潘舒琳， 何笑笑， 胡瑩瑩， 方明楚， 姜 槐， 肖 健， 林振浪△

(1溫州醫(yī)科大學(xué)附屬第二醫(yī)院、育英兒童醫(yī)院新生兒科，浙江 溫州 325027； 2溫州醫(yī)科大學(xué)藥學(xué)院，浙江 溫州 325035)

成纖維細(xì)胞生長(zhǎng)因子10抑制脂多糖刺激下BV2細(xì)胞的活化*

潘舒琳1， 何笑笑1， 胡瑩瑩1， 方明楚1， 姜 槐1， 肖 健2△， 林振浪1△

(1溫州醫(yī)科大學(xué)附屬第二醫(yī)院、育英兒童醫(yī)院新生兒科，浙江 溫州 325027；2溫州醫(yī)科大學(xué)藥學(xué)院，浙江 溫州 325035)

目的： 探索成纖維細(xì)胞生長(zhǎng)因子10(fibroblast growth factor 10，F(xiàn)GF10)對(duì)脂多糖(lipopolysaccharide，LPS)刺激下小膠質(zhì)細(xì)胞BV2活化的影響。方法: 小鼠BV2小膠質(zhì)細(xì)胞用細(xì)胞培養(yǎng)基DMEM培養(yǎng)，置于37 ℃、5% CO2、飽和濕度的培養(yǎng)箱中培養(yǎng)，1～2 d換液，4～5 d傳代。實(shí)驗(yàn)分為對(duì)照組、LPS組和FGF10組，F(xiàn)GF10組的BV2細(xì)胞預(yù)先給予FGF10 1 mg/L 30 min后，在LPS組和FGF10組中加入500 mg/L的LPS，在不同時(shí)點(diǎn)進(jìn)行檢測(cè)。用倒置顯微鏡觀察小膠質(zhì)細(xì)胞的形態(tài)學(xué)改變， RT-qPCR和ELISA分別檢測(cè)腫瘤壞死因子α(TNF-α)轉(zhuǎn)錄和蛋白表達(dá)水平的改變來(lái)觀測(cè)BV2 細(xì)胞的活化情況。結(jié)果: 靜息狀態(tài)下BV2細(xì)胞形態(tài)呈圓形或橢圓形，經(jīng)過(guò)24 h LPS刺激后，BV2細(xì)胞形狀向多極或紡錘樣改變，活化細(xì)胞數(shù)量比值明顯高于對(duì)照組；預(yù)先給予FGF10能抑制LPS刺激下的BV2細(xì)胞向活化形態(tài)改變，活化的BV2細(xì)胞明顯減少。給予LPS刺激6 h后，LPS組TNF-α的mRNA水平相比于對(duì)照組顯著升高，然而預(yù)先給予FGF10會(huì)顯著抑制TNF-α的轉(zhuǎn)錄。 LPS作用24 h后，細(xì)胞培養(yǎng)上清液內(nèi)TNF-α的表達(dá)水平與對(duì)照組相比顯著上升，而預(yù)先給予FGF10在蛋白水平顯著抑制TNF-α的表達(dá)。結(jié)論: FGF10能夠成功抑制LPS刺激下BV2細(xì)胞的活化，有望成為治療經(jīng)膠質(zhì)細(xì)胞介導(dǎo)的神經(jīng)系統(tǒng)炎癥性疾病的一種有效藥物。

