重組ProTα對(duì)肝癌小鼠Treg細(xì)胞和NKG2D陽(yáng)性細(xì)胞的影響

陳　煒,周克夫,栗　華

(1.福建中醫(yī)藥大學(xué)研究生院,福建福州350108；2.廈門(mén)大學(xué)附屬第一醫(yī)院,福建廈門(mén)361005;3.廈門(mén)大學(xué)環(huán)境與生態(tài)學(xué)院,福建廈門(mén)361102)

·研究簡(jiǎn)報(bào)·

重組ProTα對(duì)肝癌小鼠Treg細(xì)胞和NKG2D陽(yáng)性細(xì)胞的影響

陳煒1,2,周克夫3,栗華2*

(1.福建中醫(yī)藥大學(xué)研究生院,福建福州350108；2.廈門(mén)大學(xué)附屬第一醫(yī)院,福建廈門(mén)361005;3.廈門(mén)大學(xué)環(huán)境與生態(tài)學(xué)院,福建廈門(mén)361102)

摘要：為觀察不同時(shí)期應(yīng)用重組胸腺素α原(ProTα)藥物干預(yù)下肝癌荷瘤小鼠的抑瘤率,并研究其對(duì)Treg細(xì)胞和NKG2D陽(yáng)性細(xì)胞的影響,將36只昆明小鼠隨機(jī)分成空白組、荷瘤組、藥物干預(yù)組,H22小鼠肝癌細(xì)胞皮下移植建立荷瘤小鼠模型,腹腔注射重組ProTα,觀察7和14 d后肝癌荷瘤小鼠的抑瘤率,并檢測(cè)外周單個(gè)核細(xì)胞中調(diào)節(jié)性T細(xì)胞(Treg細(xì)胞)和NKG2D陽(yáng)性細(xì)胞的比例．結(jié)果表明:移植瘤7 d后,藥物干預(yù)組和荷瘤組瘤質(zhì)量對(duì)比無(wú)顯著差異；而移植瘤14 d后,藥物干預(yù)組瘤質(zhì)量較荷瘤組有顯著減小．荷瘤14 d后,荷瘤組較空白組Treg細(xì)胞比例升高,NKG2D陽(yáng)性細(xì)胞比例下調(diào),差異顯著；而不論是早期藥物干預(yù)組還是進(jìn)展期(即移植瘤7 d后)藥物干預(yù)組,Treg細(xì)胞比例均顯著降低,NKG2D陽(yáng)性細(xì)胞顯著升高．由此可見(jiàn),重組ProTα能夠抑制肝癌荷瘤小鼠腫瘤生長(zhǎng),且早期、長(zhǎng)期用藥效果更好,相關(guān)作用機(jī)制涉及下調(diào)Treg細(xì)胞數(shù)量從而抑制肝癌細(xì)胞免疫逃逸,并上調(diào)NKG2D陽(yáng)性細(xì)胞數(shù)量從而提高其介導(dǎo)的抗腫瘤效應(yīng)．

