干擾P2X7R基因?qū)AW264.7細(xì)胞增殖和吞噬的影響

干擾P2X7R基因?qū)AW264.7細(xì)胞增殖和吞噬的影響*

蘇程程1,3▲，張譯丹1▲，馬永強(qiáng)2，陳雪芬1，向國(guó)安1，周欣2，彭守春1，林志春2，魏路清1△，姬文婕1△

(1中國(guó)人民武裝警察部隊(duì)后勤學(xué)院附屬醫(yī)院呼吸與重癥醫(yī)學(xué)科,2天津市心血管重塑與靶器官損傷重點(diǎn)實(shí)驗(yàn)室，天津 300162;3河北醫(yī)科大學(xué)，河北 石家莊 050017)

[摘要]目的： 應(yīng)用RNA干擾技術(shù)抑制小鼠巨噬細(xì)胞RAW264.7細(xì)胞P2X7受體(P2X7R)基因的表達(dá)，建立穩(wěn)定干擾細(xì)胞株，并觀察其對(duì)細(xì)胞增殖和凋亡的影響。方法: 用脂質(zhì)體法將P2X7R shRNA重組質(zhì)粒轉(zhuǎn)染至RAW264.7細(xì)胞，經(jīng)G418篩選后獲得穩(wěn)定干擾細(xì)胞株。細(xì)胞分為野生型(WT)組、陰性對(duì)照(NC)組和干擾(shP2X7R)組。Real-time PCR法檢測(cè)細(xì)胞中P2X7R mRNA的表達(dá)，Western blot檢測(cè)細(xì)胞中P2X7R蛋白的表達(dá)；CCK-8方法檢測(cè)細(xì)胞生長(zhǎng)活性，5-乙炔基-2’-脫氧尿苷(5-ethynyl-2’-deoxyuridine，EdU)摻入實(shí)驗(yàn)檢測(cè)細(xì)胞增殖活性；流式細(xì)胞術(shù)分析細(xì)胞周期的分布和吞噬情況。結(jié)果: P2X7R shRNA能明顯抑制RAW264.7細(xì)胞的P2X7R mRNA和蛋白的表達(dá)，抑制率在80%以上。48 h后，shP2X7R組細(xì)胞的生長(zhǎng)速度明顯高于NC組和WT組 (P<0.05)，增殖期細(xì)胞比例明顯升高 (P<0.05)，說(shuō)明下調(diào)P2X7R基因能明顯促進(jìn)細(xì)胞增殖。shP2X7R組的細(xì)胞周期出現(xiàn)改變，S期和G2/M期的比例明顯上升，增殖指數(shù)增高 (P<0.05)。shP2X7R組的細(xì)胞吞噬活性明顯高于NC組(P<0.05)。結(jié)論: 本研究成功構(gòu)建了穩(wěn)定干擾P2X7R基因表達(dá)的小鼠巨噬細(xì)胞株RAW264.7，shP2X7R能夠明顯促進(jìn)RAW264.7細(xì)胞的增殖，改變了細(xì)胞的吞噬活性。

[關(guān)鍵詞]P2X7受體； RNA干擾； RAW264.7巨噬細(xì)胞； 細(xì)胞增殖； 細(xì)胞吞噬

[中圖分類號(hào)]R363[文獻(xiàn)標(biāo)志碼]A

doi:10.3969/j.issn.1000-4718.2015.11.023

[文章編號(hào)]1000-4718(2015)11-2070-06

[收稿日期]2015-07-20[修回日期] 2015-09-23

[基金項(xiàng)目]*廣東省科技社會(huì)發(fā)展項(xiàng)目(No. 2012B031800030)

通訊作者△Tel： 020-81332309； E-mail: qkchen@21cn.com

Effect ofP2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7SU Cheng-cheng1, ZHANG Yi-dan1, MA Yong-qiang2, CHEN Xue-fen1, XIANG Guo-an1, ZHOU Xin2, PENG Shou-chun1, LIN Zhi-chun2, WEI Lu-qing1, JI Wen-jie1

