趙娟娟　徐華林　郭萌萌　陶弋婧　周　涯　陳　超　秦娜琳　鄭　靜　田　丹　徐　林

(遵義醫(yī)學(xué)院免疫學(xué)教研室暨貴州省生物治療人才基地，遵義563000)

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microRNA-7敲減對內(nèi)毒素誘導(dǎo)小鼠急性肺損傷的影響①

趙娟娟徐華林郭萌萌陶弋婧周涯②陳超秦娜琳鄭靜田丹徐林

(遵義醫(yī)學(xué)院免疫學(xué)教研室暨貴州省生物治療人才基地，遵義563000)

目的：觀察microRNA-7(miR-7)敲減對內(nèi)毒素誘導(dǎo)小鼠急性肺損傷的影響并探討其意義。方法：利用脂多糖(LPS)腹腔注射(10 mg/kg體重)野生型(Wild type，WT)小鼠和miR-7基因敲減(Knockdown，KD)小鼠建立急性肺損傷(Acute lung injury，ALI)模型；HE染色觀察肺組織病理學(xué)變化；計算肺泡灌洗液(BAL)中的細(xì)胞總數(shù)；Real-time PCR檢測肺組織炎癥相關(guān)因子表達(dá)變化；流式細(xì)胞術(shù)(FACS)進(jìn)一步檢測BAL中固有免疫細(xì)胞γδ T細(xì)胞、F4/80巨噬細(xì)胞(Macrophages，Mφ)和適應(yīng)性免疫細(xì)胞CD4+T細(xì)胞和CD8+T細(xì)胞的比例和絕對數(shù)變化；FACS檢測CD4+T細(xì)胞活化相關(guān)分子CD62L和CD69的表達(dá)水平。結(jié)果：相對野生型(Wild type，WT)小鼠，HE染色顯示miR-7KD小鼠的肺組織小血管充血和炎性細(xì)胞浸潤明顯減少，病理性損傷明顯減輕；Real-time PCR結(jié)果顯示促炎性細(xì)胞因子IL-6的表達(dá)水平顯著降低(P<0.01)，而抗炎性細(xì)胞因子IL-4、TGF-β的表達(dá)水平明顯增加(P<0.05)；同時，BAL中總細(xì)胞數(shù)顯著減少(P<0.05)；FACS檢測進(jìn)一步顯示miR-7KD小鼠的BAL中F4/80+Mφ細(xì)胞的比例及絕對數(shù)均明顯下調(diào)(P<0.05)，而γδ T細(xì)胞的比例上調(diào)(P<0.05)，但其細(xì)胞絕對數(shù)無差異(P>0.05)；BAL中CD4+T細(xì)胞和CD8+T細(xì)胞的比例及其絕對數(shù)均顯著降低(P<0.05)；最后CD4+T細(xì)胞活化膜分子CD62L的表達(dá)水平明顯上調(diào)(P<0.05)，而CD69的表達(dá)水平顯著下調(diào)(P<0.05)。結(jié)論：miR-7敲減可減輕ALI模型小鼠的肺部損傷，提示其可能在ALI發(fā)生中發(fā)揮了重要調(diào)控作用。

miR-7基因敲減；急性肺損傷；肺泡灌洗液；免疫細(xì)胞

急性肺損傷(Acute lung injury，ALI)是以肺部嚴(yán)重的急性炎癥性反應(yīng)為特征的常見疾病[1-3]，其發(fā)生發(fā)展過程十分復(fù)雜，涉及到多種免疫細(xì)胞和免疫分子的參與。近來研究顯示微小RNA(microRNA，miRNA)在ALI的發(fā)生發(fā)展中起著重要調(diào)控作用。miR-7作為miRNA家族成員之一，新近被報道與多種臨床疾病的發(fā)生密切相關(guān)[4-6]。然而，miR-7在ALI發(fā)生中的可能作用及相關(guān)機制仍未有研究報道。因此，本研究擬利用前期構(gòu)建成功的miR-7基因敲減(Knockdown，KD)小鼠[7]，以LPS為致敏原常規(guī)建立ALI模型，初步觀察miR-7敲減后對小鼠肺組織損傷的影響，并初步探討其意義，以期為后續(xù)深入探討miR-7在ALI發(fā)生中的作用提供前期實驗基礎(chǔ)。