成纖維細(xì)胞生長(zhǎng)因子10； BV2細(xì)胞； 脂多糖； 腫瘤壞死因子α

炎癥反應(yīng)是許多中樞神經(jīng)系統(tǒng)性疾病中十分重要的病理過(guò)程。小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)炎癥和免疫反應(yīng)的主效應(yīng)細(xì)胞，約占所有膠質(zhì)細(xì)胞的10%～20%[1]。正常情況下，小膠質(zhì)細(xì)胞促進(jìn)神經(jīng)元的發(fā)育，維持中樞神經(jīng)系統(tǒng)的穩(wěn)態(tài)。當(dāng)中樞神經(jīng)系統(tǒng)受到炎癥，感染，缺氧等病理刺激時(shí)，小膠質(zhì)細(xì)胞被激活，細(xì)胞功能向巨噬細(xì)胞轉(zhuǎn)化。當(dāng)小膠質(zhì)細(xì)胞受到脂多糖(lipopolysaccharide， LPS)持續(xù)激活后將釋放大量的炎癥介質(zhì)如前列腺素E2(prostaglandin E2, PGE2)、腫瘤壞死因子α(tumor necrosis factor-α, TNF-α)、白細(xì)胞介素1β (interleukin-1β, IL-1β)、白細(xì)胞介素6 (interleukin-6, IL-6)等[2-3]，這些炎癥因子一旦過(guò)度表達(dá)將加劇神經(jīng)元損傷，同時(shí)參與一些與炎癥和神經(jīng)變性相關(guān)的中樞神經(jīng)系統(tǒng)疾病的病理過(guò)程，包括腦外傷、腦卒中、腦退行性疾病、感染等[4-5]。因此，抑制小膠質(zhì)細(xì)胞過(guò)度活化可能是治療神經(jīng)系統(tǒng)炎癥性疾病的一種有效方法。

成纖維細(xì)胞生長(zhǎng)因子10(fibroblast growth factor 10，F(xiàn)GF10)是成纖維細(xì)胞生長(zhǎng)因子家族的成員之一。FGF10與細(xì)胞的生長(zhǎng)、增殖、分化和遷移有關(guān)，能促進(jìn)四肢、肺、脂肪組織和前列腺的發(fā)育[6-8]。在對(duì)動(dòng)物模型的研究發(fā)現(xiàn)，F(xiàn)GF10可以用于治療急性肺損傷、肺炎、干眼等疾病[9-11]。FGF10的治療作用可能與病理過(guò)程的炎癥反應(yīng)被抑制有關(guān)系[9-10]，但FGF10是否能夠通過(guò)抑制小膠質(zhì)細(xì)胞活化從而減少炎癥反應(yīng)尚未完全清楚。本研究希望證實(shí)FGF10能夠抑制LPS刺激下的BV2細(xì)胞活化，為FGF10治療由膠質(zhì)細(xì)胞介導(dǎo)的神經(jīng)系統(tǒng)炎癥性疾病提供理論依據(jù)。

材 料 和 方 法

1 主要試劑以及儀器

BV2細(xì)胞購(gòu)于中國(guó)協(xié)和醫(yī)科大學(xué)細(xì)胞中心；DMEM培養(yǎng)基 (Gibco)；TNF-α ELISA試劑盒(eBioscience)；LPS (Sigma)；FGF10 (Grostre Biotech)；iQTMSYBR? Green Supermix (Bio-Rad)；TRIzol試劑(Roche)；PrimeScriptTMRT試劑盒 (TaKaRa)。CFX96 TouchTM熒光定量PCR儀(Bio-Rad)；倒置顯微鏡(NIKON)。

2 方法

2.1 細(xì)胞培養(yǎng)與分組 小鼠BV2小膠質(zhì)細(xì)胞用DMEM培養(yǎng)基(含1×105U/L青霉素、10 mg/L鏈霉素、10 mmol/L谷氨酰胺以及10%小牛血清)培養(yǎng)，置于37 ℃、5% CO2、飽和濕度的培養(yǎng)箱中培養(yǎng)，1～2 d換液，4～5 d傳代。實(shí)驗(yàn)分為對(duì)照(control)組、LPS組和FGF10組。提前一晚將細(xì)胞均勻鋪在6孔板上，F(xiàn)GF10組的BV2細(xì)胞預(yù)先給予FGF10 1 mg/L 30 min后，在LPS組和FGF10組中加入500 μg/L的LPS，刺激6 h或24 h。

2.2 ELISA試劑盒檢測(cè)TNF-α蛋白表達(dá)情況 LPS刺激24 h后，收集上清液1.5 mL于Eppendorf管中，按照ELISA檢測(cè)試劑盒的說(shuō)明書(shū)進(jìn)行操作，測(cè)定上清液中TNF-α的表達(dá)情況。