(1DepartmentofRespirologyandCriticalCareMedicine,LogisticsUniversityofChinesePeople’sArmedPoliceForces，2TianjinKeyLaboratoryofCardiovascularRemodelingandTargetOrganInjuryInstituteofCardiovascularDiseaseandHeartCenterTianjin300162,China;3HebeiMedicalUniversity,Shijazhuang050017China.E-mail:ji_wenjie@hotmail.com;wei_luqing@hotmail.com)

ABSTRACT[]AIM: To establish a cell line of stable silencing of P2X7 receptor (P2X7R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X7R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X7R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X7R at mRNA and protein levels was down-regulated by 80% in shP2X7R group compared with negative control (NC) plasmid transfection. In addition, P2X7R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X7R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X7R expression was successfully established. P2X7R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.

[KEY WORDS]P2X7receptor; RNA interference; RAW264.7 macrophages; Cell proliferation; Cell phagocytosis

巨噬細(xì)胞是天然免疫應(yīng)答的重要細(xì)胞成份，由于其組織分布、分化程度以及外界激活因子的多樣性，巨噬細(xì)胞具有復(fù)雜的異質(zhì)性與功能的多樣性。不同的環(huán)境中，巨噬細(xì)胞可以具有不同的激活途徑，主要有經(jīng)典激活的M1表型分泌促炎細(xì)胞因子，替代激活的M2表型分泌抗炎因子并與損傷修復(fù)有關(guān)。在以慢性炎癥為特征的某些疾病及其病理過(guò)程中，均觀察到巨噬細(xì)胞活化表型的變化。目前研究發(fā)現(xiàn)，P2X7受體(P2X7receptor，P2X7R)不僅通過(guò)與三磷酸腺苷(adenosine triphosphate，ATP)結(jié)合刺激細(xì)胞因子類的合成和分泌，誘導(dǎo)炎癥反應(yīng)和巨噬細(xì)胞表型的偏移等[1-3]，而且P2X7R還參與了介導(dǎo)細(xì)胞內(nèi)的信號(hào)轉(zhuǎn)導(dǎo)[4]。本研究采用RNA干擾技術(shù)，構(gòu)建穩(wěn)定干擾P2X7R基因的小鼠巨噬細(xì)胞RAW264.7，觀察干擾后其增殖和凋亡等生物學(xué)特性的變化，為進(jìn)一步探討P2X7R對(duì)巨噬細(xì)胞的調(diào)控作用及其機(jī)制，提供有力的分子生物學(xué)工具。

材料和方法

1主要試劑

RAW264.7細(xì)胞(南開(kāi)大學(xué)生命科學(xué)院韓際宏教授惠贈(zèng))；高糖DMEM培養(yǎng)基、胎牛血清、胰蛋白酶(Hyclone)；G-418(Amesco)；OPTI MEM轉(zhuǎn)染培養(yǎng)基、LipofectamineTM2000、TRIzol(Invitrogen)；CCK-8試劑盒(Dojindo)；MMLV反轉(zhuǎn)錄酶、dNTPs(Promega)；SYBR GreenPCR試劑盒、蛋白酶抑制劑(Roche)；EdU-Click 488細(xì)胞增殖檢測(cè)試劑盒、碘化丙啶(Sigma)；AnnexinV-FITC凋亡檢測(cè)試劑盒(Biolegend)；BCA蛋白定量試劑盒(Pierce)；GAPDH小鼠單克隆抗體、P2X7R小鼠單克隆抗體(Abcam)；HRP標(biāo)記山羊抗小鼠IgG(Abgent)；PVDF膜、ECL化學(xué)發(fā)光試劑盒(Millipore)；干擾表達(dá)載體系統(tǒng)(上海吉瑪制藥技術(shù)有限公司)；其余試劑均為國(guó)產(chǎn)分析純。

