NKG2D/RAE-1通路在介導(dǎo)NK/T細(xì)胞殺傷垂體瘤細(xì)胞中的作用及意義

孫其凱，馬立新，李珍珠，陳正，李澤福

(濱州醫(yī)學(xué)院附屬醫(yī)院，山東濱州256600)

NKG2D/RAE-1通路在介導(dǎo)NK/T細(xì)胞殺傷垂體瘤細(xì)胞中的作用及意義

孫其凱，馬立新，李珍珠，陳正，李澤福

(濱州醫(yī)學(xué)院附屬醫(yī)院，山東濱州256600)

目的 觀察NKG2D/RAE-1通路在介導(dǎo)自然殺傷(NK)細(xì)胞、CD8+T細(xì)胞(兩種細(xì)胞簡(jiǎn)稱NK/T細(xì)胞)殺傷垂體瘤細(xì)胞中的作用，從細(xì)胞學(xué)水平探討垂體瘤的免疫逃逸機(jī)制。方法 以小鼠垂體瘤細(xì)胞系A(chǔ)tT20為研究對(duì)象，采用免疫熒光標(biāo)記法檢測(cè)NKG2D配體RAE-1在垂體瘤細(xì)胞的表達(dá)水平及定位分布情況。采用NK/T細(xì)胞分選試劑盒分離NK/T細(xì)胞，將NK/T細(xì)胞放入Transwell小室的上室，靶細(xì)胞AtT20置入Transwell小室的下室，隨機(jī)分為對(duì)照組、實(shí)驗(yàn)組。對(duì)照組在常規(guī)條件下共培養(yǎng)0、4、8、24 h，實(shí)驗(yàn)組在培養(yǎng)液中加入阻斷RAE-1的抗體(Anti-RAE1 抗體)共培養(yǎng)0、4、8、24 h；采用MTT法檢測(cè)兩組各時(shí)間點(diǎn)細(xì)胞增殖抑制率。結(jié)果 RAE-1在AtT20細(xì)胞膜明顯表達(dá)，在細(xì)胞質(zhì)內(nèi)亦有弱表達(dá)。在AtT20細(xì)胞中RAE-1蛋白陽(yáng)性表達(dá)率為100%，其中強(qiáng)表達(dá)率為30%、中表達(dá)率為60%、弱表達(dá)率為10%，無(wú)陰性表達(dá)。對(duì)照組培養(yǎng)4、8、24 h時(shí)細(xì)胞增殖抑制率明顯高于0 h時(shí)(P均<0.05)；實(shí)驗(yàn)組培養(yǎng)0、4、8、24 h時(shí)增殖抑制率逐漸升高(P均<0.05)，培養(yǎng)8、24 h時(shí)增殖抑制率均明顯高于同時(shí)間點(diǎn)對(duì)照組(P均<0.05)。結(jié)論 NK/T細(xì)胞通過(guò)NKG2D/RAE-1通路殺滅垂體瘤細(xì)胞，RAE-1可負(fù)反饋?zhàn)饔糜贜K/T細(xì)胞，從而抑制免疫細(xì)胞對(duì)腫瘤細(xì)胞的殺傷活性。NKG2D/RAE-1通路可能作為垂體瘤的免疫治療靶點(diǎn)。

垂體瘤；NKG2D；RAE-1；自然殺傷細(xì)胞；CD8+T細(xì)胞

垂體瘤是目前臨床上最常見(jiàn)的鞍區(qū)腫瘤，患者預(yù)后較差；化療、放療和手術(shù)切除為其常用治療方法，但均不能明顯改善患者預(yù)后。免疫治療近年來(lái)應(yīng)用于腫瘤的治療，其通過(guò)影響自然殺傷(NK)細(xì)胞、T細(xì)胞等效應(yīng)細(xì)胞進(jìn)而殺滅腫瘤細(xì)胞[1,2]。尋找可靠的治療靶點(diǎn)是腫瘤免疫治療的關(guān)鍵[2]。NKG2D是一種Ⅱ型膜蛋白，屬于NK細(xì)胞特異性很高的活化性配體，在NK細(xì)胞、CD8+T細(xì)胞(兩種細(xì)胞簡(jiǎn)稱NK/T細(xì)胞)及其他免疫效應(yīng)細(xì)胞中均有表達(dá)[3]。MICA是人類免疫效應(yīng)細(xì)胞中能與NKG2D特異性結(jié)合的配體，是一種跨膜糖蛋白，具有高度糖基化[4]。目前已有研究在組織學(xué)水平證實(shí)NKG2D/MICA通路在機(jī)體抗腫瘤免疫中發(fā)揮重要作用，但尚無(wú)細(xì)胞水平的證據(jù)。小鼠細(xì)胞中RAE-1屬于NKG2D配體，可與NKG2D結(jié)合，具有類似MICA的免疫調(diào)節(jié)作用。2014年6月～2015年12月，本研究首次在細(xì)胞水平觀察NKG2D及其配體RAE-1在介導(dǎo)NK/T細(xì)胞殺傷垂體瘤細(xì)胞過(guò)程中的作用，探討垂體瘤的免疫逃逸機(jī)制。

