陳　煒,周克夫,栗　華

(1.福建中醫(yī)藥大學研究生院,福建福州350108；2.廈門大學附屬第一醫(yī)院,福建廈門361005;3.廈門大學環(huán)境與生態(tài)學院,福建廈門361102)

·研究簡報·

重組ProTα對肝癌小鼠Treg細胞和NKG2D陽性細胞的影響

陳煒1,2,周克夫3,栗華2*

(1.福建中醫(yī)藥大學研究生院,福建福州350108；2.廈門大學附屬第一醫(yī)院,福建廈門361005;3.廈門大學環(huán)境與生態(tài)學院,福建廈門361102)

摘要：為觀察不同時期應用重組胸腺素α原(ProTα)藥物干預下肝癌荷瘤小鼠的抑瘤率,并研究其對Treg細胞和NKG2D陽性細胞的影響,將36只昆明小鼠隨機分成空白組、荷瘤組、藥物干預組,H22小鼠肝癌細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,并檢測外周單個核細胞中調節(jié)性T細胞(Treg細胞)和NKG2D陽性細胞的比例．結果表明:移植瘤7 d后,藥物干預組和荷瘤組瘤質量對比無顯著差異；而移植瘤14 d后,藥物干預組瘤質量較荷瘤組有顯著減?。闪?4 d后,荷瘤組較空白組Treg細胞比例升高,NKG2D陽性細胞比例下調,差異顯著；而不論是早期藥物干預組還是進展期(即移植瘤7 d后)藥物干預組,Treg細胞比例均顯著降低,NKG2D陽性細胞顯著升高．由此可見,重組ProTα能夠抑制肝癌荷瘤小鼠腫瘤生長,且早期、長期用藥效果更好,相關作用機制涉及下調Treg細胞數(shù)量從而抑制肝癌細胞免疫逃逸,并上調NKG2D陽性細胞數(shù)量從而提高其介導的抗腫瘤效應．

關鍵詞：重組胸腺素α原;肝癌;調節(jié)性T細胞(Treg細胞);NKG2D

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

胸腺素α原(prothymosin α,ProTα)分布于哺乳動物各種組織．內源性的ProTα具有促進細胞增殖的作用,并參與細胞凋亡和腫瘤生長的調控．外源性ProTα具有較好的增強機體免疫和抗腫瘤作用,能夠促進白介素-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淋巴細胞的產生,發(fā)揮抗腫瘤作用[12-14]．我們前期的研究證實[15-16],重組ProTα對于荷瘤及癌性腹水小鼠腫瘤生長和生存周期有較好的改善作用,但其對肝癌荷瘤小鼠Treg細胞和NKG2D表達的影響尚不清楚．本實驗通過早期及進展期藥物干預,觀察重組ProTα對荷瘤小鼠抑瘤率的影響,并研究其對Treg細胞和NKG2D陽性細胞是否具有調節(jié)作用．

1材料與方法

1.1材料

實驗動物為雄性昆明(KM)小鼠,5～6周,SPF級,共36只,購買于廈門大學實驗動物中心．小鼠H22肝癌細胞由廈門大學附屬第一醫(yī)院腫瘤實驗室保種．

1.2主要試劑及儀器

重組ProTα[17]、Treg細胞流式染色試劑盒(美國eBioscience公司)、PE-antiCD314抗體(即NKG2D，美國eBioscience公司)、淋巴細胞分離液(天津灝洋有限公司)、磷酸鹽緩沖液(PBS,實驗室自配)、RPMI 1640培養(yǎng)基(美國HyClone公司)，流式細胞儀(美國BD公司，型號:LSRFortessaTM)．

1.3實驗方法

1.3.1移植瘤小鼠模型的構建

取出H22細胞凍存管,37 ℃恒溫水浴鍋速融1～2 min,1 000 r/min離心,去上清,無菌PBS洗滌細胞,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)基重懸計數(shù),細胞濃度約1×108mL-1．將36只KM雄性小鼠隨機分成3組,分為空白組(normal,N)6只、荷瘤組(tumor,T)12只、藥物干預組(drug,D)18只,其中T組和D組每只小鼠右小腿外側皮下注射0.1 mL細胞懸液,細胞數(shù)目約1×107．

