董革輝,劉福慧,韓建華,夏本杰,黃俊瓊

(1.遵義醫(yī)學(xué)院第三附屬醫(yī)院骨科,遵義 563000;2.遵義醫(yī)學(xué)院附屬醫(yī)院輸血科,遵義 563003;3.遵義醫(yī)學(xué)院附屬醫(yī)院檢驗(yàn)科,遵義 563003)

外泌體致敏樹突狀細(xì)胞體外抗骨肉瘤細(xì)胞生長(zhǎng)作用*

董革輝1,劉福慧2,韓建華1,夏本杰1,黃俊瓊3

(1.遵義醫(yī)學(xué)院第三附屬醫(yī)院骨科,遵義 563000;2.遵義醫(yī)學(xué)院附屬醫(yī)院輸血科,遵義 563003;3.遵義醫(yī)學(xué)院附屬醫(yī)院檢驗(yàn)科,遵義 563003)

目的 觀察骨肉瘤細(xì)胞分泌的外泌體體外刺激細(xì)胞毒性T細(xì)胞對(duì)骨肉瘤細(xì)胞的殺傷作用。方法采用超濾離心聯(lián)合蔗糖密度梯度超速離心的方法從骨肉瘤細(xì)胞培養(yǎng)上清液中分離外泌體(Texo)。透射電子顯微鏡鑒定Texo形態(tài),western blot分析Texo表面主要組織相容性復(fù)合體-玉(MHC-玉)類分子表達(dá)。分離培養(yǎng)小鼠骨髓來(lái)源樹突狀細(xì)胞(DC),流式細(xì)胞儀鑒定細(xì)胞表型。噻唑藍(lán)(MTT)法檢測(cè)負(fù)載Texo的DC刺激T淋巴細(xì)胞增殖情況及效應(yīng)淋巴細(xì)胞對(duì)骨肉瘤細(xì)胞的殺傷作用。結(jié)果透射電鏡下見Texo為圓形或類圓形小體,大小不等,平均直徑50～100 nm;Texo表面有MHC-玉類分子表達(dá)。骨髓來(lái)源DC邊緣有絨毛樣突起,呈典型的樹突狀細(xì)胞形態(tài),經(jīng)脂多糖誘導(dǎo)后DC的CD80、MHC-玉、MHC-Ⅱ類分子表達(dá)明顯高于未經(jīng)誘導(dǎo)的DC,陽(yáng)性率分別為77.16%,83.21%,91.26%。負(fù)載Texo的DC刺激T細(xì)胞增殖能力及T細(xì)胞對(duì)靶細(xì)胞的殺傷效應(yīng)明顯強(qiáng)于未負(fù)載Texo的DC與Texo(P＜0.05)。結(jié)論負(fù)載Texo的DC能促進(jìn)T細(xì)胞增殖,可體外誘導(dǎo)細(xì)胞毒性T細(xì)胞應(yīng)答抑制骨肉瘤細(xì)胞生長(zhǎng)。

外泌體;骨肉瘤;樹突狀細(xì)胞;T細(xì)胞;抑制作用

外泌體是由細(xì)胞分泌到細(xì)胞外的膜性小囊泡,直徑30～100 nm,具有雙層脂質(zhì)結(jié)構(gòu),攜帶大量與其來(lái)源和功能密切相關(guān)的膜性分子。淋巴細(xì)胞、樹突狀細(xì)胞(dentritic cell,DC)、腫瘤細(xì)胞等均可分泌外泌體。腫瘤細(xì)胞分泌到細(xì)胞外的外泌體(tumor-derived exosome,Texo)富含腫瘤相關(guān)抗原、主要組織相容性復(fù)合體(major histocompatibility complex,MHC)分子、共刺激分子,可誘導(dǎo)抗腫瘤細(xì)胞毒性T細(xì)胞(cytotoxic T lyphocyte,CTL)應(yīng)答[1]。Texo作為腫瘤疫苗,在實(shí)驗(yàn)性免疫治療中取得了顯著效果,筆者尚未見其應(yīng)用于骨肉瘤免疫治療的報(bào)道。筆者從骨肉瘤細(xì)胞分離Texo,體外刺激樹突狀細(xì)胞,探討Texo致敏TC誘導(dǎo)CTL對(duì)骨肉瘤細(xì)胞的生長(zhǎng)抑制作用,以期為骨肉瘤的免疫治療提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物與細(xì)胞 雌性Balb/c小鼠,6～8周齡,體質(zhì)量20～22 g,潔凈度:SPF級(jí),購(gòu)自第三軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心,生產(chǎn)許可證號(hào):SCX(渝)2012-0001,實(shí)驗(yàn)許可證號(hào):SYXK(黔)2011-003。小鼠骨肉瘤細(xì)胞K7M2購(gòu)自漢恒生物科技有限公司(批號(hào):20111219)。

