邊紅艷

(延安大學(xué) 醫(yī)學(xué)院, 陜西 延安 716000)

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·生命科學(xué)·

顆粒蛋白前體缺失型腹膜巨噬細(xì)胞的體外炎癥應(yīng)答

邊紅艷

(延安大學(xué) 醫(yī)學(xué)院, 陜西 延安 716000)

探討顆粒蛋白前體缺失型巨噬細(xì)胞的體外炎癥反應(yīng)。選取野生型C57BL/6雄性小鼠6-8周(WT)和PGRN基因敲除雄性小鼠6-8周(KO)為實(shí)驗(yàn)動(dòng)物。向小鼠腹腔內(nèi)注射1mL6%的淀粉,脫頸法處死小鼠后提取腹膜細(xì)胞。用瑞士染色后油鏡下觀察細(xì)胞,用流式細(xì)胞儀進(jìn)行細(xì)胞檢測,顯微鏡下觀察腹膜巨噬細(xì)胞吞噬功能,ELISA法檢測腹膜巨噬細(xì)胞培養(yǎng)上清中TNF-a和IL-12 因子。WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞的數(shù)目、形態(tài)和種類及巨細(xì)胞表面標(biāo)志物和巨噬細(xì)胞吞噬功能均無顯著性差異(p>0.05)。PGRN基因敲除KO小鼠來源腹膜巨噬細(xì)胞培養(yǎng)上清中前炎性因子TNF-a和IL-12的含量較WT小鼠明顯增高。PGRN對(duì)腹膜細(xì)胞的數(shù)目、形態(tài)、種類和巨噬細(xì)胞表面標(biāo)志物沒有顯著影響,但會(huì)使巨噬細(xì)胞炎癥反應(yīng)增強(qiáng)。

顆粒蛋白前體;巨噬細(xì)胞;炎癥反應(yīng)

顆粒蛋白前體(progranulin, PGRN)是由593個(gè)氨基酸殘基組成的分泌性糖蛋白,人類顆粒蛋白前體基因位于 17q21.32[1]。PGRN分子含有1個(gè)信號(hào)肽及7個(gè)半銜接重復(fù)的結(jié)構(gòu)域,每個(gè)結(jié)構(gòu)域是由 12個(gè)半胱氨酸組成[2-3]。PGRN的生理功能包括抗炎、促進(jìn)細(xì)胞增殖和營養(yǎng)等,并參與多項(xiàng)病理和生理活動(dòng),比如損傷修復(fù)、炎癥反應(yīng)、代謝調(diào)控及生長發(fā)育等。

炎癥的表現(xiàn)為紅、腫、熱、痛,是機(jī)體對(duì)內(nèi)外環(huán)境的有害刺激所產(chǎn)生的一種復(fù)雜的生理和病理反應(yīng)。炎癥可以起到保護(hù)性作用,也可以引起多種疾病[4]。以往研究表明PGRN具有抗炎作用,能抑制腫瘤壞死因子a誘導(dǎo)的中性粒細(xì)胞活化,PGRN敲除的小鼠清除胞內(nèi)菌的能力下降[5]。腹膜巨噬細(xì)胞(PM)在腹膜局部免疫中起著重要作用,本研究中利用PGRN基因敲除小鼠(KO)來源的PM作為模型,探討PGRN在PM炎癥應(yīng)答中的影響。

1 材料與方法

1.1 材 料

實(shí)驗(yàn)動(dòng)物包括野生型C57BL/6雄性小鼠6-8周(WT)和PGRN基因敲除雄性小鼠6-8周(KO)。試劑包括6%(質(zhì)量分?jǐn)?shù))淀粉、RPMI1640培養(yǎng)基、LPS、異硫氰酸硫光素、PGRN蛋白純化所選試劑、熒光標(biāo)記的抗小鼠單抗CD11b-PE、熒光標(biāo)記的抗小鼠單抗F4/80-FITC，LB培養(yǎng)基和ELISA所用試劑等。

1.2 方 法

1.2.1 小鼠腹膜細(xì)胞的提取方法[6]首先消毒小鼠腹膜皮膚,然后向腹腔內(nèi)注射1mL/6%的淀粉,脫頸法處死小鼠(72h后),用75%的酒精浸泡3min后,移至超凈臺(tái)將腹部皮膚剪開小口,用玻璃滴管將完全培養(yǎng)基打入腹腔,將腹腔清洗液離心,4℃保持備用。

