LPS handle the microglia;B.The group of LPS handle the microglia;C.Control group.*.P<0.05.

圖7　pho-JAK2-STAT與α-tubulin比值Fig.7　Ratio of pho-JAK2-STAT and α-tubulinNote: A.The group of CD200R and LPS handle the microglia;B.The group of LPS handle the microglia;C.Control group.*.P<0.05.

2.3Western blot 結(jié)果Western blot檢測(cè)三組中P38MAPK、pho-P38MAPK、pho-JAK2-STAT及α-Tubulin的表達(dá)，并進(jìn)行灰度值分析，結(jié)果如圖4～7。兩種磷酸化蛋白及半定量分析待測(cè)蛋白與內(nèi)參比而言，A組顯著高于B組、顯著高于C組，說明在CD200R抗體及LPS共刺激BV2細(xì)胞后p38MAPK級(jí)JAK2-STAT的磷酸化表達(dá)顯著增加，各組間比較差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)。

2.4加入兩條通路阻斷劑后再次檢測(cè)兩種炎癥因子的表達(dá)加入足量的兩條通路阻斷劑后，兩種炎性因子分泌均有所降低，各組間比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)說明，pho-p38MAPK及pho-JAK2-STAT通路參與了CD200R抗體及LPS誘導(dǎo)的小膠質(zhì)細(xì)胞的激活。

3討論

帕金森病是常見的神經(jīng)系統(tǒng)變性疾病，近年來其發(fā)病呈現(xiàn)逐年上升的趨勢(shì)，臨床上主要表現(xiàn)為：靜止性震顫、肌張力增高、運(yùn)動(dòng)遲緩及姿勢(shì)步態(tài)異常。病理上改變?yōu)椋汉谫|(zhì)多巴胺神經(jīng)元大量變性丟失及膠質(zhì)細(xì)胞活化。研究表明：神經(jīng)系統(tǒng)炎癥參與了PD的發(fā)病過程[12]，小膠質(zhì)細(xì)胞是神經(jīng)系統(tǒng)的免疫監(jiān)視細(xì)胞，在炎癥、創(chuàng)傷、缺血等損傷下可以被激活，釋放炎癥因子、活性氧自由基等毒性分子[12，13]，小膠質(zhì)細(xì)胞表面存在許多受體，本實(shí)驗(yàn)中的CD200R就是特異性表達(dá)于小膠質(zhì)細(xì)胞、巨噬細(xì)胞等髓系細(xì)胞表面的保護(hù)性受體，而其配體CD200則廣泛表達(dá)在神經(jīng)元、B細(xì)胞等細(xì)胞表面，正常情況下，兩者平衡可維持小膠質(zhì)細(xì)胞處于靜息狀態(tài)[14-16]。在本實(shí)驗(yàn)我們首先證實(shí)小膠質(zhì)細(xì)胞存在CD200R的表達(dá)(見圖1)，這與之前的研究結(jié)果是一致的，接下來我們應(yīng)用了CD200R的阻斷性抗體阻斷小膠質(zhì)細(xì)胞表面CD200R的表達(dá)進(jìn)而引發(fā)細(xì)胞炎性反應(yīng)。為了驗(yàn)證CD200R在細(xì)胞活化作用，我們應(yīng)用CD200R的阻斷性抗體及LPS處理小膠質(zhì)細(xì)胞，并分別檢測(cè)了三組中TNF-α及IL-1β的分泌情況,結(jié)果表明：在抗體組中炎癥因子分泌均顯著增高，組間差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)(見圖2、3)。美中不足在于:市場(chǎng)上未能找到CD200R激動(dòng)劑誘導(dǎo)其表達(dá)以抑制小膠質(zhì)細(xì)胞活化。為了驗(yàn)證細(xì)胞活化中是否有pho-p38MAPK及pho-JAK2-STAT的激活以及總的p38MAPK表達(dá)有無改變，我們用Western blot法分別檢測(cè)了上述三種蛋白的表達(dá)情況，結(jié)果：pho-p38MAPK及pho-JAK2-STAT較其余兩組發(fā)生了明顯的激活，而p38MAPK表達(dá)無差異(如圖4、6)，分別進(jìn)行灰度值分析后發(fā)現(xiàn)，抗體組中兩種磷酸化蛋白比值明顯高于藥物組及對(duì)照組，組間比較具有顯著意義(P<0.05)(見圖5、7)。

