易永祥　王　建　張　彤　王　翔　韓建波　趙　亮　張郁峰

(東南大學(xué)附屬南京市第二醫(yī)院普外科，南京210003)

[摘　要]　目的：通過檢測不同時(shí)期肝細(xì)胞癌患者外周血CD33+HLA-DR-MDSCs表達(dá)，分析其對肝細(xì)胞癌患者免疫功能的影響及臨床意義。方法：選擇肝細(xì)胞癌患者62例，并設(shè)置20名健康對照組，利用流式細(xì)胞學(xué)方法檢測外周血中CD33+HLA-DR-MDSCs的比例及Th1/Th2細(xì)胞免疫狀態(tài)，比較各組之間上述指標(biāo)有無差異。結(jié)果：與健康對照組成人相比，肝細(xì)胞癌組患者外周血中MDSCs的比例顯著增高，且進(jìn)展期肝癌組患者M(jìn)DSC的比例明顯高于早期肝癌組患者；肝細(xì)胞癌患者Th細(xì)胞向Th2漂移，與正常健康組比較具有統(tǒng)計(jì)學(xué)差異(P<0.05)。結(jié)論：肝細(xì)胞癌患者外周血CD33+HLA-DR-MDSCs含量升高，Th細(xì)胞向Th2漂移，免疫功能低下，CD33+HLA-DR-MDSCs可以作為監(jiān)測原發(fā)性患者的免疫功能和預(yù)后評估的有效指標(biāo)。

[關(guān)鍵詞]　肝癌；免疫功能；髓源抑制性細(xì)胞

doi:10.3969/j.issn.1000-484X.2015.11.017

肝細(xì)胞癌患者外周血CD33+HLA-DR-MDSCs的監(jiān)測及其臨床意義①

易永祥王建張彤②王翔②韓建波趙亮張郁峰

(東南大學(xué)附屬南京市第二醫(yī)院普外科，南京210003)

[摘要]目的：通過檢測不同時(shí)期肝細(xì)胞癌患者外周血CD33+HLA-DR-MDSCs表達(dá)，分析其對肝細(xì)胞癌患者免疫功能的影響及臨床意義。方法：選擇肝細(xì)胞癌患者62例，并設(shè)置20名健康對照組，利用流式細(xì)胞學(xué)方法檢測外周血中CD33+HLA-DR-MDSCs的比例及Th1/Th2細(xì)胞免疫狀態(tài)，比較各組之間上述指標(biāo)有無差異。結(jié)果：與健康對照組成人相比，肝細(xì)胞癌組患者外周血中MDSCs的比例顯著增高，且進(jìn)展期肝癌組患者M(jìn)DSC的比例明顯高于早期肝癌組患者；肝細(xì)胞癌患者Th細(xì)胞向Th2漂移，與正常健康組比較具有統(tǒng)計(jì)學(xué)差異(P<0.05)。結(jié)論：肝細(xì)胞癌患者外周血CD33+HLA-DR-MDSCs含量升高，Th細(xì)胞向Th2漂移，免疫功能低下，CD33+HLA-DR-MDSCs可以作為監(jiān)測原發(fā)性患者的免疫功能和預(yù)后評估的有效指標(biāo)。

[關(guān)鍵詞]肝癌；免疫功能；髓源抑制性細(xì)胞

doi:10.3969/j.issn.1000-484X.2015.11.017

中圖分類號①本文受“十二五”南京市醫(yī)學(xué)科技發(fā)展基金重點(diǎn)資助項(xiàng)目(ZDX12008)和江蘇省科技廳臨床醫(yī)學(xué)專項(xiàng)(BL2014005)資助。;②興化市人民醫(yī)院外科，興化225700。

作者簡介：易永祥(1966年-)，男，主任醫(yī)師，主要從事肝膽胰腫瘤研究。

[Abstract]Objective:The purpose of this study is to evaluate the frequency of MDSCs in peripheral blood of hepatocellular carcinoma patients and to investigate the clinical significance of change of MDSCs in the peripheral blood and provide new ways for evaluating immune state and prognosis of hepatocellular carcinoma patients.Methods: Blood samples were obtained from 62 patients with HCC and 20 healthy donors.The phenotype of CD3,CD4,CD33,HLA-DR and Th1，Th2 immune subsets in peripheral blood of each group were observed by FCM methods.Results: There were statically different frequencies in the peripheral blood between hepatocellular carcinoma and healthy control group,which the proportion of total CD3+T lymphocytes and CD3+CD4+T cells were lower and the proportion of CD33+HLA-DR-MDSCs was higher in hepatocellular carcinoma patients.(P<0.05).The increase of percentage of MDSCs was greater in patients at Stage C and D than in patients at stage A and B.Conclusion: The Th1/ Th2 ratio in the PBMC were of imbalance and MDSCs was significantly increased in peripheral blood of hepatocellular carcinoma patients.The increase of MDSCs was significantly correlated with clinical stage.CD33+HLA-DR-MDSCs may play an important role in prediction in prognosis and tumor immune status of hepatocellular carcinoma.

