龍喜貴， 李 穎， 鄭毅濤， 齊一鳴， 黃俊琪

(中山大學(xué)中山醫(yī)學(xué)院免疫教研室，免疫學(xué)研究所，教育部熱帶病防治研究重點(diǎn)實(shí)驗(yàn)室, 廣東 廣州 510080)

CXCR4抑制劑AMD3100對(duì)2型登革熱病毒誘導(dǎo)血管內(nèi)皮細(xì)胞株Eahy926凋亡的影響*

龍喜貴， 李 穎， 鄭毅濤， 齊一鳴， 黃俊琪△

(中山大學(xué)中山醫(yī)學(xué)院免疫教研室，免疫學(xué)研究所，教育部熱帶病防治研究重點(diǎn)實(shí)驗(yàn)室, 廣東 廣州 510080)

目的探討趨化因子受體CXCR4抑制劑AMD3100對(duì)2型登革熱病毒(DV2)誘導(dǎo)人臍靜脈血管內(nèi)皮細(xì)胞株 Eahy926凋亡的影響。方法免疫組織化學(xué)法檢測(cè)Eahy926細(xì)胞的Ⅷ因子。Eahy926細(xì)胞分成未感染組和DV2感染組，流式細(xì)胞術(shù)檢測(cè)兩組細(xì)胞不同時(shí)點(diǎn)(24 h、36 h、48 h和60 h)CXCR4的表達(dá)水平。流式細(xì)胞術(shù)分析未感染組、DV2感染組及DV2+AMD3100組不同時(shí)點(diǎn)的細(xì)胞凋亡率。免疫熒光法檢測(cè)細(xì)胞膜表面磷脂酰絲氨酸(PS)。結(jié)果Eahy926細(xì)胞有Ⅷ因子表達(dá)。在DV2感染Eahy926后的4個(gè)時(shí)點(diǎn)中，CXCR4的表達(dá)均有上調(diào)，其中以48 h感染組最明顯(66.13%±10.30%，Plt;0.05)。DV2感染能誘導(dǎo)Eahy926細(xì)胞凋亡，其中36 h感染組凋亡率出現(xiàn)高峰(29.85%±15.78%，Plt;0.05)。應(yīng)用AMD3100后在各時(shí)點(diǎn)均能上調(diào)DV2感染組的凋亡率，免疫熒光觀察到DV2感染組及DV2+AMD3100組綠色熒光標(biāo)記的細(xì)胞增多。結(jié)論DV2感染能誘導(dǎo)血管內(nèi)皮細(xì)胞Eahy926凋亡并上調(diào)CXCR4的表達(dá)，CXCR4抑制劑AMD3100促進(jìn)DV2誘導(dǎo)Eahy926細(xì)胞凋亡的發(fā)生。

登革熱病毒; 凋亡; 受體,CXCR4; 血管內(nèi)皮細(xì)胞

登革熱病毒(dengue virus, DV)，登革熱( dengue fever, DF)的病原體，已經(jīng)成為重要的人類蟲媒病毒，威脅到熱帶和亞熱帶超過(guò)100個(gè)國(guó)家的25億人口[1]。普遍認(rèn)為2型登革熱病毒(dengue virus type 2, DV2)流行最廣，毒力較強(qiáng)，可引起重癥DF，即登革出血熱(dengue hemorrhagic fever, DHF)和登革休克綜合征(dengue shock syndrome, DSS)，其主要的特征是血漿滲漏。有報(bào)道表明DV引起的宿主細(xì)胞凋亡在其致病機(jī)制中發(fā)揮了重要作用[2]。我們前期研究表明，DV2可通過(guò)Fas/FasL途徑誘導(dǎo)原代人臍靜脈內(nèi)皮細(xì)胞(human umbilical veins endothelium cells，HUVECs)產(chǎn)生凋亡[3]。在后續(xù)研究中我們又發(fā)現(xiàn)DV2感染HUVECs后趨化因子受體CXCR4(C-X-C motif chemokine receptor 4)的基因水平表達(dá)增加。CXCR4是人免疫缺陷病毒 (human immunodeficiency virus，HIV)感染的輔助受體，與血管生成密切相關(guān)[4]，但其在DV感染血管內(nèi)皮細(xì)胞中的作用尚未明確。本研究采用流式細(xì)胞術(shù)檢測(cè)人臍靜脈內(nèi)皮細(xì)胞株Eahy926感染DV2前后CXCR4 的表達(dá)，并研究CXCR4特異性抑制劑AMD3100對(duì)DV2誘導(dǎo)Eahy926細(xì)胞凋亡率的影響，探索CXCR4在DV2感染血管內(nèi)皮細(xì)胞中的作用。