神經(jīng)炎癥在神經(jīng)變性疾病中發(fā)揮著重要作用，那么隨著研究的深入，尋找PD等與神經(jīng)炎癥密切相關(guān)的神經(jīng)變性疾病共同的且具有特異性的病因，并從根本上阻斷這些病因或誘發(fā)因素，可以減少帕金森等疾病的發(fā)生，但該研究任重而道遠(yuǎn)，同時(shí)探索一種在基因或蛋白水平維持CD200/CD200R通路的平衡進(jìn)而維持小膠質(zhì)細(xì)胞處于靜息狀態(tài)的治療手段，必然會(huì)給帕金森患者的治療帶來福音。我們的研究也為是否可以通過預(yù)防性的、并在安全范圍內(nèi)應(yīng)用疾病相關(guān)的炎癥通路的阻斷劑，盡可能削弱神經(jīng)炎癥，似乎可能成為帕金森病的新的治療靶點(diǎn)。

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[收稿2015-08-15修回2015-08-28]

(編輯倪鵬)

Investigate whether p38MAPK and JAK2-STAT pathways can be phosphorylated activation when BV2 cells are activated

FANGYan-Yu，LUOXiao-Guang,SUYang,XIEXin,TIANXu-Cong,FANLi-Ting,RENYan.

DepartmentofNeurology,theThirdAffiliatedHospitalofQiqihaerMedicalUniversity,Qiqihaer161000,China

[Abstract]Objective:To explore the important role of CD200R in the microglia inflammation model induced by LPS as well as whether there is the activation of the pho-p38MAPK and pho-JAK2-STAT pathway or not.Methods: Microglia was used for this study.Immunofluorescence confirms there was the expression of CD200R in microglia.Enzyme-linked immunosorbent assay detected the secretory content of TNF-α and IL-1β after using CD200R-blocking antibody and LPS handle the microglia.Western blot disclose whether p38MAPK and JAK2-STAT pathway can be phosphorylated or not.Finally,we detect the secretion of TNF-α，IL-1β after applicating different doses of channel blockers.Results: We observed that CD200R expresses on the surface of microglia.To determine the relationship of CD200R and inflammatory cytokines,we tested whether blocking CD200R promotes the release of inflammatory cytokines or not.After using administrating antibodies of CD200R,TNF-α and IL-1β secreted by microglia was significantly increased and pho-p38MAPK and pho-JAK2-STAT were activated,which let us predict that pho-p38MAPK and phoJAK2-STAT participate in the microglia activation of LPS induction.To further confirm the pho-p38MAPK and pho-JAK2-STAT pathway were involved in microglia activation,we apply SB203580 and AG490 to inhibit both pathways respectively,and then we found that the number of TNF-α and IL-1β was significantly decreased,and the number of TNF-α and IL-1β was negatively correlation with the concentration of AG490 and SB203580.Conclusion: Not only pho-p38MAPK but also pho-JAK2-STAT are involved in microglia activation induced by LPS and CD200R antibody response.

[Key words]CD200R;BV2 cells;TNF-α;IL-1β;pho-p38MAPK;pho-JAK2-STAT

doi:10.3969/j.issn.1000-484X.2016.05.005

作者簡(jiǎn)介：房艷宇(1986年-)，女， 碩士，醫(yī)師，主要從事神經(jīng)病學(xué)方面研究。通訊作者及指導(dǎo)教師：羅曉光(1972年-)，女，博士后，主任醫(yī)師，博士生導(dǎo)師，主要從事帕金森病的基礎(chǔ)與臨床研究，E-mail： grace_shenyang@163.com。

中圖分類號(hào)R724.5

文獻(xiàn)標(biāo)志碼A

文章編號(hào)1000-484X(2016)05-0629-04

①本文為國(guó)家自然科學(xué)基金(81371421)、遼寧省教育廳科研項(xiàng)目計(jì)劃(L2010560)資助支持項(xiàng)目。