關(guān)鍵詞：重組胸腺素α原;肝癌;調(diào)節(jié)性T細(xì)胞(Treg細(xì)胞);NKG2D

1995年,Sakaguchi等[1]發(fā)現(xiàn)回輸CD4+CD25+T細(xì)胞可以阻止裸鼠自身免疫疾病發(fā)生,因此將這一類(lèi)具有抑制機(jī)體免疫功能的細(xì)胞命名為調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg)．大量研究發(fā)現(xiàn),Treg細(xì)胞廣泛存在于各種腫瘤微環(huán)境中[2-4],并且與腫瘤的預(yù)后呈負(fù)相關(guān)[5]．張?zhí)O等[6]發(fā)現(xiàn)肝癌荷瘤小鼠脾臟、引流淋巴結(jié)中CD4+CD25+Treg細(xì)胞比例較對(duì)照組升高,且Treg細(xì)胞數(shù)量的多少與腫瘤大小呈正相關(guān)．NKG2D是自然殺傷(natural killer,NK)細(xì)胞的活化受體,除了NK細(xì)胞外,NKG2D還表達(dá)于活化的CD8+T細(xì)胞及γδT細(xì)胞．NKG2D與特異性配體結(jié)合,發(fā)揮免疫應(yīng)答和免疫監(jiān)視作用．目前已知的配體主要是組織相容性復(fù)合體(MHC)Ⅰ類(lèi)和UL16結(jié)合蛋白(ULBP)家族[7-8]；此外,組織相容性抗原60、視黃酸早期轉(zhuǎn)錄因子1也參與NKG2D的免疫調(diào)控[9]．NKG2D配體主要在受感染細(xì)胞和惡性腫瘤細(xì)胞表面表達(dá),健康細(xì)胞很少表達(dá)．在腫瘤細(xì)胞和效應(yīng)性T細(xì)胞表面,Groh等[10]發(fā)現(xiàn)NKG2D與可溶性配體MIC結(jié)合后發(fā)生內(nèi)化降解,導(dǎo)致效應(yīng)T細(xì)胞抗腫瘤作用減弱．Tomohiro等[11]發(fā)現(xiàn)在胃癌患者體內(nèi),CD8+T細(xì)胞表面NKG2D的表達(dá)顯著下調(diào),并且NKG2D的表達(dá)與癌組織的侵潤(rùn)深度呈負(fù)相關(guān)．

胸腺素α原(prothymosin α,ProTα)分布于哺乳動(dòng)物各種組織．內(nèi)源性的ProTα具有促進(jìn)細(xì)胞增殖的作用,并參與細(xì)胞凋亡和腫瘤生長(zhǎng)的調(diào)控．外源性ProTα具有較好的增強(qiáng)機(jī)體免疫和抗腫瘤作用,能夠促進(jìn)白介素-2(IL-2)、腫瘤壞死因子-α(TNF-α)、干擾素(IFN)的分泌,刺激腫瘤特異性T淋巴細(xì)胞的產(chǎn)生,發(fā)揮抗腫瘤作用[12-14]．我們前期的研究證實(shí)[15-16],重組ProTα對(duì)于荷瘤及癌性腹水小鼠腫瘤生長(zhǎng)和生存周期有較好的改善作用,但其對(duì)肝癌荷瘤小鼠Treg細(xì)胞和NKG2D表達(dá)的影響尚不清楚．本實(shí)驗(yàn)通過(guò)早期及進(jìn)展期藥物干預(yù),觀察重組ProTα對(duì)荷瘤小鼠抑瘤率的影響,并研究其對(duì)Treg細(xì)胞和NKG2D陽(yáng)性細(xì)胞是否具有調(diào)節(jié)作用．

1材料與方法

1.1材料

實(shí)驗(yàn)動(dòng)物為雄性昆明(KM)小鼠,5～6周,SPF級(jí),共36只,購(gòu)買(mǎi)于廈門(mén)大學(xué)實(shí)驗(yàn)動(dòng)物中心．小鼠H22肝癌細(xì)胞由廈門(mén)大學(xué)附屬第一醫(yī)院腫瘤實(shí)驗(yàn)室保種．

1.2主要試劑及儀器

重組ProTα[17]、Treg細(xì)胞流式染色試劑盒(美國(guó)eBioscience公司)、PE-antiCD314抗體(即NKG2D，美國(guó)eBioscience公司)、淋巴細(xì)胞分離液(天津?yàn)笥邢薰?、磷酸鹽緩沖液(PBS,實(shí)驗(yàn)室自配)、RPMI 1640培養(yǎng)基(美國(guó)HyClone公司)，流式細(xì)胞儀(美國(guó)BD公司，型號(hào):LSRFortessaTM)．