1.3.2重組ProTα干預

將上述T組小鼠隨機分成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組小鼠隨機分成3小組(D-7、D-14、D進-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7和14 d后分別脫脊處死,而D進-7組小鼠于移植瘤后第8天開始每只每天腹腔注射同等劑量藥物,干預7 d后處死；N組小鼠每只每天腹腔注射等體積無菌生理鹽水,于14 d后處死．

1.3.3計算抑瘤率

無菌條件下剝離小鼠腫瘤,稱取小鼠瘤質量(m),計算抑瘤率,比較小鼠腫瘤大小及抑瘤率．

1.3.4流式細胞術檢測Treg細胞及NKG2D陽性細胞比例

無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)過濾,PBS洗滌3次,淋巴細胞分離液分離外周單個核淋巴細胞(PBMC),適量PBS重懸細胞,使細胞濃度約1×107mL-1.每個流式上樣管中加入100 μL準備好的細胞懸液,細胞數(shù)目約1×106,根據(jù)Treg細胞流式染色試劑盒及NKG2D流式試劑說明書加入相應膜表面染色抗體,4 ℃避光孵育30 min,PBS洗滌細胞,另于Treg細胞標記管中加入固定/破膜工作液,避光孵育30 min,PBS洗滌,加入核內標記抗體避光孵育30 min,PBS洗滌細胞后2 000 r/min離心去上清,加入適量PBS重懸細胞,上機檢測并分析．

1.3.5統(tǒng)計分析

實驗數(shù)據(jù)用SPSS 18.0軟件分析,統(tǒng)計結果用平均值±標準差表示,組間比較采用配對或成組t-檢驗,p<0.05表示差異顯著．

2結果與分析

2.1藥物干預的抑瘤效果

T組及D組的腫瘤大小及瘤質量如圖1和2所示．藥物干預7 d后,D-7組較T-7組的小鼠瘤質量略有減小,計算抑瘤率為30.41%,但差異并不顯著(p=0.148 9)；而藥物干預14 d后,D-14組較T-14組的小鼠瘤質量顯著減小(p=0.003 1),抑瘤率為54.59%．同T-14組小鼠相比,進展期藥物干預(D進-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組的瘤質量Fig.2The tumor mass of tumor-bearing group and drug intervention group

2.2Treg細胞及NKG2D陽性細胞比例變化

圖3　CD25+Foxp3+ Treg細胞占CD4+細胞比例Fig.3The proportion of CD25+Foxp3+Treg cells in CD4+ cells

運用流式細胞術檢測CD25+Foxp3+Treg細胞占CD4+細胞比例,以及PBMC中NKG2D陽性細胞的比例,結果分別如圖3和4所示．與N組小鼠對比,T-7及T-14組小鼠的Treg細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陽性細胞比例均顯著降低(p<0.05)．藥物干預7 d后,D-7組較T-7組Treg細胞比例略有降低,NKG2D陽性細胞比例略有升高,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-7組小鼠的Treg細胞比例均顯著降低(p<0.05),NKG2D陽性細胞比例顯著升高(p<0.05)．

圖4　NKG2D陽性細胞占PBMC比例Fig.4The proportion of NKG2D-positive cells in PBMC

3討論

Treg細胞是促進腫瘤免疫逃逸的重要細胞．Treg細胞通過識別免疫細胞表面的MHC,并經(jīng)T細胞受體(TCR)介導提呈,抑制CD4+和CD8+T細胞的活化和增殖．Foxp3是Treg細胞特異的轉錄因子,Treg細胞可以通過IL-2/STAT、轉化生長因子-β(TGF-β)/Smad通路誘導Foxp3表達,促進腫瘤免疫逃逸[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細胞誘導的促腫瘤免疫逃逸[20-21]．Zhang等[22]發(fā)現(xiàn),利用Foxp3 siRNA轉染Treg細胞,通過下調Foxp3表達,能夠顯著抑制Treg細胞增殖,呈現(xiàn)出明顯的抗腫瘤效應．本研究發(fā)現(xiàn),T組小鼠Treg細胞比例較N組小鼠不同程度地升高,并且T-14組小鼠Treg細胞比例較T-7組小鼠升高,瘤質量明顯增加,提示Treg細胞參與肝癌細胞免疫逃逸,促進腫瘤生長,這與已有的研究結果是一致的[6]．而應用重組ProTα干預后,小鼠Treg細胞比例降低,并且D-14組小鼠Treg細胞比例較D-7組小鼠進一步降低,抑瘤率明顯增大,可見,重組ProTα能夠通過抑制Treg細胞增殖,阻止腫瘤生長．