1　材料與方法

1.1材料8周FVB背景的SPF級雌性野生型小鼠和miR-7KD小鼠[7]；顯微鏡(Olympus)；流式細(xì)胞儀(Beckman Coulter)；LPS(Sigma)；SYBRPremix Ex Taq Ⅱ (2×) (TAKARA.RR820A)；10%水合氯醛(遵醫(yī)附院研制)；R-Phycoerythrin(PE)標(biāo)記的CD4、CD62L抗鼠的單克隆抗體，Percp-Cy5.5標(biāo)記的CD4、F4/80抗鼠單克隆抗體，Allophycocyain(APC)標(biāo)記的CD8、CD69、γδT細(xì)胞抗鼠單克隆抗體，PE-Cy7標(biāo)記的CD8細(xì)胞抗鼠單克隆抗體，F(xiàn)ixation/Permeabilization Diluent(Ebioscience)；血細(xì)胞計數(shù)板(上海求精生化試劑儀器有限公司)。

1.2方法

1.2.1 以LPS為致敏原建立ALI模型按照每只小鼠每千克體重10 mg LPS的劑量，進(jìn)行腹腔注射，48 h后，觀察兩組小鼠肺組織炎性損傷的情況。

1.2.2HE染色觀察肺臟組織的病理學(xué)改變?nèi)iR-7KD小鼠和WT小鼠肺臟組織，置于4%的多聚甲醛中固定24 h，然后用石蠟包埋，制成5 μm切片并做HE染色，最后在倒置顯微鏡下觀察肺臟組織的病理學(xué)變化。

1.2.3利用Real-time PCR手段檢測肺組織中炎性因子的表達(dá)水平提取ALI模型下，WT和miR-7 KD小鼠的肺組織的RNA，逆轉(zhuǎn)為cDNA，進(jìn)行實時定量Real-time PCR檢測。Real-time PCR檢測體系為20 μl，其中SYBR Premix Ex Taq Ⅱ(×2) 10 μl，cDNA 2 μl，上下游引物混合液(1 μmol/L)1 μl，高壓的去離子水7 μl。反應(yīng)條件：95℃ 30 s；95℃ 5 s，60℃ 30 s，45個循環(huán)。每個反應(yīng)設(shè)置3復(fù)孔，復(fù)孔間Cq值差異不超過0.5者用于數(shù)據(jù)分析。

1.2.4獲取小鼠的肺泡灌洗液并記取所含細(xì)胞的總數(shù)首先以5%水合氯醛(10%水合氯醛加PBS配置而成)，按照0.005 ml/g小鼠體重經(jīng)行腹腔注射，2 min后小鼠進(jìn)入麻醉狀態(tài)；接著將麻醉狀態(tài)中的小鼠置于手術(shù)板上，固定四肢。用剪刀、鑷子對小鼠的頸部經(jīng)行解剖，暴露出氣管，用穿刺針進(jìn)行插管，所有小鼠均用0.8 ml的PBS進(jìn)行2次灌洗，收集灌洗液入2 ml離心管中，4℃ 1 500 r/min離心15 min，小心棄去上清，用1 ml PBS重懸細(xì)胞沉淀，取10 μl重懸液進(jìn)行細(xì)胞計數(shù)。將剩余液體再次4℃離心，待流式細(xì)胞術(shù)(FACS)的檢測。