2方法

2.1細(xì)胞培養(yǎng)小鼠巨噬細(xì)胞株RAW264.7用高糖DMEM培養(yǎng)基(含12%胎牛血清、4 mmol/L 谷氨酰胺)，置于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)，取對(duì)數(shù)生長(zhǎng)期細(xì)胞用于實(shí)驗(yàn)。

2.2P2X7R穩(wěn)定干擾細(xì)胞株的建立分別將攜帶P2X7R短發(fā)夾RNA(short hairpin RNA，shRNA)和陰性對(duì)照(negative control，NC)的重組質(zhì)粒pGPU6/GFP/Neo，按照LipofectamineTM2000說(shuō)明書進(jìn)行細(xì)胞轉(zhuǎn)染，簡(jiǎn)述如下：將對(duì)數(shù)生長(zhǎng)期的RAW264.7細(xì)胞計(jì)數(shù)后接種于6孔板，每孔4×105個(gè)，使轉(zhuǎn)染時(shí)細(xì)胞達(dá)到80%～90%的融合。細(xì)胞分為野生型(wild type，WT)組、NC組和轉(zhuǎn)染質(zhì)粒pGPU6/GFP/Neo-P2X7R(RNA interference by P2X7R-shRNA, shP2X7R)組。質(zhì)粒DNA用量為每孔4 μg，轉(zhuǎn)染后36 h用G418(500 mg/L)篩選轉(zhuǎn)染細(xì)胞，然后挑取單克隆進(jìn)行擴(kuò)大培養(yǎng)，傳代3次以上，real-time PCR法和Western blot法檢測(cè)P2X7R的干擾效果，若細(xì)胞狀態(tài)穩(wěn)定，則表明穩(wěn)定轉(zhuǎn)染細(xì)胞系構(gòu)建成功。

2.3Real-time PCR法檢測(cè)細(xì)胞中P2X7R mRNA的表達(dá)情況按照TRIzol說(shuō)明書提取各組細(xì)胞的總RNA，反轉(zhuǎn)錄成cDNA，然后用SYBR Green法進(jìn)行PCR擴(kuò)增，同一樣本設(shè)2個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。P2X7R正義鏈為5’-CAGTCACTGGAGGAACTGGAA-3’，反義鏈為5’-CCAAAGGAAACACACCGATT-3’，擴(kuò)增產(chǎn)物長(zhǎng)度為77 bp ；β-actin正義鏈為5’-CTAAGGCCAACCGTGAAAAG-3’，反義鏈為5’-ACCAGAGGCATACAGGGACA-3’，擴(kuò)增產(chǎn)物長(zhǎng)度為104 bp 。擴(kuò)增條件為：50 ℃ 2 min， 95 ℃ 10 min；95 ℃ 15 s，60 ℃ 1min，共40個(gè)循環(huán)。以上引物均由北京三博遠(yuǎn)志生物技術(shù)有限公司合成。用2-ΔΔCt法對(duì)擴(kuò)增結(jié)果進(jìn)行分析。

2.4Western blot法檢測(cè)細(xì)胞中P2X7R蛋白的表達(dá)情況分別提取各組細(xì)胞總蛋白，采用BCA法定量蛋白濃度，將經(jīng)過(guò)95 ℃ 10 min變性處理的蛋白樣品進(jìn)行SDS-PAGE，半干法轉(zhuǎn)膜；5%的脫脂奶粉溶液封閉，分別加入GAPDH小鼠單克隆抗體(1∶1 000)、P2X7R小鼠單克隆抗體(1∶2 000)，4 ℃過(guò)夜；次日加入HRP標(biāo)記山羊抗小鼠IgG，室溫孵育1 h，PBST洗滌15 min 3次；ECL化學(xué)底物發(fā)光法顯色，Image Lab 4.0軟件進(jìn)行圖像灰度分析， P2X7R的蛋白相對(duì)表達(dá)量用其與GAPDH的灰度比值表示。