NKG2D/NKG2DL通路在機體的免疫監(jiān)視中起重要作用．Sha等[23]對比原發(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、NK細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細胞比例和NKG2D表達均明顯低于癌旁組織,并且與腫瘤的臨床分期呈負相關．在體內,腫瘤細胞通過多種途徑下調NKG2D表達,逃脫免疫監(jiān)視和免疫清除．Clayton等[25]發(fā)現(xiàn)腫瘤細胞產生大量的TGF-β,通過降低NKG2D在CD8+T細胞及NK細胞的表達,減弱NKG2D介導的抗腫瘤效應,而應用TGF-β中和抗體可恢復NKG2D的表達,呈現(xiàn)出顯著的抗腫瘤作用．在一項關于卵巢癌的研究中,Mathias等[26]發(fā)現(xiàn)不論是體內還是體外實驗,巨噬細胞遷移抑制因子通過下調NKG2D表達,可以抑制NK細胞對腫瘤細胞的殺傷作用,促進腫瘤細胞的免疫逃逸．本研究中發(fā)現(xiàn),與N組小鼠相比,T組小鼠NKG2D陽性細胞減少．同時我們觀察到通過腹腔注射重組ProTα,肝癌小鼠NKG2D陽性細胞比例逐漸升高,且干預時間越長,NKG2D陽性細胞升高越明顯,對腫瘤的抑制效果越顯著,但重組ProTα上調NKG2D陽性細胞的作用機制尚不清楚．Ghiringhelli等[27]的研究表明,Treg細胞可以通過表達膜表面蛋白TGF-β,抑制NK細胞表面NKG2D的表達,降低NK細胞的殺傷作用,降低機體的抗腫瘤作用．Smyth等[28]將Treg細胞轉輸至重組激活基因(RAG-1)缺陷的小鼠中,也發(fā)現(xiàn)Treg細胞能夠抑制NKG2D的表達,降低NK細胞的抗腫瘤活性．本實驗中是否存在相似的機制還需要深入研究．

綜上所述,本研究通過腹腔注射重組ProTα,觀察不同時期重組ProTα干預對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展期進行藥物干預,重組ProTα對腫瘤均有一定抑制作用,但早期、較長時間應用重組ProTα能夠明顯抑制腫瘤生長，對于進一步的臨床應用具有指導意義．通過7和14 d取樣對比,在一定程度上動態(tài)監(jiān)測了Treg細胞、NKG2D陽性細胞的變化情況，發(fā)現(xiàn)腹腔注射重組ProTα 14 d后,D-14組小鼠Treg細胞比例較T-14組明顯降低,NKG2D陽性細胞比例上調,抗腫瘤效應增加．以上研究結果提示,重組ProTα可通過下調Treg細胞比例而抑制肝癌細胞免疫逃逸,并上調NKG2D陽性細胞比例而提高NKG2D介導的抗腫瘤效應,具體的信號通路需要后續(xù)的課題進一步深入研究．

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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

基金項目：福建省自然科學基金(2013J01383)；福建省醫(yī)學創(chuàng)新課題(2009-CXB-51)

*通信作者：endohlihua@126.com

中圖分類號:R 735.7

文獻標志碼：A

文章編號：0438-0479(2016)03-0456-05

引文格式：陳煒,周克夫,栗華.重組ProTα對肝癌小鼠Treg細胞和NKG2D陽性細胞的影響.廈門大學學報(自然科學版)，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)