1.2.2 瑞士染色 將PEC懸液進(jìn)行離心濃縮20倍后,用完全培養(yǎng)基重懸細(xì)胞,將懸液涂片自然風(fēng)干,干燥后用瑞士染色,干燥后油鏡下觀察細(xì)胞。

1.2.3 流式細(xì)胞術(shù) 用1×106/管提取后進(jìn)行離心,棄上清后進(jìn)行重懸,分別加入小鼠巨噬細(xì)胞抗體F4/80和CD11b進(jìn)行避光孵育,再離心,去上清,重懸,最后用流式細(xì)胞儀進(jìn)行檢測。

1.2.4 腹膜巨噬細(xì)胞吞噬功能檢測 取PEC接種于培養(yǎng)皿中,然后放于孵箱中,等到腹膜巨噬細(xì)胞貼壁后,將未貼壁的洗去,將每皿均加入做過標(biāo)記的大腸桿菌,避光孵育后,洗去胞外大腸桿菌,熒光顯微鏡下進(jìn)行觀察。

1.2.5 ELISA法檢測腹膜巨噬細(xì)胞培養(yǎng)上清中TNF-a和IL-12 用去離子水進(jìn)行稀釋,捕獲抗體溶于胞被液中,然后包被過夜,棄去胞被液,酶標(biāo)板在吸水紙上扣干后,洗7遍,進(jìn)行封閉,棄掉封閉液,重復(fù)上述操作,最后加終止液,上機(jī)進(jìn)行檢測。

2 結(jié) 果

2.1 WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞的數(shù)目、形態(tài)和種類比較結(jié)果

分別提取WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞進(jìn)行細(xì)胞數(shù)目、形態(tài)和種類的觀察。WT小鼠腹膜細(xì)胞總數(shù)為11.42×106,PGRN基因敲除KO小鼠腹膜細(xì)胞總數(shù)為8.84×106,兩組間無顯著性差異(p>0.05),見圖1。腹膜細(xì)胞圖片進(jìn)行瑞士染色,油鏡下進(jìn)行觀察發(fā)現(xiàn)PGRN基因敲除KO小鼠腹膜細(xì)胞中主要含有單核細(xì)胞、中細(xì)粒細(xì)胞、淋巴細(xì)胞和肥大細(xì)胞,其中單核細(xì)胞呈現(xiàn)圓形或橢圓形,呈弱嗜堿性,內(nèi)有嗜天青顆粒等。WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞進(jìn)行對(duì)比,在形態(tài)、種類比例中均無顯著性差異(p>0.05),見圖1和圖3。

2.2 巨細(xì)胞表面標(biāo)志物檢測

用流式細(xì)胞術(shù)檢測巨細(xì)胞表面標(biāo)志物發(fā)現(xiàn)KO組腹膜細(xì)胞F4/80和CD11b陽性細(xì)胞含量分別是49.7%和91.6%,WT組腹膜細(xì)胞F4/80和CD11b陽性細(xì)胞含量分別是52.68%和89.44%,兩組之間無顯著性差異(p>0.05),見圖4和圖5。

圖1 WT小鼠和PGRN基因敲除Fig.1 Number of peritoneal cells of WT mice

圖2 WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞數(shù)目KO小鼠腹膜細(xì)胞種類比較Fig.2 Comparison of peritoneal cell types in and PGRN knockout KO mice WT mice and PGRN knockout KO mice WT KO

圖3 WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞形態(tài)比較Fig.3 Morphological comparison of peritoneal cells of WT mice and PGRN knockout KO mice

圖4 巨細(xì)胞表面標(biāo)志物F4/80Fig.4 Giant cell surface marker F4/80

圖5 巨細(xì)胞表面標(biāo)志物CD11bFig.5 Giant cell surface marker CD11b

2.3 巨噬細(xì)胞吞噬能力比較

在熒光顯微鏡下觀察吞噬陽性的巨噬細(xì)胞數(shù)量,兩組間無顯著差異p>0.05),見圖6。

圖6 巨噬細(xì)胞吞噬能力比較Fig.6 Comparison of phagocytic capacity of macrophages

2.4 炎性應(yīng)答結(jié)果

ELISA法結(jié)果顯示,PGRN基因敲除KO小鼠來源腹膜巨噬細(xì)胞培養(yǎng)上清中前炎性因子TNF-a和IL-12的含量較WT小鼠明顯增高,見圖7和圖8。