1.2.5FACS檢測BAL中F4/80巨噬細(xì)胞、γδT細(xì)胞以及T淋巴細(xì)胞亞群及相關(guān)膜分子表達(dá)的比例變化將1.2.4中獲得的miR-7KD小鼠和WT小鼠BAL中的細(xì)胞，經(jīng)PBS 1 200 r/min離心10 min洗滌2次后，棄掉上清液，在余下細(xì)胞沉淀中加入200 μl PBS，然后彈散，經(jīng)篩網(wǎng)過濾后分別加入PE標(biāo)記的CD4、CD62L熒光抗體，Percp-Cy5.5標(biāo)記F4/80、CD4、CD8熒光抗體，APC標(biāo)記的γδT、CD8、CD69熒光抗體各1 μl，冰上避光孵育30 min，PBS洗滌2遍，最后通過FACS進(jìn)行檢測。

1.3統(tǒng)計學(xué)分析本實驗中對細(xì)胞因子表達(dá)水平的分析采用2-ΔΔCt法進(jìn)行。反應(yīng)體系中熒光信號達(dá)到閾值的循環(huán)數(shù)為Ct，ΔΔCt=(Ct實驗組目的基因-Ct實驗組內(nèi)參基因)-(Ct對照組目的基因-Ct對照組內(nèi)參基因)，計算ΔΔCt后再計算2-ΔΔCt值，2-ΔΔCt為該基因的相對表達(dá)量。

2　結(jié)果

2.1 HE染色觀察肺組織的病理學(xué)改變HE染色結(jié)果顯示，與WT小鼠相比，miR-7KD小鼠的肺間質(zhì)中血管充血明顯減輕，且炎性細(xì)胞浸潤顯著減少(圖1)。

2.2 肺組織中炎性因子IL-6、IL-4和TGF-β的表達(dá)水平Real-time PCR檢測結(jié)果顯示，miR-7KD小鼠的肺組織中促炎性細(xì)胞因子IL-6的表達(dá)水平顯著降低(圖2C，P<0.01)?？寡仔约?xì)胞因子IL-4，TGF-β的表達(dá)水平明顯增高(圖2C，P<0.01)。

2.3 ALI模型下miR-7KD小鼠的BAL細(xì)胞總數(shù)變化結(jié)果顯示miR-7KD小鼠的BAL中總細(xì)胞數(shù)明顯低于WT小鼠BAL中總細(xì)胞數(shù)(圖3，P<0.01)。

2.4 BAL中γδT細(xì)胞、F4/80+Mφ細(xì)胞的比例和絕對數(shù)變化如圖4A、B所示，與WT小鼠相比，miR-7KD小鼠BAL中F4/80+Mφ細(xì)胞的比例顯著下調(diào)(P<0.05)、而γδT細(xì)胞的比例卻明顯上調(diào)(P<0.05)；進(jìn)一步統(tǒng)計分析BAL中F4/80+Mφ細(xì)胞和γδT細(xì)胞的絕對數(shù)，結(jié)果顯示，F(xiàn)4/80+Mφ細(xì)胞的絕對數(shù)顯著減少(圖4C，P<0.01)，而γδT細(xì)胞的絕對數(shù)則無明顯差異(P>0.05)。

圖1　ALI模型下的WT小鼠和miR-7KD小鼠的肺臟組織病理學(xué)變化(HE染色)Fig.1　Lung pathology of LPS treated-miR-7KD mice and WT mice(HE staining)

圖2　ALI模型下miR-7KD小鼠肺組織中炎性因子表達(dá)變化Fig.2　Relative expression of cytokines in lung of LPS treated-miR-7KD miceNote: A.Amplification curves；B.Melt peak；C.Relative expression level of cytokine including IL-6,IL-4,TGF-β；*.P<0.05,**.P<0.01 compared with WT mice.