1 材料與方法

1.1 材料 小鼠垂體瘤細(xì)胞系A(chǔ)tT20，購(gòu)自中國(guó)科學(xué)院上海細(xì)胞所。主要試劑：RAE-1抗體(Abcam)，熒光標(biāo)記二抗(武漢博士德生物工程有限公司)，NK/T細(xì)胞分選試劑盒、細(xì)胞磁珠分選試劑盒(德國(guó)美天旎公司)，Transwell小室(美國(guó)康寧公司)等。

1.2 AtT20細(xì)胞培養(yǎng)及RAE-1蛋白表達(dá)檢測(cè) 將小鼠垂體瘤細(xì)胞系A(chǔ)tT20置于F-12K培養(yǎng)基(含15%馬血清、2.5%胎牛血清)，于37 ℃、5% CO2環(huán)境下半貼壁培養(yǎng)，0.25%胰酶消化傳代。取第二代細(xì)胞常規(guī)條件下培養(yǎng)，采用免疫熒光標(biāo)記法檢測(cè)RAE-1蛋白表達(dá)：將半貼壁培養(yǎng)的細(xì)胞消化吹打成細(xì)胞懸液；取細(xì)胞懸液涂于載玻片上，滴加羊血清室溫封閉1 h，滴加RAE-1抗體(1∶1 000)4 ℃過(guò)夜，滴加熒光標(biāo)記二抗(1∶500)室溫孵育1 h，滴加DAPI標(biāo)記細(xì)胞核，封片，顯微鏡下觀察細(xì)胞。RAE-1蛋白熒光信號(hào)位于細(xì)胞膜和細(xì)胞質(zhì)。免疫熒光標(biāo)記的分級(jí)標(biāo)準(zhǔn)(以熒光強(qiáng)度分級(jí))：0級(jí)即陰性：高倍鏡下隱約可見(jiàn)熒光信號(hào)，低倍鏡下不顯示；1級(jí)即弱表達(dá)：高倍鏡下可見(jiàn)熒光信號(hào)，低倍鏡下隱約可見(jiàn)；2級(jí)即中表達(dá)：高倍鏡下清晰可見(jiàn)熒光信號(hào)，低倍鏡下亦可見(jiàn)；3級(jí)即強(qiáng)表達(dá)：高倍鏡下可見(jiàn)耀眼熒光信號(hào)，低倍鏡下清晰可見(jiàn)；4級(jí)即超強(qiáng)表達(dá)：高倍鏡及低倍鏡下均可見(jiàn)刺眼熒光信號(hào)。1～4級(jí)均為陽(yáng)性表達(dá)，3～4級(jí)為強(qiáng)表達(dá)。