1.3實(shí)驗(yàn)方法

1.3.1移植瘤小鼠模型的構(gòu)建

取出H22細(xì)胞凍存管,37 ℃恒溫水浴鍋速融1～2 min,1 000 r/min離心,去上清,無(wú)菌PBS洗滌細(xì)胞,加入適量RPMI 1640培養(yǎng)基,吸打成懸液,取適量注射入小鼠體腔,飼育1周后脫脊處死.無(wú)菌條件下取小鼠腹水,加入適量培養(yǎng)基稀釋,1 000 r/min離心,去上清,PBS洗滌,加入適量培養(yǎng)基重懸計(jì)數(shù),細(xì)胞濃度約1×108mL-1．將36只KM雄性小鼠隨機(jī)分成3組,分為空白組(normal,N)6只、荷瘤組(tumor,T)12只、藥物干預(yù)組(drug,D)18只,其中T組和D組每只小鼠右小腿外側(cè)皮下注射0.1 mL細(xì)胞懸液,細(xì)胞數(shù)目約1×107．

1.3.2重組ProTα干預(yù)

將上述T組小鼠隨機(jī)分成2小組(T-7、T-14),各6只,分別于荷瘤7和14 d后脫脊處死；D組小鼠隨機(jī)分成3小組(D-7、D-14、D進(jìn)-7),各6只,其中D-7、D-14組小鼠于移植瘤第2天每只每天腹腔注射300 ng重組ProTα[15,17],并于藥物干預(yù)7和14 d后分別脫脊處死,而D進(jìn)-7組小鼠于移植瘤后第8天開(kāi)始每只每天腹腔注射同等劑量藥物,干預(yù)7 d后處死；N組小鼠每只每天腹腔注射等體積無(wú)菌生理鹽水,于14 d后處死．

1.3.3計(jì)算抑瘤率

無(wú)菌條件下剝離小鼠腫瘤,稱取小鼠瘤質(zhì)量(m),計(jì)算抑瘤率,比較小鼠腫瘤大小及抑瘤率．

1.3.4流式細(xì)胞術(shù)檢測(cè)Treg細(xì)胞及NKG2D陽(yáng)性細(xì)胞比例

無(wú)菌條件下分離小鼠脾臟,加入適量培養(yǎng)基研磨,200目篩網(wǎng)過(guò)濾,PBS洗滌3次,淋巴細(xì)胞分離液分離外周單個(gè)核淋巴細(xì)胞(PBMC),適量PBS重懸細(xì)胞,使細(xì)胞濃度約1×107mL-1.每個(gè)流式上樣管中加入100 μL準(zhǔn)備好的細(xì)胞懸液,細(xì)胞數(shù)目約1×106,根據(jù)Treg細(xì)胞流式染色試劑盒及NKG2D流式試劑說(shuō)明書(shū)加入相應(yīng)膜表面染色抗體,4 ℃避光孵育30 min,PBS洗滌細(xì)胞,另于Treg細(xì)胞標(biāo)記管中加入固定/破膜工作液,避光孵育30 min,PBS洗滌,加入核內(nèi)標(biāo)記抗體避光孵育30 min,PBS洗滌細(xì)胞后2 000 r/min離心去上清,加入適量PBS重懸細(xì)胞,上機(jī)檢測(cè)并分析．

1.3.5統(tǒng)計(jì)分析

實(shí)驗(yàn)數(shù)據(jù)用SPSS 18.0軟件分析,統(tǒng)計(jì)結(jié)果用平均值±標(biāo)準(zhǔn)差表示,組間比較采用配對(duì)或成組t-檢驗(yàn),p<0.05表示差異顯著．