2.5BAL中CD4+T細(xì)胞、CD8+T細(xì)胞的比例和絕對數(shù)變化結(jié)果顯示，miR-7KD小鼠BAL中的 CD4+T細(xì)胞和CD8+T細(xì)胞比例均較WT小鼠BAL中顯著下調(diào)(圖5A、B，P<0.05)；與此同時，BAL中的 CD4+T細(xì)胞和CD8+T細(xì)胞的絕對細(xì)胞數(shù)也均明顯減少(圖5C，P<0.05)。

2.6BAL中CD4+T細(xì)胞中CD62L、CD69的表達(dá)變化結(jié)果顯示，與WT小鼠組相比，miR-7KD小鼠BAL中CD4+T細(xì)胞的CD69+細(xì)胞的比例和絕對數(shù)均明顯減少(圖6，P<0.05)；而CD62L+細(xì)胞的比例增加(P<0.05)，而絕對數(shù)仍顯著減少(圖6，P<0.05)。

圖3　ALI模型下miR-7KD小鼠BAL中總細(xì)胞數(shù)的變化Fig.3　Change of total cell counts of BAL in LPS-treated miR-7KD miceNote: *.P<0.01.

圖4　ALI模型下miR-7KD小鼠BAL中γδT細(xì)胞、F4/80巨噬細(xì)胞的比例和絕對數(shù)變化Fig.4　Change on proportion and cell counts of γδT and F4/80+Macrophages(Mφ) cells in BAL of LPS-treated miR-7KD miceNote: A.FACS analysis;B.The percentage;C.Cell counts;*.P<0.05,**.P<0.01 compared with WT mice.

圖5　ALI模型下miR-7KD小鼠BAL中CD4+ T細(xì)胞和CD8+ T細(xì)胞的比例和絕對數(shù)變化Fig.5　Change on proportion and cell counts of CD4+ T cells and CD8+ T cells in BAL of LPS-treated miR-7KDNote: A.FACS analysis;B.The percentage;C.Cell counts;*.P<0.05,**.P<0.01 compared with WT mice.

圖6　ALI模型下miR-7KD小鼠BAL中CD4+T細(xì)胞中CD62L+細(xì)胞和CD69+細(xì)胞的比例和絕對數(shù)變化Fig.6　Change on proportion and cell counts of CD4+CD62L+ T cells and CD4+CD69+ T cells in BAL of LPS-treated miR-7KDNote: A.The percentage;B.Cell counts;*.P<0.05,**.P<0.01 compared with WT mice.

3　討論

微小RNA(microRNAs，miRNAs)是一大類由內(nèi)源性基因編碼、成熟體長度約在20～25個核苷酸序列的非編碼短小單鏈RNA，主要通過作用靶基因的3′非編碼端參與真核基因的轉(zhuǎn)錄后水平的調(diào)節(jié)。已有大量研究數(shù)據(jù)表明，miRNAs分子在ALI的發(fā)生中發(fā)揮了重要作用。如Xie等[8]研究發(fā)現(xiàn)miR-127可通過下調(diào)M1巨噬細(xì)胞極化的重要分子CD64的水平，從而減少M1巨噬細(xì)胞的功能來減弱ALI的病理性損傷；Yang等[9]的實驗數(shù)據(jù)也表明高水平的miR-320可通過下調(diào)沉默調(diào)節(jié)蛋白1(Sirtuin type 1，SIRT1)引起肺泡上皮的損傷，易于誘發(fā)ALI；新近，我們也發(fā)現(xiàn)miR-155的反義寡核苷酸(miR-155ASO)可以通過增加M2型Mφ細(xì)胞分泌IL-10的水平，上調(diào)的IL-10隨后又可調(diào)節(jié)CD4+CD25+regulatory T (Tregs)細(xì)胞的擴增，從而加快ALI損傷的恢復(fù)[10]。因此，深入探討特定miRNA分子在ALI發(fā)生中的作用及相關(guān)機制不僅對于ALI發(fā)生機理的認(rèn)識，且對于后續(xù)臨床診治均具有積極意義。