材 料 和 方 法

1材料

1.1細(xì)胞系 Eahy926 是由人肺腺癌細(xì)胞株A549與原代培養(yǎng)的人臍靜脈血管內(nèi)皮細(xì)胞融合構(gòu)建而成的永生化細(xì)胞株，具有血管內(nèi)皮細(xì)胞的特性[5]，為中山醫(yī)學(xué)院干細(xì)胞與組織工程研究中心惠贈(zèng)。

1.2病毒 DV2病毒株(new guinea Cstrain)來(lái)自中山醫(yī)學(xué)院微生物學(xué)教研室， 本實(shí)驗(yàn)室已進(jìn)行病毒擴(kuò)增和定量保存。

1.3主要試劑 DMEM/F12培養(yǎng)基、胎牛血清、青鏈霉素雙抗等為Gibco產(chǎn)品。凋亡檢測(cè)試劑盒為南京凱基(含F(xiàn)ITC-AnnexinⅤ組份)產(chǎn)品，CXCR4流式抗體為BD產(chǎn)品，Ⅷ因子Ⅰ抗為Thermo產(chǎn)品，Ⅱ抗為上?；蚩萍籍a(chǎn)品。

2方法

2.1細(xì)胞培養(yǎng) Eahy926 用100 mL/L胎牛血清及青鏈霉素雙抗為1.0 ×105U/L DMEM/F12培養(yǎng)液， 37 ℃、5%CO2孵育箱中培養(yǎng)。

2.2Ⅷ因子染色 取對(duì)數(shù)期生長(zhǎng)的Eahy926細(xì)胞，經(jīng)4%多聚甲醛固定20 min后，0.2%Triton打孔10 min，3%H2O2處理10 min，5%BSA封閉20 min，加抗Ⅷ因子 Ⅰ 抗，37 ℃ 60 min，Ⅱ 抗37 ℃ 30 min，DAB顯色。

2.3登革病毒感染及抑制劑應(yīng)用 取已定量好的2 型登革病毒液( 109PFU/L) 加入Eahy926 細(xì)胞培養(yǎng)板中吸附2 h，棄去上清，加入培養(yǎng)液，應(yīng)用抑制劑AMD3100(1 mg/L)于感染2 h后棄去多余病毒時(shí)，加入培養(yǎng)基，37℃、5%CO2培養(yǎng)，在各時(shí)點(diǎn)棄去培養(yǎng)液, 收集細(xì)胞。CXCR4的流式細(xì)胞術(shù)檢測(cè)分4個(gè)時(shí)間點(diǎn)(24 h、36 h、48 h和60 h)，每個(gè)時(shí)點(diǎn)設(shè)未感染組和DV2感染組。Eahy926的凋亡檢測(cè)時(shí)點(diǎn)設(shè)置同CXCR4檢測(cè)，每時(shí)點(diǎn)設(shè)未感染組、DV2感染組及DV2+AMD3100組，病毒及試劑濃度參照文獻(xiàn)[6]。

2.4流式細(xì)胞術(shù)檢測(cè)Eahy926細(xì)胞的CXCR4表達(dá) 取對(duì)數(shù)期生長(zhǎng)的Eahy926 細(xì)胞,500×g離心5 min， 用PBS 洗滌2 遍。用buffer重懸并調(diào)整細(xì)胞數(shù)為1×109cells/L 吸取100 μL 轉(zhuǎn)移至5 mL 的流式離心管中，加CXCR4的流式抗體12 μL，室溫孵育30min， PBS 洗滌2遍后用buffer 200 μL重懸，流式細(xì)胞儀檢測(cè)。