1.2 試劑與儀器 細(xì)胞培養(yǎng)液RPMI1640購(gòu)自Hyclone公司,胎牛血清(fetal bovine serum,FBS)購(gòu)自Gibco公司,鼠MHC-玉單克隆抗體購(gòu)自Senta Cruz公司,辣根過(guò)氧化物酶標(biāo)記羊抗鼠二抗購(gòu)自Sigma公司, 100 kD Amicon Ultra-15離心超濾管購(gòu)自美國(guó)Millipore公司,超速離心管購(gòu)自美國(guó)Beckman公司,增強(qiáng)化學(xué)發(fā)光(enhanced chemiluminescence,ECL)試劑盒(批號(hào):20120123)、蛋白預(yù)染Marker購(gòu)自碧云天生物技術(shù)研究所。

1.3 Texo的分離與純化 將K7M2細(xì)胞用含10%胎牛血清的RPMI1640培養(yǎng)液于37℃、5%二氧化碳條件下培養(yǎng),隔天換液,待單層細(xì)胞生長(zhǎng)達(dá)到80%融合時(shí),用胰蛋白酶消化細(xì)胞,進(jìn)行傳代。選取對(duì)數(shù)生長(zhǎng)期細(xì)胞,收集細(xì)胞培養(yǎng)上清液200～500 mL,4℃、300×g離心10 min,取上清液于4℃、800×g離心30 min,再取上清液4℃、10 000×g離心30 min,吸取上清液加入100 kD Amicon Ultra-15離心超濾管,4℃、1 500×g離心30 min,至濃縮液體積＜8 mL;將濃縮液、30 g·L-1蔗糖/重水墊和磷酸鹽緩沖液(phosphate buffered solution,PBS) (0.01 mol·L-1)以3∶3∶4的比例依次加入Beckman專用離心管,4℃、100 000×g超速離心60 min,取出承載Texo的蔗糖/重水墊,用PBS50倍體積稀釋,再經(jīng)100 000×g超速離心60 min,得到的沉淀即為Texo。取適量PBS重懸,孔徑0.22 μm濾膜過(guò)濾除菌,分裝, -80℃保存。

1.4 透射電鏡觀察Texo形態(tài)特征 取Texo重懸液10 μL滴于載樣銅網(wǎng)上,室溫靜置1 min,用濾紙從側(cè)面吸干液體,滴加20 g·L-1磷鎢酸溶液(pH6.8)約30 μL于銅網(wǎng)上,室溫負(fù)染1 min,吸干負(fù)染液,白熾燈下烤干,透射電子顯微鏡(日立高新技術(shù)國(guó)際貿(mào)易有限公司生產(chǎn),H-7650型)觀察Texo形態(tài)。

1.5 Western blot分析MHC-Ⅰ類分子表達(dá) 以體外培養(yǎng)的骨肉瘤細(xì)胞作為對(duì)照,調(diào)整細(xì)胞濃度為1× 1011·L-1,于-20℃反復(fù)快速凍融4次后,1 000×g離心30 min,上清液即為細(xì)胞裂解物。取Texo 40 μg及骨肉瘤細(xì)胞裂解物40 μg,分別加入適量電泳上樣緩沖液,在12%聚丙烯酰胺凝膠上進(jìn)行電泳分離,用半干電轉(zhuǎn)儀將蛋白質(zhì)轉(zhuǎn)移至醋酸纖維膜上,室溫用5%脫脂奶粉包被2 h后,加入一抗(1∶400)4℃過(guò)夜, PBS洗滌3次,加入辣根過(guò)氧化物酶標(biāo)記的二抗(1∶5 000),37℃孵育30 min,加入發(fā)光試劑室溫放置1 min,PBS洗滌3次,暗室曝光顯影。