2.5CCK-8測(cè)定細(xì)胞生長(zhǎng)水平分別將對(duì)數(shù)生長(zhǎng)期的WT組、NC組和shP2X7R組RAW264.7細(xì)胞計(jì)數(shù)后，接種于96孔板，每孔103個(gè)，每組均設(shè)6個(gè)復(fù)孔，分別于培養(yǎng)24 h、48 h、72 h、96 h后，每孔加入CCK-8 10 μL，繼續(xù)培養(yǎng)4 h后，測(cè)定450 nm波長(zhǎng)處吸光度(A)值。實(shí)驗(yàn)重復(fù)3次，計(jì)算平均值，根據(jù)各組細(xì)胞每天的A值繪制細(xì)胞生長(zhǎng)曲線。

2.6EdU法檢測(cè)細(xì)胞增殖活性按照EdU細(xì)胞檢測(cè)試劑盒的操作說(shuō)明進(jìn)行，簡(jiǎn)述如下：分別接種對(duì)數(shù)生長(zhǎng)期的WT組、NC組和shP2X7R組RAW264.7細(xì)胞于12孔細(xì)胞培養(yǎng)板中，每孔接種2×105個(gè)細(xì)胞，在培養(yǎng)24 h、48 h、72 h、96 h后，換含10 μmol/L EdU的培養(yǎng)基1 mL，孵育1 h后，4%多聚甲醛固定15 min，0.5% Triton X-100室溫破膜20 min，加入click-reaction mixture(含1 mol/L pH 8.5 Tris-HCl 50 μL，25 mmol/L CuSO420 μL，10 mmol/L 6-FAM-Azide 2.5 μL，0.5 mol/L抗壞血酸50 μL，去離子水補(bǔ)足體系至500 μL)室溫避光孵育30 min，然后用DAPI染DNA，最后用Leica DMZ 3000熒光顯微鏡觀察并采集圖像。每個(gè)樣本隨機(jī)選取10個(gè)視野，細(xì)胞增殖率按照高倍視野下EdU陽(yáng)性細(xì)胞數(shù)占總細(xì)胞數(shù)的百分比計(jì)算。

2.8流式細(xì)胞術(shù)檢測(cè)細(xì)胞吞噬活性按照熒光素標(biāo)記大腸桿菌吞噬試劑盒操作說(shuō)明進(jìn)行測(cè)定。簡(jiǎn)述如下：取各組對(duì)數(shù)生長(zhǎng)期細(xì)胞，調(diào)整細(xì)胞密度為1×109/L，接種于96孔板，每孔105個(gè)細(xì)胞，每組設(shè)3個(gè)復(fù)孔。0.5 mL HBSS與熒光素標(biāo)記的E.colik-12微粒超聲混勻后，移入含4.5 mL Milli Q水的棕色小瓶中，再次超聲充分混勻。每孔加入100 μL微粒懸液，輕輕混勻，CO2培養(yǎng)箱中孵育2 h后，去除上清，加入100 μL 0.25 g/L臺(tái)盼藍(lán)溶液1 min，吸出上清，加入培養(yǎng)基，收集細(xì)胞懸液，Beckman Coulter FC500流式細(xì)胞儀分析，計(jì)算各組細(xì)胞的平均熒光強(qiáng)度(mean fluorescence intensity，MFI)，表示吞噬活性的強(qiáng)弱。

3統(tǒng)計(jì)學(xué)處理

采用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)分析，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)誤(mean±SEM)表示。組間的比較采用t檢驗(yàn)和單因素方差分析，各組均數(shù)多重比較采用Tukey’s法。單變量配對(duì)資料之間的比較采用配對(duì)樣本t檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

1P2X7R mRNA和蛋白的表達(dá)水平

用2-ΔΔCt法對(duì)PCR結(jié)果進(jìn)行分析，shRNA組的P2X7R mRNA水平明顯低于WT組和NC組(P<0.05)，抑制率為80%左右，而NC組和WT組之間差異不顯著；Western blot結(jié)果表明，與NC組相比，shP2X7R組的P2X7R蛋白表達(dá)水平明顯下調(diào)(P<0.05)，而NC組和WT組之間的P2X7R蛋白表達(dá)水平無(wú)明顯變化。上述結(jié)果說(shuō)明成功構(gòu)建了P2X7R穩(wěn)定干擾細(xì)胞株，見(jiàn)圖1、2。

Figure 1.The mRNA expression of P2X7R in the RAW264.7 cells determined by real-time PCR analysis. Mean±SEM.n=3.*P<0.05vsNC and WT.