2.5流式細(xì)胞術(shù)檢測(cè)Eahy926細(xì)胞的凋亡 取對(duì)數(shù)期生長(zhǎng)的Eahy926 細(xì)胞離心，500×g離心5 min，用PBS 洗滌2 遍。用binding buffer 重懸并調(diào)整細(xì)胞數(shù)為1×109cells/L 吸取100 μL 轉(zhuǎn)移至5 mL 的流式離心管中， 加入FITC-AnnexinⅤ 5 μL、PI 5 μL 混勻， 室溫避光放置15 min，加入binding buffer 400 μL，流式細(xì)胞儀檢測(cè)。

2.6熒光顯微鏡觀察Eahy926細(xì)胞表面的磷脂酰絲氨酸(PS) 細(xì)胞經(jīng)DV2刺激及AMD3100處理后，PBS漂洗2次，加含有FITC-AnnexinⅤ的buffer，室溫反應(yīng)5 min，熒光顯微鏡下觀察。

3統(tǒng)計(jì)學(xué)處理

結(jié) 果

1Eahy926細(xì)胞Ⅷ因子染色呈陽(yáng)性

Eahy926細(xì)胞經(jīng)內(nèi)皮細(xì)胞特性Ⅷ因子染色后出現(xiàn)棕黃色的陽(yáng)性反應(yīng)，見(jiàn)圖1，證實(shí)其具有內(nèi)皮細(xì)胞的特性。

Figure 1. Immunohistochemistry staining of factor Ⅷ in Eahy926 cells(DAB staining,×200).

2DV2感染上調(diào)了Eahy926的CXCR4表達(dá)

Eahy926細(xì)胞未感染組設(shè)4個(gè)時(shí)點(diǎn)(24 h、36 h、48 h和60 h)，各時(shí)點(diǎn)表面表達(dá)CXCR4的Eahy926細(xì)胞百分比分別是:47.23% ±17.85%、 55.90%±11.48%、45.65%±20.93%和46.23%±21.47%。DV2感染Eahy926細(xì)胞各個(gè)相應(yīng)時(shí)點(diǎn)細(xì)胞表面CXCR4表達(dá)的百分比分別為52.90%±16.28%，58.93%±11.3%， 66.13%±10.30%，46.23%±21.47%，與感染前比均上升，其中48 h組感染前后差異最明顯(Plt;0.05,n=3) ，見(jiàn)圖2、3。

Figure 2. Expression of CXCR4 in Eahy926 cells before or after DV2 infection at 24 h, 36 h, 48 h and 60 h detected by flow cytometry.

Figure 3. CXCR4 expression in Eahy926 cells before or after DV2 infection at 24 h, 36 h, 48 h and 60 h .±s.n=3 *Plt;0.05 vs untreated cells

3DV2感染與應(yīng)用AMD3100后增加了Eahy926的凋亡率

圖4結(jié)果顯示DV2感染組4個(gè)時(shí)點(diǎn)(24 h、36 h、48 h和60 h)Eahy926細(xì)胞凋亡率分別為：25.18% ±8.82%、29.85% ±15.78%、19.95% ±4.96%和29.29% ±10.08%，均高于未感染組的Eahy926細(xì)胞凋亡率(18.95% ±9.00%、22.92% ±13.06%、18.91%±2.37%和25.73% ±9.35%)。而DV2+AMD3100組各時(shí)點(diǎn)Eahy926細(xì)胞凋亡率為39.39% ±15.94%、30.45% ±16.88%、27.46% ±8.26%和36.37% ±18.86%。48 h DV2+AMD3100組Eahy926細(xì)胞凋亡率高于DV2相應(yīng)時(shí)點(diǎn)組(Plt;0.05,n=3)，見(jiàn)圖4、 5。

Figure 4. Apoptosis of Eahy926 cells treated with or without AMD3100 before or after DV2 infection at 24 h,36 h,48 h and 60 h detected by flow cytometry.