Study on detection and clinical significance of CD33+HLA-DR-MDSCs cells in hepatocellular carcinoma

YIYong-Xiang,WANGJian,ZHUANGTong,WANGXiang,HANJian-Bo,ZHAOLiang,ZHANGYu-Feng.DepartmentofGeneralSurgerytheSecondHospitalofNanjing,AffiliatedtoSoutheastUniversity,Nanjing210003,China

[Key words]Hepatocellular carcinoma;Immune function;Myeloid derived suppressor cells

肝細(xì)胞癌(Hepatocellular carcinoma,HCC)是最常見的惡性腫瘤，其臨床治療預(yù)后較差[1,2]。原發(fā)性肝癌在體內(nèi)的發(fā)生、發(fā)展與機(jī)體免疫功能受抑制和紊亂有關(guān)，同時(shí)腫瘤細(xì)胞也可以通過分泌細(xì)胞因子而引起機(jī)體免疫抑制[3]。MDSCs被證實(shí)是一類通過抑制機(jī)體免疫功能促進(jìn)腫瘤生長的免疫抑制細(xì)胞，其在外周血中的水平與疾病的進(jìn)展程度和腫瘤分期有關(guān)[4,5]。通過降低腫瘤患者體內(nèi)MDSCs的水平，改善腫瘤患者的免疫抑制狀態(tài)已被作為抗腫瘤藥物的開發(fā)靶點(diǎn)[6,7]。本研究通過檢測62例肝癌患者外周血Th細(xì)胞的漂移狀態(tài)評估機(jī)體的免疫功能，并追蹤其疾病惡化過程的關(guān)系，同時(shí)監(jiān)測外周血CD33+HLA-DR-MDSCs含量變化與肝癌患者免疫功能的關(guān)系，以期探討CD33+HLA-DR-MDSCs在肝癌免疫功能監(jiān)測的意義。

1資料與方法

1.1一般資料納入2012年5月～2014 年9月間在我院住院的HCC患者62例，其中男性48例，女性14例，年齡32~69歲，平均年齡(58.78±10.11)歲，診斷參照《原發(fā)性肝癌臨床診斷標(biāo)準(zhǔn)》；巴塞羅那臨床肝癌(BCLC)分期，A 期患者11例、B 期患者13例、C 期患者28例、D 期患者10例；本研究取血樣獲得本院倫理委員會批準(zhǔn)，并簽署知情同意書。排除合并其他惡性腫瘤及免疫性疾病的患者；排除嚴(yán)重精神障礙患者；排除合并神經(jīng)系統(tǒng)疾病。

1.2方法

1.2.1外周血Th1細(xì)胞和Th2細(xì)胞分析分離外周血單個(gè)核細(xì)胞，加入20 ng/ml PMA和500 ng/ml離子霉素刺激4 h后，收集細(xì)胞。將細(xì)胞分為兩份，稱為對照管和實(shí)驗(yàn)管，分別加入CD3-APC，CD8-PercP 10 μl，混勻，室溫放置20 min，加入紅細(xì)胞裂解液1 ml，混勻，室溫放置10 min，2 789 r/min離心5 min，棄上清。加入Cytofix/Cytoperm液200 μl，4℃放置20 min，使細(xì)胞固定穿孔。加入Perm/Wash液1 ml，3 416 r/min離心5 min，棄上清。實(shí)驗(yàn)管加入IFN-γ-FITC、IL-4-PE各5 μl，對照管加入同型對照抗體，4℃避光孵育30 min。加入Perm/Wash液500 μl洗滌兩次。最后以300 μl洗滌液重懸細(xì)胞。采用EXPO32 ADC軟件及CD3-APC、CD4-PercP 、IFN-γ-FITC和IL4-PE抗體單雙標(biāo)記的淋巴細(xì)胞調(diào)節(jié)儀器的補(bǔ)償，以前向角(FSC)和側(cè)向角(SSC)散射光散點(diǎn)圖設(shè)門區(qū)分淋巴細(xì)胞，以CD3和CD4散點(diǎn)圖設(shè)門區(qū)分Th細(xì)胞(CD3+CD4-)。最終以IFN-γ和IL-4的散點(diǎn)圖表示Th1細(xì)胞和Th2細(xì)胞。