2.3 RT-qPCR檢測(cè)TNF-α的轉(zhuǎn)錄水平 收集經(jīng)LPS刺激6 h后的BV2細(xì)胞，用TRIzol抽提總RNA，抽提步驟根據(jù)生產(chǎn)商提供的方法進(jìn)行，然后將總RNA逆轉(zhuǎn)錄成cDNA。逆轉(zhuǎn)錄后得到的cDNA通過(guò)熒光定量PCR的方法，檢測(cè)熒光信號(hào)強(qiáng)度，然后轉(zhuǎn)化成為Ct值。β-actin作為管家基因用以消除因標(biāo)本處理、PCR反應(yīng)和逆轉(zhuǎn)錄不同而出現(xiàn)的差異。因此，用TNF-α與內(nèi)參照β-actin mRNA含量比值作為T(mén)NF-α的mRNA表達(dá)水平指標(biāo)。TNF-α的正義鏈?zhǔn)?’-TGATCCGCGACGTGGAA-3’， 反義鏈?zhǔn)?’-ACCGCCTGGAGTTCTGGAA-3’； β-actin的正義鏈?zhǔn)?’-CCGTGAAAAGATGACCCAGA-3’，反義鏈?zhǔn)?’-TACGACCAGAGGCATACAG-3’。

2.4 倒置顯微鏡觀察細(xì)胞形態(tài)學(xué)變化 LPS刺激24 h后，將細(xì)胞放在倒置顯微鏡下觀察，放大倍數(shù)200倍。每組樣品取3個(gè)視野計(jì)數(shù)，重復(fù)3次。活化的BV2細(xì)胞的相對(duì)值=(多極型細(xì)胞+紡錘樣雙極細(xì)胞)/總細(xì)胞數(shù)量。

3 統(tǒng)計(jì)學(xué)處理

用GraphPad Prism 5軟件中單因素方差分析的Tukey法來(lái)分析數(shù)據(jù)。計(jì)量數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 FGF10對(duì)LPS刺激下BV2細(xì)胞形態(tài)學(xué)的影響

我們?cè)?00倍顯微鏡下觀察了對(duì)照組、LPS組和FGF10組BV2細(xì)胞的形態(tài)學(xué)改變。處于靜息狀態(tài)下的BV2細(xì)胞形態(tài)呈圓形或橢圓形，體積較小。經(jīng)過(guò)LPS刺激24 h后，LPS組的BV2細(xì)胞因受到激活體積相對(duì)于對(duì)照組增大，且形狀從圓形或橢圓形向分枝樣改變?；罨腂V2細(xì)胞中一些向多極細(xì)胞轉(zhuǎn)變，一些向兩極延長(zhǎng)、呈紡錘樣改變。然而，F(xiàn)GF10組在預(yù)先給予FGF10 (1 mg/L)的情況下，活化的BV2細(xì)胞相對(duì)于LPS組明顯減少。通過(guò)統(tǒng)計(jì)活化的小膠質(zhì)細(xì)胞數(shù)占總細(xì)胞數(shù)量的比值我們可以得到相同的結(jié)果。LPS組活化的小膠質(zhì)細(xì)胞占總細(xì)胞數(shù)量的比值明顯高于對(duì)照組(P<0.01)，而預(yù)先給予FGF10可以降低活化細(xì)胞比值(P<0.05)，見(jiàn)圖1。

Figure 1.The effect of FGF10 on the morphological changes of LPS-induced BV2 microglia. The scale bar=200 μm. Mean±SD. n=3. **P<0.001 vs control group; #P<0.05 vs LPS group.

2 FGF10抑制LPS刺激下BV2細(xì)胞TNF-α的轉(zhuǎn)錄水平

給予LPS刺激6 h后，用RT-qPCR檢測(cè)促炎癥因子TNF-α的轉(zhuǎn)錄水平。如圖2所示，予以500 μg/L LPS刺激后，LPS組TNF-α的mRNA水平相比于對(duì)照組顯著升高(P<0.01)，然而FGF10組TNF-α的mRNA表達(dá)量相比LPS組顯著下降(P<0.05)。

Figure 2.The effect of FGF10 on the mRNA expression of TNF-α in the BV2 microglial cells stimulated with LPS. Mean±SD. n=3. **P<0.01 vs control group; #P<0.05 vs LPS group.