Figure 5. Apoptosis of Eahy926 cells treated with or without AMD3100 24 h, 36 h, 48 h and 60 h after DV2 infection±s.n=3. *Plt;0.05 vs untreated group; △Plt;0.05 vs DV2 group.

4免疫熒光法檢測(cè)發(fā)現(xiàn)DV2感染組和DV2+AMD3100組Eahy926細(xì)胞表面磷脂酰絲氨酸表達(dá)增多

結(jié)果顯示未感染組綠色熒光標(biāo)記的磷脂酰絲氨酸極少，DV2感染組及DV2+AMD3100組綠色熒光標(biāo)記的細(xì)胞增多，見(jiàn)圖6。

討 論

細(xì)胞凋亡(apoptosis) 或細(xì)胞程序性死亡( programmed cell death, PCD)在維持機(jī)體正常發(fā)育、清除病毒感染細(xì)胞、腫瘤細(xì)胞等有著極其重要的意義。Clarke等[7]認(rèn)為呼腸病毒引起的細(xì)胞凋亡可能是由腫瘤壞死因子相關(guān)誘導(dǎo)凋亡配體 (tumor necrosis factor-related apoptosis-inducing ligand，TRAIL) 介導(dǎo)的， Kim等[8]研究表明HIV病毒可上調(diào)獲得性免疫缺陷綜合征 (acquired immune deficiency syndrome,AIDS) 易感病人體內(nèi)抗原提呈細(xì)胞的TRAIL從而介導(dǎo)凋亡，Larochelle等[9]曾報(bào)道EB病毒 (Epstein-Barr virus,EBV)引起細(xì)胞凋亡可能是FasL的作用，我們研究發(fā)現(xiàn)DV2感染原代HUVECs可通過(guò)Fas/FasL途徑誘導(dǎo)凋亡的發(fā)生[3]，HUVECs感染DV2后CXCR4的基因水平表達(dá)增加。

Figure 6. Direct immunofluorescence staining of phosphatidylserine in Eahy926 cells with FITC-Annexin V(×100)

為了進(jìn)一步研究CXCR4在DV2感染血管內(nèi)皮細(xì)胞中的作用，我們選擇人靜脈血管內(nèi)皮細(xì)胞株Eahy926細(xì)胞作為細(xì)胞模型。Eahy926細(xì)胞來(lái)源于HUVECs[5]，與原代HUVECs相比易于培養(yǎng)且無(wú)原代細(xì)胞分離培養(yǎng)過(guò)程中雜細(xì)胞的影響。但Eahy926細(xì)胞在長(zhǎng)期不斷傳代培養(yǎng)后是否依然具有內(nèi)皮細(xì)胞特性？我們對(duì)其進(jìn)行了內(nèi)皮細(xì)胞特性Ⅷ因子染色，證實(shí)了這一點(diǎn)。

CXCR4是基質(zhì)細(xì)胞衍生因子-1 (stromal cell derived factor 1,SDF-1)的特異性受體[10]，屬于7次跨膜的G蛋白偶聯(lián)受體家族。CXCR4與配體SDF-1結(jié)合傳遞的細(xì)胞間信號(hào)在造血干細(xì)胞歸巢、淋巴細(xì)胞遷移、促腫瘤細(xì)胞侵襲[11]、促血管細(xì)胞增殖生長(zhǎng)[ 12,13]等方面發(fā)揮著重要作用。有報(bào)道表明下調(diào)CXCR4的表達(dá)抑制了腫瘤細(xì)胞的代謝和侵襲性[14]，增加CXCR4的表達(dá)抑制了過(guò)敏性紫癜血小板的自身凋亡[15]。有報(bào)道應(yīng)用CXCR4的單克隆抗體或其特異性的抑制劑AMD3100可上調(diào)Fas等凋亡相關(guān)因子的表達(dá)[16]。AMD3100是SDF-1受體CXCR4的非肽類阻斷劑，能特異性與CXCR4結(jié)合而不產(chǎn)生激動(dòng)作用，阻斷SDF-1/CXCR4軸參與的生理過(guò)程。本文初步研究了AMD3100對(duì)DV2誘導(dǎo)血管內(nèi)皮細(xì)胞Eahy926凋亡的影響。