1.6 小鼠骨髓來(lái)源DC的培養(yǎng) CO2窒息法處死小鼠,無(wú)菌條件下取小鼠股骨、脛骨,PBS沖洗骨髓至髓腔變白,收集沖洗液,1 500×g離心5 min,收集細(xì)胞用PBS洗2次。三羥甲基氨基甲烷-氯化氨紅細(xì)胞裂解液(Tris-NH4Cl)裂解紅細(xì)胞,用含20 μg·L-1粒細(xì)胞-巨噬細(xì)胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)、1 μg·L-1白細(xì)胞介素4 (interlukin 4,IL-4)的DMEM培養(yǎng)基重懸細(xì)胞至2× 109·L-1,按每孔4 mL加入24孔板,置37℃、5%CO2培養(yǎng)箱中培養(yǎng)。第3天更換培養(yǎng)液,輕輕晃動(dòng)培養(yǎng)板,棄去上清液,補(bǔ)入含細(xì)胞因子的DMEM完全培養(yǎng)液。第6天細(xì)胞換液,用含20 μg·L-1GM-CSF、1 μg·L-1IL-4和15 μg·L-1腫瘤壞死因子(tumor necrosis factor,TNF)的DMEM培養(yǎng)液重懸細(xì)胞,繼續(xù)培養(yǎng)24 h后收集細(xì)胞,換無(wú)血清的AIM-V培養(yǎng)基,加入10 μg·mL-1脂多糖(lipopolysaccharide,LPS),置37℃、5%CO2培養(yǎng)箱中培養(yǎng)過(guò)夜,次日收集細(xì)胞進(jìn)行表型鑒定。

1.7 T淋巴細(xì)胞增殖實(shí)驗(yàn) CO2窒息法處死Balb/c小鼠,無(wú)菌分離脾臟,經(jīng)200目鋼網(wǎng)研磨制成單個(gè)細(xì)胞懸液,PBS洗2次,用RPMI 1640培養(yǎng)液重懸細(xì)胞,加入尼龍毛柱中,常規(guī)培養(yǎng)1 h,收集流出的T淋巴細(xì)胞。將DC與T淋巴細(xì)胞(T)按1∶3比例混合培養(yǎng)。實(shí)驗(yàn)分為4組,A組:Texo-DC+T;B組:DC+T;C組:Texo (10 μg·mL-1)+T;D組:T(對(duì)照組)。將上述各組細(xì)胞加入96孔培養(yǎng)板,每孔100 μL,每組設(shè)復(fù)孔3個(gè),將培養(yǎng)板置于37℃、5%CO2培養(yǎng)箱中培養(yǎng)3 d后,每孔加入噻唑藍(lán)(methylthiazolyldiphenyl-tetrazoliumbromide, MTT)溶液(5 mg·mL-1)20 μL,繼續(xù)于培養(yǎng)箱中孵育4 h,每孔再加入二甲基亞砜(dimethyl sulfoxide,DMSO) 150 μL,震蕩混勻,酶標(biāo)儀(北京普朗新技術(shù)有限公司生產(chǎn),型號(hào):DNM-9600G)于波長(zhǎng)570 nm測(cè)定吸光度(A)值。按下列公式計(jì)算刺激指數(shù)(stimulating index,SI), SI=實(shí)驗(yàn)組平均A值/對(duì)照組平均A值。

1.8 細(xì)胞毒性實(shí)驗(yàn) 設(shè)實(shí)驗(yàn)組、效應(yīng)細(xì)胞對(duì)照組、DC對(duì)照組及靶細(xì)胞對(duì)照組,實(shí)驗(yàn)組為Texo-DC+T,T淋巴細(xì)胞為效應(yīng)細(xì)胞,實(shí)驗(yàn)組按效靶比12.5∶1,25∶1,50∶1,加入已鋪好骨肉瘤細(xì)胞的96孔培養(yǎng)板,每孔100 μL,每組設(shè)復(fù)孔3個(gè)。于37℃、5%CO2培養(yǎng)箱中培養(yǎng)48 h,加入5 mg·mL-1MTT溶液20 μL,繼續(xù)培養(yǎng)4 h后加入DMSO150 μL,于波長(zhǎng)570 nm測(cè)定A值,計(jì)算殺傷率。殺傷率(%)=[靶細(xì)胞對(duì)照組A值-(實(shí)驗(yàn)組A值-效應(yīng)細(xì)胞對(duì)照組A值)]/靶細(xì)胞對(duì)照組A值×100%。

2 結(jié)果

2.1 Texo的分離鑒定 細(xì)胞培養(yǎng)上清液經(jīng)300×g,800 ×g,10 000×g梯度離心后,管內(nèi)可見體積大小不等的沉淀物,去沉淀物后上清液經(jīng)超濾離心管100 000×g離心濃縮60 min,離心管底見塊狀黃白色沉淀。吸取蔗糖/重水墊,PBS稀釋后再經(jīng)100 000×g離心60 min,離心管底可見少量黃白色沉淀,該沉淀即為Texo。電子顯微鏡觀察見Texo呈圓形或類圓形,大小不等,平均直徑50～100 nm,散在分布或聚集成團(tuán)(圖1)。Texo和細(xì)胞裂解物均表達(dá)MHC-玉類分子,腫瘤細(xì)胞來(lái)源的Texo MHC-玉灰度值較腫瘤細(xì)胞裂解物高(圖2)。