3 FGF10抑制LPS刺激下BV2細(xì)胞TNF-α的蛋白表達(dá)

我們分別用100 μg/L、250 μg/L、500 μg/L、1.0 mg/L和1.5 mg/L LPS濃度刺激BV2小膠質(zhì)細(xì)胞，然后通過(guò)檢測(cè)24 h后TNF-α的蛋白表達(dá)量來(lái)選擇合適的LPS劑量。結(jié)果如圖3所示，隨著LPS劑量增加TNF-α表達(dá)量與對(duì)照組相比明顯增加(P<0.05)，且當(dāng)LPS濃度為500 μg/L時(shí)達(dá)到最大值，而1.0 mg/L組和1.5 mg/L組與500 μg/L組比較TNF-α表達(dá)量的差異無(wú)統(tǒng)計(jì)學(xué)顯著性。因此，我們將LPS濃度500 μg/L作為最適刺激濃度。LPS作用24 h后，細(xì)胞培養(yǎng)上清液內(nèi)TNF-α的水平與對(duì)照組相比顯著上升(P<0.01)，這種表現(xiàn)與轉(zhuǎn)錄水平的上升趨勢(shì)是一致的。而預(yù)先給予FGF10在蛋白水平顯著抑制TNF-α的表達(dá)(P<0.01)，見(jiàn)圖4。

討 論

成纖維細(xì)胞生長(zhǎng)因子家族具有多種生化和生物學(xué)功能。它們?cè)谂咛グl(fā)育，維持穩(wěn)態(tài)，應(yīng)對(duì)損傷反應(yīng)，調(diào)節(jié)細(xì)胞的電生理興奮性等方面中扮演著十分重要的角色[12]。FGF10是成纖維細(xì)胞生長(zhǎng)因子家族中的成員，參與了多種生理和病理過(guò)程，包括了修復(fù)和再生等[13]。FGF10在治療肺部疾病中的效果明顯且應(yīng)用廣泛，包括了博來(lái)霉素引起的肺纖維化[14]，高原肺水腫[15]，缺血再灌注肺損傷[16]等。Tong等[9]在FGF10對(duì)LPS引起的急性肺損傷的影響的研究中發(fā)現(xiàn)，F(xiàn)GF10促進(jìn)肺組織的修復(fù)，明顯抑制支氣管肺泡灌洗液和肺組織中的IL1β、TNFα等促炎癥因子表達(dá)。Feng 等[10]用FGF10治療銅綠假單胞菌肺炎不但可以增加7 d生存率而且肺部的IL-6和TNF-α水平明顯降低。以上的研究結(jié)果提示，F(xiàn)GF10可能通過(guò)抑制炎癥反應(yīng)從而改善肺部疾病的修復(fù)情況。

Figure 3.The effect of LPS at different concentrations on protein expression of TNF-α in the activated BV2 microglia. Mean±SD. n=3. **P<0.01 vs 0 μ/L.

Figure 4.The effect of FGF10 on the protein of TNF-α in the BV2 microglial cells stimulated with LPS. Mean±SD. n=3. **P<0.01 vs control group; ##P<0.01 vs LPS group.