2結(jié)果與分析

2.1藥物干預(yù)的抑瘤效果

T組及D組的腫瘤大小及瘤質(zhì)量如圖1和2所示．藥物干預(yù)7 d后,D-7組較T-7組的小鼠瘤質(zhì)量略有減小,計(jì)算抑瘤率為30.41%,但差異并不顯著(p=0.148 9)；而藥物干預(yù)14 d后,D-14組較T-14組的小鼠瘤質(zhì)量顯著減小(p=0.003 1),抑瘤率為54.59%．同T-14組小鼠相比,進(jìn)展期藥物干預(yù)(D進(jìn)-7)組也有顯著效果(p=0.018 4),抑瘤率為39.15%．

圖1　T組與D組的腫瘤大小Fig.1The tumor size of tumor-bearing group and drug intervention group

*p>0.05,**p<0.05，下同.圖2　T組與D組的瘤質(zhì)量Fig.2The tumor mass of tumor-bearing group and drug intervention group

2.2Treg細(xì)胞及NKG2D陽(yáng)性細(xì)胞比例變化

圖3　CD25+Foxp3+ Treg細(xì)胞占CD4+細(xì)胞比例Fig.3The proportion of CD25+Foxp3+Treg cells in CD4+ cells

運(yùn)用流式細(xì)胞術(shù)檢測(cè)CD25+Foxp3+Treg細(xì)胞占CD4+細(xì)胞比例,以及PBMC中NKG2D陽(yáng)性細(xì)胞的比例,結(jié)果分別如圖3和4所示．與N組小鼠對(duì)比,T-7及T-14組小鼠的Treg細(xì)胞比例有不同程度的升高(其中N組與T-7組相比p>0.05,而N組與T-14組相比p<0.05),而NKG2D陽(yáng)性細(xì)胞比例均顯著降低(p<0.05)．藥物干預(yù)7 d后,D-7組較T-7組Treg細(xì)胞比例略有降低,NKG2D陽(yáng)性細(xì)胞比例略有升高,但差異均不顯著(p>0.05)．而與T-14組小鼠相比，D-14組及D進(jìn)-7組小鼠的Treg細(xì)胞比例均顯著降低(p<0.05),NKG2D陽(yáng)性細(xì)胞比例顯著升高(p<0.05)．

圖4　NKG2D陽(yáng)性細(xì)胞占PBMC比例Fig.4The proportion of NKG2D-positive cells in PBMC

3討論

Treg細(xì)胞是促進(jìn)腫瘤免疫逃逸的重要細(xì)胞．Treg細(xì)胞通過(guò)識(shí)別免疫細(xì)胞表面的MHC,并經(jīng)T細(xì)胞受體(TCR)介導(dǎo)提呈,抑制CD4+和CD8+T細(xì)胞的活化和增殖．Foxp3是Treg細(xì)胞特異的轉(zhuǎn)錄因子,Treg細(xì)胞可以通過(guò)IL-2/STAT、轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)/Smad通路誘導(dǎo)Foxp3表達(dá),促進(jìn)腫瘤免疫逃逸[18-19]．此外,腺苷/前列腺素E2以及凋亡途徑Fas/Fasl也可能參與Treg細(xì)胞誘導(dǎo)的促腫瘤免疫逃逸[20-21]．Zhang等[22]發(fā)現(xiàn),利用Foxp3 siRNA轉(zhuǎn)染Treg細(xì)胞,通過(guò)下調(diào)Foxp3表達(dá),能夠顯著抑制Treg細(xì)胞增殖,呈現(xiàn)出明顯的抗腫瘤效應(yīng)．本研究發(fā)現(xiàn),T組小鼠Treg細(xì)胞比例較N組小鼠不同程度地升高,并且T-14組小鼠Treg細(xì)胞比例較T-7組小鼠升高,瘤質(zhì)量明顯增加,提示Treg細(xì)胞參與肝癌細(xì)胞免疫逃逸,促進(jìn)腫瘤生長(zhǎng),這與已有的研究結(jié)果是一致的[6]．而應(yīng)用重組ProTα干預(yù)后,小鼠Treg細(xì)胞比例降低,并且D-14組小鼠Treg細(xì)胞比例較D-7組小鼠進(jìn)一步降低,抑瘤率明顯增大,可見(jiàn),重組ProTα能夠通過(guò)抑制Treg細(xì)胞增殖,阻止腫瘤生長(zhǎng)．