1.3 NK/T細(xì)胞對(duì)AtT20細(xì)胞的殺傷活性檢測(cè) 采用Transwell共培養(yǎng)技術(shù)。采用NK/T細(xì)胞分選試劑盒，利用免疫磁珠負(fù)性篩選法分離NK/T細(xì)胞，并采用流式細(xì)胞儀進(jìn)行鑒定，使分離的細(xì)胞純度分別達(dá)到80%以上，步驟參照試劑盒說(shuō)明書。將NK/T細(xì)胞放入Transwell小室的上室，AtT20細(xì)胞(靶細(xì)胞置入Transwell小室的下室(效靶比20∶1，效應(yīng)細(xì)胞密度為2×106個(gè)/mL、靶細(xì)胞密度為1×105個(gè)/mL)；每批次樣本設(shè)置3個(gè)復(fù)孔，中間以0.4 μm孔徑聚碳酸酯膜隔開(kāi)，重復(fù)3次；隨機(jī)分為對(duì)照組、實(shí)驗(yàn)組，對(duì)照組在常規(guī)條件下共培養(yǎng)0、4、8、24 h，實(shí)驗(yàn)組在培養(yǎng)液中加入阻斷RAE-1的抗體(Anti-RAE1抗體)共培養(yǎng)0、4、8、24 h；采用MTT法分別檢測(cè)兩組NK/T細(xì)胞在540 nm波長(zhǎng)處的光密度(OD)值，計(jì)算細(xì)胞增殖抑制率。增殖抑制率(%)=(1-實(shí)驗(yàn)組OD值/對(duì)照組OD值)×100%。

2 結(jié)果

2.1AtT20細(xì)胞RAE-1蛋白表達(dá)RAE-1在AtT20細(xì)胞膜明顯表達(dá)，在細(xì)胞質(zhì)內(nèi)亦有弱表達(dá)。在AtT20細(xì)胞中RAE-1蛋白陽(yáng)性表達(dá)率為100%，其中強(qiáng)表達(dá)率為30%、中表達(dá)率為60%、弱表達(dá)率為10%，無(wú)陰性表達(dá)。見(jiàn)插頁(yè)Ⅰ圖1。

2.2NK/T細(xì)胞對(duì)AtT20細(xì)胞的殺傷活性 對(duì)照組培養(yǎng)4、8、24h時(shí)NK/T細(xì)胞增殖抑制率均較培養(yǎng)0h時(shí)增加(P均<0.05)，但培養(yǎng)4、8、24h時(shí)增殖抑制率比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P均>0.05)；實(shí)驗(yàn)組培養(yǎng)0、4、8、24h時(shí)增殖抑制率逐漸升高(P均<0.05)；實(shí)驗(yàn)組培養(yǎng)8、24h時(shí)增殖抑制率均明顯高于同時(shí)間點(diǎn)對(duì)照組(P均<0.05)。見(jiàn)表1。

3 討論

垂體瘤是鞍區(qū)最常見(jiàn)的良性腫瘤，在顱內(nèi)腫瘤中其發(fā)病率僅次于腦膠質(zhì)瘤和腦膜瘤， 垂體瘤可發(fā)生在任何年齡，并無(wú)明顯性別差異(除泌乳素腺瘤多見(jiàn)于女性外)[5]。目前，臨床主要采用腫瘤全切并輔以放療和化療的方法來(lái)治療垂體瘤，但侵襲性垂體瘤患者術(shù)后復(fù)發(fā)率較高的難題仍未解決[6]。

表1 兩組培養(yǎng)不同時(shí)間點(diǎn)NK/T細(xì)胞增殖抑制率比較

注：與對(duì)照組同時(shí)間點(diǎn)比較，*P<0.05；與同組0 h比較，△P<0.05；與同組4 h比較，#P<0.05；與同組8 h比較，▲P<0.05。

近年研究發(fā)現(xiàn)，腫瘤的發(fā)生、發(fā)展與機(jī)體免疫系統(tǒng)紊亂有關(guān)。NK細(xì)胞是一群大顆粒淋巴細(xì)胞，屬于一類與T、B淋巴細(xì)胞不同的大顆粒淋巴細(xì)胞[7]。NK細(xì)胞具有其他淋巴細(xì)胞無(wú)法比擬的優(yōu)點(diǎn)，即腫瘤殺傷活性(無(wú)需抗原致敏)[8]，可以快速、靈敏、直接殺傷腫瘤細(xì)胞[9，10]。目前利用NK細(xì)胞殺傷腫瘤細(xì)胞的方法正逐漸應(yīng)用于臨床，如IL-2激活NK細(xì)胞殺傷腫瘤，IL-2能夠增加NK細(xì)胞數(shù)量，提高NK細(xì)胞殺傷腫瘤細(xì)胞(轉(zhuǎn)移性腎細(xì)胞癌、多發(fā)性骨髓瘤)的活性。但是，NK細(xì)胞殺傷腫瘤細(xì)胞的具體機(jī)制及其信號(hào)通路尚不清楚。NKG2D是一種Ⅱ型膜蛋白，屬于C型凝集素樣受體家族，NKG2D能夠結(jié)合特異的轉(zhuǎn)接蛋白通過(guò)跨膜區(qū)域的帶電殘基完成信號(hào)轉(zhuǎn)導(dǎo)[11]；同時(shí)，NKG2D屬于NK細(xì)胞的特異性很高的活化性配體，在NK細(xì)胞、CD8+T細(xì)胞及其他免疫效應(yīng)細(xì)胞中均有表達(dá)；但在正常生理?xiàng)l件下，NKG2D在人和小鼠CD4+T細(xì)胞中不表達(dá)[12]。本研究選用NK細(xì)胞、CD8+T細(xì)胞用于共培養(yǎng)實(shí)驗(yàn)。