許多中樞神經(jīng)系統(tǒng)疾病的病理過(guò)程中均有炎癥反應(yīng)的參與，如帕金森綜合征，阿爾茲海默疾病，多發(fā)性硬化，腦外傷，感染，腦缺血再灌注損傷等。而中樞神經(jīng)系統(tǒng)中的小膠質(zhì)細(xì)胞在炎癥反應(yīng)過(guò)程中占據(jù)著重要的位置。正常情況下，小膠質(zhì)細(xì)胞具有神經(jīng)保護(hù)的作用，但是一旦受到病理因素的刺激后，它的形態(tài)會(huì)從靜息態(tài)轉(zhuǎn)變成活化態(tài)[17]。LPS是一種常見(jiàn)的細(xì)胞內(nèi)毒素，它能夠激活細(xì)胞中重要的病理機(jī)制，并且這些病理機(jī)制常常存在于炎癥反應(yīng)的病理過(guò)程中。LPS被廣泛應(yīng)用于模擬革蘭氏陰性菌感染引起的小膠質(zhì)細(xì)胞活化。因此，LPS激活小膠質(zhì)細(xì)胞的模型可用于研究與炎癥反應(yīng)有關(guān)的中樞神經(jīng)系統(tǒng)疾病中的小膠質(zhì)細(xì)胞活化的機(jī)制以及小膠質(zhì)細(xì)胞對(duì)神經(jīng)元的損傷機(jī)制。

本研究采用LPS作為刺激BV2細(xì)胞活化的病理刺激原。在LPS激活BV2小膠質(zhì)細(xì)胞的相關(guān)文獻(xiàn)中，LPS的參考劑量一般在100 μg/L～1 mg/L范圍內(nèi)[1, 18]。我們用不同濃度 LPS刺激BV2小膠質(zhì)細(xì)胞,然后通過(guò)檢測(cè)24 h后TNF-α的蛋白表達(dá)量來(lái)選擇合適的LPS劑量。結(jié)果顯示濃度500 μg/L LPS作為最適刺激濃度。激活狀態(tài)下的小膠質(zhì)細(xì)胞會(huì)大量轉(zhuǎn)錄和翻譯促炎癥因子，如一氧化氮(nitric oxide，NO)、PGE2、TNF-α、IL-1β和IL-6 等。本研究發(fā)現(xiàn)，BV2細(xì)胞在500 μg/L LPS刺激時(shí)細(xì)胞形態(tài)發(fā)生明顯的改變，細(xì)胞胞體增大且細(xì)胞向紡錘樣的雙極細(xì)胞或多極細(xì)胞轉(zhuǎn)變。在給予LPS刺激后，BV2細(xì)胞的TNF-α轉(zhuǎn)錄和蛋白表達(dá)水平明顯提高了。促炎癥因子TNF-α在轉(zhuǎn)錄和蛋白表達(dá)水平顯著升高以及小膠質(zhì)細(xì)胞形態(tài)上向活化態(tài)改變的表現(xiàn)均提示了小膠質(zhì)細(xì)胞在LPS刺激下發(fā)生了明顯的活化轉(zhuǎn)變。而大量促炎癥因子的產(chǎn)生將大大加重中樞神經(jīng)系統(tǒng)的炎癥反應(yīng)，加劇神經(jīng)元的損傷。

有研究顯示促炎癥因子過(guò)表達(dá)與一些中樞神經(jīng)系統(tǒng)退行性疾病有關(guān)，如阿爾茲海默、帕金森綜合癥、多發(fā)性硬化[19-20]等。因此，抑制炎癥因子產(chǎn)生成為控制中樞神經(jīng)系統(tǒng)炎癥反應(yīng)的重要機(jī)制。本研究發(fā)現(xiàn)，在給予LPS刺激后，BV2細(xì)胞的TNF-α轉(zhuǎn)錄和蛋白表達(dá)水平明顯提高了，而預(yù)先給予FGF10則明顯減少了在形態(tài)學(xué)上向活化態(tài)轉(zhuǎn)變的小膠質(zhì)細(xì)胞，同時(shí)抑制了促炎癥因子TNF-α的轉(zhuǎn)錄和蛋白表達(dá)。說(shuō)明FGF10具有抑制小膠質(zhì)細(xì)胞活化的作用。這個(gè)結(jié)果提示，F(xiàn)GF10可能具有抑制中樞神經(jīng)系統(tǒng)炎癥反應(yīng)的功能。一項(xiàng)支氣管肺發(fā)育不良的研究發(fā)現(xiàn)，F(xiàn)GF10與Toll樣受體的炎癥信號(hào)通路有關(guān)[21]。既往的研究已經(jīng)證實(shí)，中樞神經(jīng)系統(tǒng)中的炎癥反應(yīng)與Toll樣受體信號(hào)通路激活有關(guān)[22]。因此我們推測(cè)，F(xiàn)GF10有可能通過(guò)作用于BV2細(xì)胞上Toll樣受體信號(hào)通路對(duì)炎癥反應(yīng)產(chǎn)生影響，這需要我們?cè)诤罄m(xù)實(shí)驗(yàn)中進(jìn)一步證實(shí)。