圖1各組RAW264.7細(xì)胞中P2X7R mRNA的表達(dá)

Figure 2.The protein expression of P2X7R in the RAW264.7 cells determined by Western blot. Mean±SEM.n=3.*P<0.05vsNC and WT.

圖2各組RAW264.7細(xì)胞中P2X7R 蛋白的表達(dá)

2P2X7R shRNA對(duì)RAW264.7細(xì)胞生長(zhǎng)的影響

如細(xì)胞生長(zhǎng)曲線所示，WT組和NC組之間的細(xì)胞增殖活性沒(méi)有顯著差異，而48 h后，shP2X7R組細(xì)胞的生長(zhǎng)明顯高于前2組(P<0.05)，說(shuō)明下調(diào)P2X7R基因能明顯促進(jìn)細(xì)胞生長(zhǎng)，見(jiàn)圖3。

Figure 3.Cell growth curve of RAW264.7 cells obtained by CCK-8 assay. Mean±SEM.n=3.**P<0.01vsNC and WT.

圖3P2X7R shRNA對(duì)RAW264.7細(xì)胞生長(zhǎng)水平的影響

3P2X7R shRNA對(duì)RAW264.7細(xì)胞增殖活性的影響

EdU法檢測(cè)結(jié)果如圖4所示，與NC組相比，shP2X7R組的細(xì)胞增殖活性明顯升高(P<0.05)，NC組和WT組比較無(wú)明顯差異。說(shuō)明下調(diào)P2X7R基因能明顯提高細(xì)胞的增殖活性。

4P2X7R shRNA對(duì)RAW264.7細(xì)胞周期的影響

與WT組和NC組相比，shP2X7R組的細(xì)胞周期分布出現(xiàn)改變，G0/G1期比例降低，S期和G2/M期的比例增高，增殖指數(shù)明顯升高(P<0.05)；而WT組與NC組的細(xì)胞周期分布無(wú)明顯差異，見(jiàn)表1。

Figure 4.Detection of EdU incorporated into the DNA of RAW264.7 cells by fluorescence microscopy. Mean±SEM.n=3.*P<0.05，**P﹤0.01vsNC and WT.

圖4EdU法檢測(cè)P2X7R shRNA對(duì)RAW264.7細(xì)胞增殖活性的影響

表1　各組細(xì)胞周期的分布及增殖指數(shù)的變化

*P<0.05vsWT and NC.

5P2X7R shRNA對(duì)RAW264.7細(xì)胞吞噬活性的影響

小鼠巨噬細(xì)胞RAW264.7吞噬FITC標(biāo)記的大腸桿菌后流式分析結(jié)果顯示，各組細(xì)胞的平均熒光強(qiáng)度分別為：WT組(120.0±3.61)、NC組(47.66±6.78)和shP2X7R組(82.97±8.69)。WT組的細(xì)胞吞噬活性最強(qiáng)，其次是shP2X7R組，而NC組的吞噬活性最弱，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)，見(jiàn)圖5。由于NC轉(zhuǎn)染引起細(xì)胞吞噬功能下降。因此該結(jié)果尚需后續(xù)實(shí)驗(yàn)進(jìn)一步驗(yàn)證。