NKG2D/NKG2DL通路在機(jī)體的免疫監(jiān)視中起重要作用．Sha等[23]對(duì)比原發(fā)性肝癌、乙型肝炎肝硬化、慢性乙型肝炎患者和健康人,發(fā)現(xiàn)肝癌患者中NKG2D的表達(dá)、NK細(xì)胞的活性明顯低于其他人群．Jiang 等[24]也發(fā)現(xiàn)原發(fā)性肝癌組織中NK細(xì)胞比例和NKG2D表達(dá)均明顯低于癌旁組織,并且與腫瘤的臨床分期呈負(fù)相關(guān)．在體內(nèi),腫瘤細(xì)胞通過(guò)多種途徑下調(diào)NKG2D表達(dá),逃脫免疫監(jiān)視和免疫清除．Clayton等[25]發(fā)現(xiàn)腫瘤細(xì)胞產(chǎn)生大量的TGF-β,通過(guò)降低NKG2D在CD8+T細(xì)胞及NK細(xì)胞的表達(dá),減弱NKG2D介導(dǎo)的抗腫瘤效應(yīng),而應(yīng)用TGF-β中和抗體可恢復(fù)NKG2D的表達(dá),呈現(xiàn)出顯著的抗腫瘤作用．在一項(xiàng)關(guān)于卵巢癌的研究中,Mathias等[26]發(fā)現(xiàn)不論是體內(nèi)還是體外實(shí)驗(yàn),巨噬細(xì)胞遷移抑制因子通過(guò)下調(diào)NKG2D表達(dá),可以抑制NK細(xì)胞對(duì)腫瘤細(xì)胞的殺傷作用,促進(jìn)腫瘤細(xì)胞的免疫逃逸．本研究中發(fā)現(xiàn),與N組小鼠相比,T組小鼠NKG2D陽(yáng)性細(xì)胞減少．同時(shí)我們觀察到通過(guò)腹腔注射重組ProTα,肝癌小鼠NKG2D陽(yáng)性細(xì)胞比例逐漸升高,且干預(yù)時(shí)間越長(zhǎng),NKG2D陽(yáng)性細(xì)胞升高越明顯,對(duì)腫瘤的抑制效果越顯著,但重組ProTα上調(diào)NKG2D陽(yáng)性細(xì)胞的作用機(jī)制尚不清楚．Ghiringhelli等[27]的研究表明,Treg細(xì)胞可以通過(guò)表達(dá)膜表面蛋白TGF-β,抑制NK細(xì)胞表面NKG2D的表達(dá),降低NK細(xì)胞的殺傷作用,降低機(jī)體的抗腫瘤作用．Smyth等[28]將Treg細(xì)胞轉(zhuǎn)輸至重組激活基因(RAG-1)缺陷的小鼠中,也發(fā)現(xiàn)Treg細(xì)胞能夠抑制NKG2D的表達(dá),降低NK細(xì)胞的抗腫瘤活性．本實(shí)驗(yàn)中是否存在相似的機(jī)制還需要深入研究．