圖1 K7M2細(xì)胞分泌的Texo電鏡圖(×50 000)Fig.1 Electron microscope results of exosome purified fromM7K2 cell(×50 000)

圖2 Texo的Western blot分析結(jié)果Fig.2 Western blot analysis on MHC-1 expression

2.2 DC表型分析 高倍鏡下可見部分細(xì)胞表面有2或3個(gè)毛刺樣突起。培養(yǎng)第7天加入LPS 24 h后(即第8天),細(xì)胞全部脫壁,呈懸浮生長(zhǎng),細(xì)胞邊緣有突起2～5個(gè),呈星形,為典型的樹突狀細(xì)胞形態(tài)。未經(jīng)LPS誘導(dǎo)的DC表面CD80、MHC-玉類分子、MHC-Ⅱ類分子表達(dá)較低,陽(yáng)性率分別為42.53%,54.96%, 33.44%;經(jīng)LPS誘導(dǎo)后的DC表面分子的表達(dá)較未經(jīng)LPS誘導(dǎo)的DC明顯增高,陽(yáng)性率分別為77.16%, 83.21%,91.26%,為成熟的DC(圖3)。

圖3 流式細(xì)胞儀分析DC表面標(biāo)志Fig.3 FCManalysis on surface marker of DC

2.3 Texo致敏DC刺激T淋巴細(xì)胞增殖 Texo-DC+T組、DC+T組和Texo+T組對(duì)T淋巴細(xì)胞的SI分別為(1.51±0.25)(0.76±0.06)(1.15±0.08)。Texo組SI＜1,說(shuō)明沒有刺激T細(xì)胞活化的能力;負(fù)載Texo的Texo-DC組和未負(fù)載Texo的DC組均＞1,說(shuō)明有刺激T細(xì)胞活化的能力;負(fù)載Texo的DC與未負(fù)載Texo的DC相比,刺激T細(xì)胞活化的能力明顯增強(qiáng)(P＜0.05)。

2.4 Texo致敏DC誘導(dǎo)CTL的特異性殺傷作用 負(fù)載Texo的DC在效靶比為12.5∶1時(shí),其抗腫瘤活性與未負(fù)載Texo的DC、Texo、T細(xì)胞對(duì)照組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義;在效靶比25∶1,50∶1時(shí),負(fù)載Texo的DC誘導(dǎo)CTL抗腫瘤活性明顯高于其他3組,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05),見圖4。

3 討論

骨肉瘤是一種來(lái)源于間葉組織的惡性腫瘤,易轉(zhuǎn)移,手術(shù)治療5年生存率較低,目前多采用手術(shù)切除聯(lián)合化學(xué)治療(化療),但抗癌藥物常常導(dǎo)致骨髓抑制、肝功能損害等嚴(yán)重不良反應(yīng)[2]。對(duì)于那些化療藥物耐藥或無(wú)法忍受化療藥物不良反應(yīng)的患者,骨肉瘤的治療束手無(wú)策,因而需要研發(fā)新的治療策略。腫瘤患者腫瘤細(xì)胞表面MHC-玉類分子表達(dá)減少或缺失,導(dǎo)致抗原呈遞功能障礙,從而影響腫瘤特異性的CTL活化和抗腫瘤效應(yīng),患者抗腫瘤免疫功能低下。Texo作為新型亞細(xì)胞腫瘤疫苗,是近年來(lái)疫苗研究的熱點(diǎn)。Texo是由活細(xì)胞分泌的膜性微囊結(jié)構(gòu),其功能根據(jù)細(xì)胞來(lái)源的不同而不同[3-5]。目前研究得較多的是DC來(lái)源的Texo和腫瘤細(xì)胞來(lái)源的Texo。Texo含有MHC-玉類分子、四跨膜蛋白、腫瘤相關(guān)抗原等[6-7],被樹突狀細(xì)胞攝取后,可激活T淋巴細(xì)胞,發(fā)揮抗腫瘤免疫效應(yīng)[8-9]。