討論

巨噬細(xì)胞是研究細(xì)胞吞噬、細(xì)胞免疫和分子免疫學(xué)的重要對(duì)象，在不同環(huán)境中，巨噬細(xì)胞可以發(fā)生不同性質(zhì)的活化，成為具有不同分子表型和功能特征的亞群，從而決定了其在各種環(huán)境和不同疾病中發(fā)揮的作用[5]。目前認(rèn)為巨噬細(xì)胞至少存在2種活化狀態(tài)，經(jīng)典活化(M1)和替代活化(M2)。M1巨噬細(xì)胞通常認(rèn)為是經(jīng)干擾素γ激活，主要表現(xiàn)為促進(jìn)炎癥和氧化應(yīng)激反應(yīng)、增強(qiáng)的吞噬活性；M2巨噬細(xì)胞主要經(jīng)IL-4和IL-13激活，主要表現(xiàn)為抑制炎癥反應(yīng)，并與組織的修復(fù)和纖維化過(guò)程有關(guān)[6]。兩種狀態(tài)的平衡對(duì)于機(jī)體維持正常的免疫功能是必須的，任何一種活化失衡狀態(tài)都可能對(duì)組織炎癥的恢復(fù)產(chǎn)生不良的影響。

P2X7R是一種ATP門控型離子通道，在巨噬細(xì)胞、樹(shù)突狀細(xì)胞和小膠質(zhì)細(xì)胞高表達(dá)[7]，參與了細(xì)胞的多種病理生理過(guò)程，包括細(xì)胞增殖、分化和凋亡、單核巨噬細(xì)胞分泌細(xì)胞因子以及細(xì)胞介導(dǎo)的細(xì)胞毒性作用等[8-11]。細(xì)胞外ATP與P2X7R結(jié)合引發(fā)的寡聚化反應(yīng)，使離子通道迅速開(kāi)放，產(chǎn)生K+外流，引起胞內(nèi)K+的迅速耗竭，進(jìn)而激活NALP3炎癥復(fù)合體。正常情況下，胞外ATP的濃度受到胞外ATP酶和ADP酶的嚴(yán)格調(diào)控而處于低水平，出現(xiàn)組織損傷時(shí)，ATP大量釋放至細(xì)胞外間隙，并以旁分泌的形式通過(guò)臨近細(xì)胞的P2X7R進(jìn)一步促發(fā)炎癥反應(yīng)[7, 12]。因此，通過(guò)對(duì)P2X7R的調(diào)控，調(diào)節(jié)巨噬細(xì)胞功能，可能成為潛在的慢性炎癥及相關(guān)疾病的治療和預(yù)防靶點(diǎn)之一。

Figure 5.Evaluation of phagocytic function by flow cytometry withE.coli-FITC. Representative histograms of count (y-axis)vsFITC (x-axis) were showed. Mean±SEM.n=3.*P<0.05vsNC.

圖5shP2X7R對(duì)RAW264.7細(xì)胞吞噬功能的影響

本研究中采用基于質(zhì)粒載體的shRNA系統(tǒng)，用脂質(zhì)體將重組質(zhì)粒pGPU6/GFP/Neo-P2X7R shRNA轉(zhuǎn)染至小鼠巨噬細(xì)胞RAW264.7，獲得穩(wěn)定干擾細(xì)胞株。實(shí)驗(yàn)結(jié)果表明P2X7R shRNA有效地抑制了RAW264.7細(xì)胞中P2X7R的 mRNA和蛋白表達(dá)，明顯促進(jìn)了RAW264.7細(xì)胞的生長(zhǎng)和增殖活性，S期和G2/M期的比例明顯升高。但是目前尚未闡明P2X7R對(duì)巨噬細(xì)胞的調(diào)控途徑和機(jī)制，以及在此過(guò)程中與其它信號(hào)轉(zhuǎn)導(dǎo)通路之間的相互關(guān)系等。因此，本實(shí)驗(yàn)中成功構(gòu)建的穩(wěn)定干擾細(xì)胞株將為進(jìn)一步探討P2X7R在巨噬細(xì)胞相關(guān)的病理生理過(guò)程的作用和機(jī)制，提供有力的研究工具和奠定初步的實(shí)驗(yàn)基礎(chǔ)。

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(責(zé)任編輯： 陳妙玲， 余小慧)