本實(shí)驗(yàn)結(jié)果顯示DV2感染Eahy926細(xì)胞后能上調(diào)CXCR4的表達(dá)，48 h升高最為明顯。根據(jù)SDF-1/CXCR4軸參與的生理過(guò)程，其CXCR4于感染后表達(dá)增加可能為DV2誘導(dǎo)Eahy926細(xì)胞凋亡后的保護(hù)性上調(diào)。DV2感染可以誘導(dǎo)Eahy926細(xì)胞凋亡，其中36 h凋亡率增高最為明顯；AMD3100能上調(diào)DV2誘導(dǎo)的Eahy926細(xì)胞的凋亡率，48 h DV2感染組與DV2+AMD3100組凋亡率存在顯著差異，其原因可能為48 h時(shí)點(diǎn)CXCR4的表達(dá)增加最為明顯，從而AMD3100對(duì)SDF-1/CXCR4軸產(chǎn)生的抑制作用也會(huì)更加明顯。綜上可以看出AMD3100在DV2感染血管內(nèi)皮細(xì)胞株Eahy926中可以促進(jìn)病毒誘導(dǎo)的細(xì)胞凋亡的發(fā)生，但其作用機(jī)制還有待進(jìn)一步研究。

[1] Guzmán MG, Kourí G. Dengue: an update [J].Lancet Infect Dis, 2002, 2(1): 33-42.

[2] Matsuda T, Almasan A, Tomita M, et al. Dengue virus-induced apoptosis in hepatic cells is partly mediated by Apo2 ligand/tumour necrosis factor-related apoptosis-inducing ligand [J] . Gen Virol, 2005, 86(4): 1055-1065.

[3] Liao H, Xu J, Huang J. FasL/Fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells [J]. Med Virol, 2010,82(8):1392-1399.

[4] Yang SX, Chen JH, J iang XF, et al. Activation of chemokine receptor CXCR4 in malignant glioma cells promotes the production of vascular endothelial growth factor [J]. Biochem Biophys Res Commun,2005, 335 (2): 523 - 528.

[5] Lidinqton EA , Moves DL , McCormack AM, et al . A comparison of primary endothelial cells and endothelial cell lines for studies of immune interactions [J] . Transpl Immunol , 1999 , 7 (4): 239-246.

[6] 廖紅舞,黃俊琪. 2型登革病毒誘導(dǎo)ED25細(xì)胞凋亡及死亡受體的表達(dá)變化[J]. 中國(guó)病理生理雜志,2007,23(12):2401-2404.

[7] Clarke P, Tyler KL. Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis [J]. Apoptosis,2007, 12(1): 211-223.

[8] Kim N,Dabrowska A, Jenner RG, et al. Human and simian immunodeficiency virus-mediated upregulation of the apoptotic factor TRAIL occurs in antigen-presenting cells from AIDS-susceptible but not from AIDS-resistant species [J] . J Virol, 2007, 81(14): 7584-7597.

[9] Larochelle B, Flamand L, Gourde P, et al. Epstein-Barr virus infects and induces apoptosis in human neutrophils [J] . Blood, 1998, 92(1): 291-299.

[10]Altundag K, Morandi P, Altundag O, et al. Possible role of CXCR4 - mediated chemotaxis in breast cancer patients with central nervous system metastases[J]. Breast Cancer Res Treat, 2005 , 89(3): 317-321.

[11]Juarez J, Bendall L, Bradstock K. Chemokines and their receptors as therapeutic targets: the role of the CXCL12/CXCR4 axis[J]. Curr Pharm Des, 2004, 10(11): 1245-1259.

[12]Brand S, Dambacher J, Beigel F, et al. CXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP29 activation[J]. Exp Cell Res, 2005,310 (1): 117 - 130.