miR-7作為miRNA家族成員之一，在多個組織、器官及細(xì)胞中存在表達(dá)[11,12]，且與組織器官的發(fā)育、分化及損傷修復(fù)密切相關(guān)[13-15]。我們前期實驗結(jié)果顯示miR-7在肺組織高表達(dá)[11]。在本研究中，我們利用miR-7KD小鼠，首次以LPS為致敏原建立ALI模型，值得注意的是，經(jīng)HE染色發(fā)現(xiàn)miR-7KD小鼠的肺組織炎性損傷明顯減輕；同時，也發(fā)現(xiàn)BAL的細(xì)胞總數(shù)也顯著減少。進(jìn)一步Real-time PCR檢測顯示，miR-7KD小鼠肺組織中促炎性細(xì)胞因子IL-6的表達(dá)水平明顯降低，而IL-4、TGF-β抗炎性細(xì)胞因子水平顯著增加。以上數(shù)據(jù)均提示miR-7敲減后可減輕ALI模型的肺組織損傷。類似的，Rao等[16]發(fā)現(xiàn)miR-155可以通過對靶基因細(xì)胞因子信號抑制物1(Suppressor of cytokine signaling 1，Socs1)的抑制，增加肺組織炎性因子IFN-γ的分泌，使肺組織的損傷加重。這些研究提示特定miRNAs分子對于ALI的發(fā)生具有重要調(diào)控作用。值得一提的是，我們前期發(fā)現(xiàn)miR-7KD小鼠的肺組織中存在炎癥細(xì)胞的浸潤[7]。因此，miR-7在ALI發(fā)生中的確切調(diào)控機制仍有待后續(xù)深入闡明。

現(xiàn)有的研究顯示，固有免疫細(xì)胞巨噬細(xì)胞、γδT細(xì)胞以及適應(yīng)性免疫細(xì)胞中的T細(xì)胞等參與了ALI的發(fā)生、發(fā)展過程[17-19]。如Murdoch等[20]研究顯示，γδT參與調(diào)節(jié)急性炎癥下組織的完整和穩(wěn)態(tài)，主要在過敏性氣道炎癥和氣道免疫應(yīng)答中高表達(dá)；Holub等[21]利用腸毒素構(gòu)建ALI模型發(fā)現(xiàn)，BAL中高水平IFN-γ主要是被肺部來源CD11b+Mφ分泌，且這群細(xì)胞被明顯激活，然而CD4+T細(xì)胞及整個T細(xì)胞群卻明顯減少。本研究中，我們進(jìn)一步分析了模型小鼠BAL的細(xì)胞組成變化。結(jié)果顯示miR-7KD小鼠BAL中固有免疫細(xì)胞F4/80+Mφ細(xì)胞的比例和絕對數(shù)均明顯降低；而γδT細(xì)胞的比例盡管增加，但總細(xì)胞數(shù)未見明顯改變。適用性免疫細(xì)胞方面，miR-7KD小鼠BAL中適應(yīng)性免疫細(xì)胞CD4+T細(xì)胞、CD8+T細(xì)胞的比例及絕對數(shù)均顯著降低。盡管由于未建模的對照小鼠BAL細(xì)胞數(shù)過少難以分析各亞群組成，但結(jié)合到前述BAL總細(xì)胞數(shù)的變化，我們的研究也提示miR-7KD小鼠ALI發(fā)生減輕可能與BAL中免疫細(xì)胞的組成改變有關(guān)。最后，我們進(jìn)一步檢測了BAL中CD4+T細(xì)胞活化和歸巢能力變化。結(jié)果顯示miR-7KD小鼠中CD4+T細(xì)胞低表達(dá)CD69分子，高表達(dá)CD62L分子，提示其活化程度降低，且歸巢能力減弱。這些研究結(jié)果提示，miR-7KD后ALI發(fā)生的改變，可能與免疫細(xì)胞功能的變化有關(guān)。類似地，Guenin-Macé等[22]研究發(fā)現(xiàn)過表達(dá)Let-7b可上調(diào)T細(xì)胞的CD62L表達(dá)水平，從而阻止結(jié)核桿菌內(nèi)脂引發(fā)的T細(xì)胞的歸巢效應(yīng)。我們前期還發(fā)現(xiàn)miR-7KD小鼠的腸系膜淋巴結(jié)增生且多種免疫細(xì)胞的組成比例也顯著改變[23]。因此，后續(xù)深入探討miR-7在各免疫細(xì)胞功能中的調(diào)控作用，不僅對于ALI發(fā)生機制，且對于機體免疫自穩(wěn)的認(rèn)識均具有重要意義。總之，本研究中我們首次發(fā)現(xiàn)miR-7敲減后可顯著減輕ALI模型小鼠肺損傷，為后續(xù)深入探討miR-7在ALI發(fā)生中的作用及機制，以及后續(xù)臨床相關(guān)生物治療新靶標(biāo)開發(fā)提供了重要前期實驗依據(jù)。