綜上所述,本研究通過(guò)腹腔注射重組ProTα,觀察不同時(shí)期重組ProTα干預(yù)對(duì)移植瘤小鼠抑瘤率的影響,發(fā)現(xiàn)不論是腫瘤早期還是進(jìn)展期進(jìn)行藥物干預(yù),重組ProTα對(duì)腫瘤均有一定抑制作用,但早期、較長(zhǎng)時(shí)間應(yīng)用重組ProTα能夠明顯抑制腫瘤生長(zhǎng)，對(duì)于進(jìn)一步的臨床應(yīng)用具有指導(dǎo)意義．通過(guò)7和14 d取樣對(duì)比,在一定程度上動(dòng)態(tài)監(jiān)測(cè)了Treg細(xì)胞、NKG2D陽(yáng)性細(xì)胞的變化情況，發(fā)現(xiàn)腹腔注射重組ProTα 14 d后,D-14組小鼠Treg細(xì)胞比例較T-14組明顯降低,NKG2D陽(yáng)性細(xì)胞比例上調(diào),抗腫瘤效應(yīng)增加．以上研究結(jié)果提示,重組ProTα可通過(guò)下調(diào)Treg細(xì)胞比例而抑制肝癌細(xì)胞免疫逃逸,并上調(diào)NKG2D陽(yáng)性細(xì)胞比例而提高NKG2D介導(dǎo)的抗腫瘤效應(yīng),具體的信號(hào)通路需要后續(xù)的課題進(jìn)一步深入研究．

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Influence of Proportion of Treg Cells and NKG2D-positive Cells by Recombinant ProTα in Mice Bearing Liver Cancer

CHEN Wei1,2,ZHOU Kefu3,LI Hua2*

(1．Graduate School of Fujian University of Traditional Chinese Medicine,Fuzhou 350108,China;2．The First Affiliated Hospital of Xiamen University,Xiamen 361005,China;3．College of the Environment & Ecology, Xiamen University,Xiamen 361102,China)

Abstract:To observe the tumor inhibition rate by recombinant ProTα in liver cancer-bearing mice model,and to study the influence of the proportion of Treg cells and NKG2D-positive cells,36 Kunming mice were randomly divided into control group,tumor-bearing group and drug intervention group．Then a tumor-bearing mice model was established by transplanting H22 cells subcutaneously．By medicating recombinant ProTα through intraperitoneal injection,we observed the tumor inhibition rate after 7 days and 14 days,and detected the proportion of Treg cells and NKG2D-positive cells in PBMCs．Results showed that the difference in tumor mass between the tumor-bearing group and the drug intervention group was not significant after bearing tumor for 7 days．However,after bearing tumor for 14 days,the difference in tumor mass was significant．Additionally,the proportion of Treg cells increased and the number of NKG2D-positive cells declined significantly in the tumor-bearing group after bearing tumor for 14 days．No matter 14 days intraperitoneal injection of recombinant ProTα or drug intervention from the advanced stage,the proportion of Treg cells declined and the number of NKG2D-positive cells increased significantly．The results suggest that recombinant ProTα inhibits tumor growing,and the early and long-term drug intervention benefits the most．It is believed that the putative mechanism is related to that recombinant ProTα down-regulates the proportion of Treg cells,inhibiting the immune escape of hepatocellular carcinoma cells,and up-regulates the number of NKG2D-positive cells,improving the NKG2D-mediated anti-tumor effect.

Key words:recombinant ProTα;liver cancer;regulatory T cell (Treg cells);NKG2D

doi：10.6043/j.issn.0438-0479.2016.03.026

收稿日期：2015-12-16錄用日期：2016-02-16

基金項(xiàng)目：福建省自然科學(xué)基金(2013J01383)；福建省醫(yī)學(xué)創(chuàng)新課題(2009-CXB-51)

*通信作者：endohlihua@126.com

中圖分類(lèi)號(hào):R 735.7

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：0438-0479(2016)03-0456-05

引文格式：陳煒,周克夫,栗華.重組ProTα對(duì)肝癌小鼠Treg細(xì)胞和NKG2D陽(yáng)性細(xì)胞的影響.廈門(mén)大學(xué)學(xué)報(bào)(自然科學(xué)版)，2016,55(3)：456-460.

Citation：CHEN Wei,ZHOU Kefu,LI Hua．Influence of proportion of Treg cells and NKG2D-positive cells by recombinant ProTα in mice bearing liver cancer.Journal of Xiamen University(Natural Science)，2016,55(3)：456-460．(in Chinese)