NKG2D可結(jié)合許多不同的配體，這些配體均屬于主要組織相容性復(fù)合體Ⅰ類(MHC-Ⅰ)相關(guān)蛋白，其中人NKG2D配體家族包括MICA和MICB。MICA是人類免疫效應(yīng)細(xì)胞中能與NKG2D特異性結(jié)合的配體，是一種跨膜糖蛋白，具有高度糖基化。目前已有研究在組織學(xué)水平證明NKG2D/MICA通路在機(jī)體抗腫瘤免疫中發(fā)揮重要作用。Chen等[13]發(fā)現(xiàn)，相對(duì)于正常腦組織，MICA蛋白在垂體瘤組織中高表達(dá)，提示MICA蛋白高表達(dá)可能參與垂體瘤的發(fā)生、發(fā)展；MICA與NKG2D受體特異性結(jié)合激活NK/T細(xì)胞等效應(yīng)細(xì)胞可能是其抑制腫瘤發(fā)生、發(fā)展的機(jī)制。

本研究以小鼠垂體瘤細(xì)胞系A(chǔ)tT20為研究對(duì)象，現(xiàn)已證實(shí)小鼠和人之間氨基酸序列同源性高達(dá)99%，因此該細(xì)胞系能夠充分替代人源垂體瘤細(xì)胞系[14]。但小鼠細(xì)胞缺少人類MICA同源基因，在其細(xì)胞中RAE-1基因充當(dāng)MICA基因的角色。RAE-1屬于NKG2D配體，可與NKG2D結(jié)合，并且具有類似MICA的免疫調(diào)節(jié)作用。鑒于RAE-1在AtT20細(xì)胞的表達(dá)情況及其對(duì)垂體瘤生物學(xué)行為的影響尚不清楚，本研究首先觀察RAE-1在小鼠細(xì)胞系A(chǔ)tT20中的表達(dá)情況，結(jié)果發(fā)現(xiàn)RAE-1在垂體瘤細(xì)胞膜上明顯表達(dá)，同時(shí)細(xì)胞質(zhì)內(nèi)亦有弱表達(dá)；提示RAE-1高表達(dá)有可能參與垂體瘤的發(fā)生、發(fā)展，該結(jié)論為下一步檢測(cè)NKG2D/RAE-1通路在NK/T細(xì)胞殺傷垂體瘤細(xì)胞中的作用提供了前提。本研究在阻斷RAE-1配體后NK/T細(xì)胞對(duì)垂體瘤細(xì)胞的殺傷活性明顯增加，提示NK/T細(xì)胞可能通過(guò)NKG2D/RAE-1通路殺滅垂體瘤細(xì)胞；阻斷RAE-1配體后NK/T細(xì)胞的殺傷活性較前明顯增加，提示RAE-1負(fù)反饋?zhàn)饔糜贜K/T細(xì)胞，進(jìn)而抑制免疫細(xì)胞對(duì)腫瘤細(xì)胞的殺傷活性。