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[收稿2015-12-01修回2015-12-14]

(編輯倪鵬)

Effect of microRNA-7 knockdown on pathology of Enterotoxin-induced murine acute lung injury

ZHAO Juan-Juan,XU Hua-Lin,GUO Meng-Meng,TAO Yi-Jing,ZHOU Ya,CHEN Chao,QIN Na-Lin,ZHENG Jing,TIAN Dan，XU Lin.

Department of Immunology,Zunyi Medical University,Talent Base of Biological Therapy of Guizhou Province,Zunyi 563000,China

Objective:To detect the effect of microRNA-7 (miR-7) knockdown on pathology in murine acute lung injury (ALI) model,and preliminarily explore its significance.Methods: Murine ALI model was performed by intraperitoneal injection of Lipopolysaccharide (LPS) (10 mg/kg) into miR-7KD mice and wild-type (wild type,WT) mice respectively.Then,the pathologic injury of lung tissue were observed by HE staining.And total cell count of bronchoalveolarlavage(BAL) was calculated.The relative expression of related cytokines in lung tissue was analyzed by Real-time PCR assay.Furthermore,the changes on proportion of innate immune cells (γδT cell and F4/80 macrophages cell) and adaptive immune cell (CD4+T cell and CD8+T cell) were analyzed by FACS.Meanwhile,the expression of CD62L and CD69,as well as the absolute number,in CD4+T cell were also analyzed.Results: Compared with WT mice,pathological damage in lung tissues was significantly alleviated in miR-7KD mice.Real-time PCR analysis showed that the relative expression of IL-6 was obviously reduced (P<0.01),conversely,relative expression of IL-4 and TGF-β were obviously increased (P<0.05).Furthermore,the total cell number in BAL also reduced significantly (P<0.05).Importantly,FACS analysis showed that the proportion and the absolute number of F4/80+Mφ cells obviously reduced (P<0.05);however,the proportion of γδT cells increased (P<0.05).Moreover,the proportion and the absolute number of CD4+T cells and CD8+T cells were significantly reduced (P<0.05).Finally, the proportion and the absolute number of CD62L+in CD4+T cells were upregulated vigorously,contrastly,the proportion and the absolute number of CD69+in CD4+T cells were notably up-regulated (P<0.05).Conclusion: miR-7 defeciency could significantly ameliorate the pathology of murine ALI,suggesting that it may play an important regulatory role in the development of ALI.

miR-7 knockdown;Acute lung injury;Bronchoalveolar lavage；Immune cell

10.3969/j.issn.1000-484X.2016.09.003

趙娟娟(1987年-)，女，在讀碩士，初級檢驗師，主要從事分子免疫學(xué)方面的研究。

及指導(dǎo)教師：徐林(1977年-)，男，博士，教授，主要從事腫瘤免疫學(xué)和分子免疫學(xué)方面研究，E-mail：xulinzhouya@163.com。

R392

A

1000-484X(2016)09-1257-05

①本文受教育部“新世紀(jì)優(yōu)秀人才計劃”項目(NCET-12-0661)和國家自然科學(xué)基金(No.31370918)資助。

②遵義醫(yī)學(xué)院醫(yī)學(xué)物理學(xué)教研室，遵義 563000。