[13]Smith MC, Luker KE, Garbow JR, et al. CXCR4 regulates growth of both primary and metastatic breast cancer[J]. Cancer Res, 2004,64 (23): 8604 - 8612.

[14]Yadav VR, Sung B,Prasad S, et al. Celastrol suppresses invasion of colon and pancreatic cancer cells through the downregulation of expression of CXCR4 chemokine receptor [J]. J Mol Med, 2010,88(12):1243-1253.

[15]Wang JD, Ou TT,Wang CJ, et al. Platelet apoptosis resistance and increased CXCR4 expression in pediatric patients with chronic immune thrombocytopenic purpura[J].Thromb Res,2010, 126(4):311-318.

[16]魏 立,孔佩艷,陳幸華,等. 抗CXCR4 單克隆抗體對(duì)HL260細(xì)胞粘附性及Bcl-2、Fas 蛋白表達(dá)的影響[J].中國(guó)實(shí)驗(yàn)血液學(xué)雜志,2004,12(4):436-440.

EffectofAMD3100ondenguevirustype2-inducedapoptosisinEahy926cells

LONG Xi-gui, LI Ying, ZHENG Yi-tao, QI Yi-ming, HUANG Jun-qi

(DepartmentofImmunology,InstituteofImmunology,ZhongshanSchoolofMedicine,SunYet-senUniversity,KeyLaboratoryofTropicalDiseaseControl,MinistryofEducation,Guangzhou510080,China.E-mail:junqi_huang@yahoo.com.cn)

AIM: To investigate the role of AMD3100 (an inhibitor of CXCR4) in dengue virus type 2 (DV2)-induced apoptosis in human umbilical vein endothelial cell line Eahy926.METHODSThe expression of factor Ⅷ in Eahy926 cells was examined by immunohistochemistry staining. The cells were divided into untreated group, DV2 infection group and DV2+AMD3100 group. Flow cytometric analysis was used to detect the expression of CXCR4 in Eahy926 cells 24 h, 36 h, 48 h and 60 h after DV2 infection. In addition, the percentage of apoptotic cells was also analyzed by flow cytometry. Immunofluorescence was performed to detect the phosphatidylserine (PS) on the surface of Eahy926 cells.RESULTSEahy926 cells were factor Ⅷ-positive. Compared with untreated group, the expression of CXCR4 increased in DV2 infection group, most markedly 48 h after infection (66.13%±10.30%,Plt;0.05). The percentage of apoptotic Eahy926 cells after DV2 infection was the highest at 36 h (29.85%±15.78%,Plt;0.05). The percentage of DV2-induced apoptotic cells in DV2+AMD3100 group was higher than that in DV2 infection group. The green fluorescence-labeled cells in DV2 infection group and DV2+AMD3100 group were more than those in untreated group.CONCLUSIONDV2 infection induces apoptosis and increases the expression of CXCR4 in Eahy926 cells. AMD3100, the inhibitor of CXCR4, may be a promoter of apoptosis in Eahy926 cells after DV2 infection.

Dengue virus; Apoptosis; Receptors,CXCR4; Vascular endothelial cells

1000-4718(2011)04-0632-06

R373.33

A

10.3969/j.issn.1000-4718.2011.04.002

2010-09-05

2011-02-24

國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 30872350;No. 30400371;No. 30571736 )；廣東省自然科學(xué)基金資助項(xiàng)目(No.8151008901000017)；廣東省團(tuán)隊(duì)項(xiàng)目(No. 06201946)；廣東省科技計(jì)劃項(xiàng)目(No.2007B031516008；No. 2010B050700008)；廣州市科技計(jì)劃項(xiàng)目(No. 2006Z3-E4081; No.2008Z1-E221)；高?；究蒲袠I(yè)務(wù)費(fèi)中山大學(xué)青年教師培育項(xiàng)目(No.10YKPY31)

△通訊作者Tel: 020-87330068-838;E-mail: junqi_huang@yahoo.com.cn