有研究發(fā)現(xiàn)，在免疫力正常的動(dòng)物體內(nèi)表達(dá)NKG2D配體的腫瘤仍然能繼續(xù)發(fā)生、發(fā)展[15]，故腫瘤發(fā)生、發(fā)展過(guò)程中可能存在依賴NKG2D/配體通路的免疫逃逸。本研究從細(xì)胞水平證明RAE-1不僅可與NKG2D結(jié)合介導(dǎo)NK/T細(xì)胞殺傷腫瘤細(xì)胞，也可負(fù)反饋?zhàn)饔糜贜K/T細(xì)胞，降低NK/T細(xì)胞的殺傷腫瘤細(xì)胞活性，上述結(jié)果為研究垂體瘤免疫逃逸機(jī)制提供了依據(jù)。NKG2D配體(包括MICA和RAE-1)在機(jī)體內(nèi)有兩種存在形式，一種是存在于細(xì)胞膜表面的膜型配體，一種是活化脫落后被分泌到血液或腫瘤微環(huán)境中的分泌型配體。在大多數(shù)腫瘤，如骨肉瘤[16]、口腔鱗狀細(xì)胞癌[17]，雖然NKG2D配體明顯表達(dá)，但由于膜型配體脫落為分泌型配體，分泌型配體與NKG2D結(jié)合后使NKG2D發(fā)揮消極作用，使NK/T細(xì)胞殺傷腫瘤細(xì)胞的活性降低，促進(jìn)腫瘤細(xì)胞的免疫逃逸。

綜上所述，NK/T細(xì)胞通過(guò)NKG2D/RAE-1通路殺滅垂體瘤細(xì)胞，RAE-1可負(fù)反饋?zhàn)饔糜贜K/T細(xì)胞，從而抑制免疫細(xì)胞對(duì)腫瘤細(xì)胞的殺傷活性。NKG2D/RAE-1通路可能作為垂體瘤的免疫治療靶點(diǎn)。

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Effect and significance of NKG2D/RAE-1 pathway in the process of NK/T cells killing hypophysoma cells

SUNQikai,MALixin,LIZhenzhu,CHENZheng,LIZefu

(AffiliatedHospitalofBinzhouMedicalCollege,Binzhou256600,China)

Objective To observe the effect of NKG2D/RAE-1 pathway on mediating natural killer (NK) cell and CD8+T cell (both cells were referred to as NK/T cells) killing hypophysoma cells and to investigate the immune escape mechanism of hypophysoma from the cytological level. Methods The hypophysoma cell line AtT20 was selected. The immunofluorescent labeling method was used to detect the expression level of NKG2D ligand RAE-1, its position and distribution in the hypophysoma cells. NK/T cells were isolated by NK/T cell sorting kit, and NK/T cells were placed in the upper chamber of the Transwell chamber, the target cells AtT20 were placed in the lower chamber of the Transwell chamber and then were randomly divided into the control group and experimental group. The control group was co-cultured under normal conditions for 0, 4, 8, and 24 h. In the experimental group, the anti-RAE1 antibody was added to the culture medium, and the cells were co-cultured for 0, 4, 8, and 24 h. MTT assay was used to detect the inhibitory rate of cell proliferation at each time point. Results RAE-1 was clearly expressed in AtT20 cell membrane, and there was a weak expression in the cytoplasm. The positive expression rate of RAE-1 protein in AtT20 cells was 100%, among which the strong expression rate was 30% (15/50), the moderate expression rate was 60%, the weak expression rate was 10%, and there was no negative expression. In the control group, at 4, 8 and 24 h after culture, NK/T cell proliferation inhibition rate increased as compared with that at 0 h (allP<0.05). In the experimental group, the proliferation inhibition rate gradually increased at 0, 4, 8 and 24 h (allP<0.05), and at 8 and 24 h, the proliferation inhibition rates in the experimental group were significantly higher than those of the control group (allP<0.05). Conclusions NK/T cells kill the hypophysoma cells through NKG2D/ RAE-1 pathway, meanwhile, RAE-1 has a negative feedback effect on NK/T cells and consequently inhibits the killing activity of immune cells against the tumor cells. NKG2D/RAE1 pathway can be used as immunotherapy target for hypophysoma.

hypophysoma; NKG2D; RAE-1; natural killer cells; CD8+T cells

國(guó)家自然科學(xué)基金資助項(xiàng)目(81171119)。

孫其凱(1989-)，男，在讀碩士研究生，研究方向?yàn)榇贵w瘤的免疫治療。E-mail: sunqikai4453@163.com

簡(jiǎn)介：李澤福(1969-)，男，主任醫(yī)師，研究方向?yàn)榇贵w瘤的免疫治療。E-mail: lizefu163@163.com

10.3969/j.issn.1002-266X.2016.44.002

R736.4

A

1002-266X(2016)44-0004-04

2016-04-04)