圖7 炎性因子TNF-a比較Fig.7 Comparison of inflammatory factors TNF-a

圖8 炎性因子IL-12比較Fig.8 Comparison of inflammatory factors IL-12

3 討 論

PGRN是免疫調(diào)節(jié)分子中的其中之一,對(duì)免疫反應(yīng)中的關(guān)鍵性信號(hào)通路進(jìn)行調(diào)節(jié),對(duì)炎癥性疾病有抗炎和促炎作用。國外學(xué)者Tang等發(fā)現(xiàn)PGRN是腫瘤壞死因子受體的新配體,可以結(jié)合TNFR的胞外域,而且能夠阻斷TNF-α介導(dǎo)的 NFκB 等信號(hào)通路,從而在類風(fēng)濕性關(guān)節(jié)炎中發(fā)揮重要的抗炎作用[7-8]。

WT小鼠和PGRN基因敲除KO小鼠腹膜細(xì)胞的數(shù)目、形態(tài)、種類和巨噬細(xì)胞表面標(biāo)志物比較無顯著性差異,說明PGRN基因內(nèi)源性缺失對(duì)小鼠腹膜細(xì)胞的數(shù)目、形態(tài)、種類和巨噬細(xì)胞表面標(biāo)志物無明顯影響。而且PGRN基因缺失也不會(huì)明顯影響巨噬細(xì)胞吞噬功能。但是，PGRN基因敲除KO小鼠來源腹膜巨噬細(xì)胞培養(yǎng)上清中前炎性因子TNF-a和IL-12的含量較WT小鼠明顯增高。說明PGRN基因缺失會(huì)使炎性因子產(chǎn)生更強(qiáng)的炎癥反應(yīng)。TNF-a是一種重要的免疫調(diào)控因子,在炎癥反應(yīng)中處于核心位置[9-10]。本次研究發(fā)現(xiàn),PGRN基因敲除KO小鼠來源腹膜巨噬細(xì)胞TNF-a和IL-12因子明顯增多,表現(xiàn)出更強(qiáng)的炎癥反應(yīng)。

綜上所述,PGRN對(duì)腹膜細(xì)胞的數(shù)目、形態(tài)、種類和巨噬細(xì)胞表面標(biāo)志物沒有顯著影響。但是，會(huì)影響巨噬細(xì)胞炎癥反應(yīng),使巨噬細(xì)胞炎癥反應(yīng)增強(qiáng)。

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(編 輯 徐象平)

The in vitro inflammatory response of granular protein precursor deleted peritoneal macrophages

BIAN Hongyan

(School of Medicine, Yan′an University, Yan′an 716000， China)

To investigate the in vitro inflammatory response of the granular protein precursor. Selection of wild-type 6-8 male mice (6-8) and PGRN (WT) C57BL/6 weeks (KO) in male mice as experimental animals.After intraperitoneal injection of 1ml6% into the mice, the mice were sacrificed and the peritoneal cells were extracted.Switzerland after staining under oil microscope to observe the cell, by flow cytometry for cell detection, under a microscope observation of peritoneal macrophage phagocytic function and ELISA method for the detection of peritoneal macrophage culture supernatant TNF-and IL-12 factor. There were no significant differences in the number, shape, type, and phagocytic function of peritoneal cells of WT mice and PGRN knockout KO mice(p>0.05).The contents of TNF-a and IL-12 were significantly higher in PGRN mice than in KO mice, and the contents of and WT were significantly higher in the supernatant of peritoneal macrophages. PGRN has no significant effect on the number, shape, type of peritoneal cells and the surface markers of macrophages, but it can enhance the inflammatory response of macrophages.

PGRN; macrophage; inflammatory reaction

2015-11-17

陜西省自然科學(xué)基金資助項(xiàng)目(2013K12-01-23)

邊紅艷，女，陜西延安人，從事生命科學(xué)及疾病相關(guān)性研究。

R364.5

A

10.16152/j.cnki.xdxbzr.2016-05-016