圖4 Texo致敏DC誘導(dǎo)特異性CTL活性Fig.4 Activity of CTL induced by Texo-pulsed DC

筆者在本實(shí)驗(yàn)中采用超濾離心聯(lián)合蔗糖密度梯度超速離心的方法從骨肉瘤細(xì)胞提取Texo,電鏡下見骨肉瘤細(xì)胞分泌的Texo呈圓形或類圓形,平均直徑50～100 nm,表面有MHC-玉類分子表達(dá),與文獻(xiàn)報(bào)道一致。小鼠骨髓來(lái)源的DC經(jīng)LPS誘導(dǎo)成熟后,采用Texo進(jìn)行刺激,觀察Texo致敏DC對(duì)T細(xì)胞的活化增殖效應(yīng)及活化T細(xì)胞對(duì)骨肉瘤細(xì)胞的殺傷效應(yīng),探討Texo的抗腫瘤作用。結(jié)果顯示,單獨(dú)Texo不能刺激T淋巴細(xì)胞增殖,負(fù)載Texo的DC和未負(fù)載Texo的DC均能刺激T細(xì)胞增殖,負(fù)載Texo的DC刺激T細(xì)胞活化的能力較未負(fù)載Texo的DC強(qiáng)。細(xì)胞毒性實(shí)驗(yàn)中,負(fù)載Texo的DC在高濃度時(shí)有較強(qiáng)的殺瘤作用。未負(fù)載Texo的DC、Texo與效應(yīng)細(xì)胞對(duì)照組相比,無(wú)明顯抑瘤作用。以上結(jié)果表明,Texo致敏DC可誘導(dǎo)T細(xì)胞活化,活化后的CTL發(fā)揮抗腫瘤作用抑制骨肉瘤細(xì)胞生長(zhǎng)。Texo雖然含有MHC-玉類分子和腫瘤抗原,但不能直接誘導(dǎo)CTL反應(yīng),T細(xì)胞的活化除了需要腫瘤抗原及MHC分子刺激外,還需要DC等抗原提呈細(xì)胞提供共刺激信號(hào),只有當(dāng)其負(fù)載到DC上時(shí),才能高效誘導(dǎo)CTL發(fā)揮抗腫瘤作用。

本研究成功地從骨肉瘤細(xì)胞分離了Texo,并證實(shí)了骨肉瘤細(xì)胞來(lái)源的Texo可誘導(dǎo)CTL反應(yīng),增強(qiáng)抗腫瘤免疫效應(yīng),為骨肉瘤的免疫治療提供依據(jù)。

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DOI 10.3870/yydb.2014.07.009

Texo-pulsed Dendritic Cells Inhibited the Growth of Osteosarcoma Cells by Stimulating CTL Response in Vitro

DONG Ge-hui1,LIU Fu-hui2,HAN Jian-hua1,XIA Ben-jie1,HUANG Jun-qiong3
(1.Department of Orthopeadics,the Third Affiliated Hospital of Zunyi Medical College,Zunyi 563000,China;2.Department of Blood Transfusion,Affiliated Hospital of Zunyi Medical College,Zunyi 563003,China;3.Clinical Laboratory, Affiliated Hospital of Zunyi Medical College,Zunyi 563003,China)

ObjectiveTo investigate the stimulation of exosome derived fromosteosarcoma cells suppressing cytotoxic T cells and the inhibitory effect of active T cells for surpressing osteosarcoma cells.MethodsExosome derived fromtumor cells was isolated and purified by ultracentrifugation.Its morphology was observed with transmission electron microscope,and the major histocompatibility complex-I(MHC-I)molecules were analyzed by western blot.Mice bone marrow-derived dendritic cells were pulsed with exosome.Surface membrane MHC-I molecules were analyzed with flowcytometry.The effect of active T cells on the growth of osteosarcoma cells were detected by MTT assay after the T cells being stimulated by exosome-pulsed dendritic cells.ResultsThe exosome was round or near round corpuscle,and the diameter was about 50-100 nmby transmission electron microscope.The size was relatively homogeneous.Western blot showed that the exosome expressed MHC-玉molecules.Surface membrane CD80,MHC-I and II molecules were expressed on 77.16%,83.21%,and 91.26%of LPS-treated dendritic cells, respectively,which were up-regulated compared to untreated cells.Dendritic cells pulsed with exosome derived fromosteosarcoma cells caused significantly higher T cells stimulation and osteosarcoma cells inhibition as compared to un-pulsed dendritic cells.(P＜0.05).ConclusionT cells can inhibit the growth of osteosarcoma cells after being stimulated by exosome-pulsed dendritic cellsin vitro.

Exosome;Osteosarcoma;Dendritic cells;T cells;Inhibition

R979.6;R965

A

1004-0781(2014)07-0874-04

2013-09-01

2013-10-11

*遵義市科技計(jì)劃項(xiàng)目(遵市科合社字[2008] 4號(hào))

董革輝(1968-),男,貴州遵義人,副主任醫(yī)師,學(xué)士,主要從事骨腫瘤的研究。電話:0852-8925271,E-mail: 1468195302@qq.com。