綜上所述，預(yù)先給予FGF10夠能抑制LPS刺激下的BV2細(xì)胞形態(tài)向活化方向改變，同時(shí)減少小膠質(zhì)細(xì)胞BV2細(xì)胞對(duì)促炎癥因子TNF-α的轉(zhuǎn)錄和蛋白表達(dá)，表明FGF10有可能對(duì)膠質(zhì)細(xì)胞介導(dǎo)的神經(jīng)系統(tǒng)炎癥性疾病具有保護(hù)作用。FGF10有望成為治療經(jīng)膠質(zhì)細(xì)胞介導(dǎo)的神經(jīng)系統(tǒng)炎癥性疾病的一種有效藥物。

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(責(zé)任編輯： 林白霜， 羅 森)

Fibroblast growth factor 10 inhibits lipopolysaccharide-induced microglial activation

PAN Shu-lin1, HE Xiao-xiao1, HU Ying-ying1, FANG Ming-chu1, JIANG Huai1, XIAO Jian2, LIN Zhen-lang1

(1DepartmentofNeonatology,TheSecondAffiliatedHospital&YuyingChildren’sHospitalofWenzhouMedicalUniversity,Wenzhou325027,China;2SchoolofPharmacy,WenzhouMedicalUniversity,Wenzhou325035,China.E-mail:xfxj2000@126.com;linzhenlang@hotmail.com)

AIM: To investigate the effects of fibroblast growth factor 10 (FGF10) on lipopolysaccharide (LPS)-induced microglial activation. METHODS: Mouse BV2 microglial cells were maintained in DMEM in a humidified incubator with 95%/5% (V/V) mixture of air and CO2at 37 ℃. The medium was changed every 1 or 2 d. The cells were digested and passaged every 4 or 5 d. The BV2 microglial cells were first pretreated with FGF10 (1 mg/L) for 30 min and then stimulated with LPS (500 μg/L). The medium and the cells were collected at different time points. The morphological changes of microglia were visualized under microscope. To evaluate the microglial activation, the transcription and production of proinflammatory factor tumor necrosis factor-α (TNF-α) were examined by real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: The morphology of control BV2 microglia showed circular or oval shape. After exposure to LPS for 24 h, the microglia revealed spindle shaped or multipolar morphology, and the percentage of activated cells was significantly increased compared with control group. Pretreatment with FGF10 successfully inhibited the morphological change from normal to activated shape. LPS sti-mulation for 6 h significantly increased the transcription of TNF-α, while FGF10 pretreatment remarkably reversed the effect. In addition, the production of TNF-α increased in the presence of LPS stimulation for 24 h compared with control group. Pretreatment with FGF10 suppressed LPS-induced TNF-α expression.CONCLUSION: Pretreatment with FGF10 inhibits the morphological change from normal to activated shape, and remarkably suppressed the transcription and production of TNF-α. FGF10 successfully suppresses LPS-induced BV2 microglial activation, indicating that FGF10 is a therapeutic agent for the treatment of glia-mediated neuroinflammatory diseases.

Fibroblast growth factor 10; BV2 cells; Lipopolysaccharide; Tumor necrosis factor-α

1000- 4718(2017)03- 0534- 05

2016- 10- 09

2016- 12- 15

2014年度溫州市科技局公益性科技計(jì)劃(No. Y20140681)

△通訊作者 肖健 Tel: 0577-85773087; E-mail: xfxj2000@126.com； 林振浪 Tel: 0577-88002798; E-mail: linzhenlang@hotmail.com

R363.2

A

10.3969/j.issn.1000- 4718.2017.03.025