1.2.2外周血CD33±HLA-DR-MDSCs檢測分離外周血單個(gè)核細(xì)胞， 加入APC-HLA-DR抗體和FITC-CD33抗體，4℃避光孵育30 min。加入PBS液500 μl洗滌兩次。最后以300 μl洗滌液重懸細(xì)胞。采用EXPO32 ADC軟件分析CD33+HLA-DR-MDSCs所占的比例，并分析其與臨床分期的相關(guān)性。

1.3統(tǒng)計(jì)分析采用SPSS 13.0統(tǒng)計(jì)軟件，計(jì)量資料數(shù)據(jù)以±s表示，兩組間的比較采用LSD-t檢驗(yàn)，資料多組間比較采用單因素方差分析(ANOVA)檢驗(yàn)，P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1外周血輔助Th細(xì)胞Th1/Th2分化檢測結(jié)果正常健康人群Th1細(xì)胞占輔助Th細(xì)胞的(18.24±5.26)%，Th2細(xì)胞占(5.35±2.26)%，其比值(3.40±1.25)；而HCC患者Th細(xì)胞向Th2漂移，Th1細(xì)胞為(9.65±4.12)%，Th2細(xì)胞為(7.49±2.18)%，Th1/ Th2其比值(1.28±0.86)，P<0.05，肝癌患者相比于健康人，外周循環(huán)中輔助性T 細(xì)胞中的Th1型細(xì)胞明顯減低，Th2 型細(xì)胞明顯增高；在HCC的BCLC分期中，各期Th1/Th2比值分別為A期(2.03±1.12)，B期(1.50±0.89)，C期(1.06±0.68) 和D期(0.88±0.54)，C和D期患者外周血Th細(xì)胞向Th2漂移更為顯著，隨著病情的進(jìn)展，機(jī)體免疫功能狀態(tài)進(jìn)一步惡化(見圖1、表1) 。

2.2CD33+HLA-DR-MDSCs在外周血中的比例肝癌患者外周血CD33+HLA-DR-MDSCs細(xì)胞在單個(gè)核細(xì)胞中所占的比例為(2.25±0.41)%，明顯高于健康志愿者(1.38±0.29)%，P=0.009<0.05；在HCC的BCLC分期中，C和D期患者外周血CD33+-HLA-DR-MDSCs細(xì)胞在單個(gè)核細(xì)胞中所占的比例分別為(2.43±0.39)%和(2.50±0.48)%，顯著高于A和B期患者外周血CD33+HLA-DR-MDSCs細(xì)胞在單個(gè)核細(xì)胞中所占的比例(1.82±0.21)%和(2.01±0.28)%，P=0.007<0.05。隨著病情的進(jìn)展，CD33+HLA-DR-MDSCs在外周血的比例增高(見圖2、3) 。

圖1　流式細(xì)胞儀檢測肝癌患者外周血細(xì)胞Th1、Th2免疫表型Fig.1　Immunophenotype of Th1 and Th2 in peripheral blood of patients of hepatocellular carcinoma by flow cytometry

表1　不同BCLC分期肝癌患者的Th1和Th2亞群分布情況(±s)Tab.1　Th1 and Th2 subsets in patients with different BCLC stage in HCC patients(±s)

表1　不同BCLC分期肝癌患者的Th1和Th2亞群分布情況(±s)Tab.1　Th1 and Th2 subsets in patients with different BCLC stage in HCC patients(±s)

GroupsnCD3+(%)CD3+CD4+(%)Th1(%)Th2(%)Th1/Th2Healthyvolunteers2070.21±6.6946.56±5.6918.24±5.265.35±2.263.40±1.25P=0.001<0.5HCCpatients6256.15±6.2726.98±5.079.65±4.127.49±2.181.28±0.86BCLCstageAstage1159.61±5.2528.14±6.0212.24±4.536.02±1.862.03±1.12P=0.0117<0.5Bstage1358.15±5.2526.87±5.1410.29±4.376.84±1.951.50±0.89Cstage2855.25±4.8924.17±5.028.64±4.038.12±2.051.06±0.68Dstage852.16±4.6422.12±4.927.56±3.938.95±2.360.88±0.54

圖2　流式細(xì)胞儀檢測血液循環(huán)中MDSCs免疫表型Fig.2　 Immunophenotype of MDSCs in circulating blood measured by flow cytometry

圖3　肝癌患者血液系統(tǒng) CD33+HLA-DR-MDSCs細(xì)胞分布情況Fig.3　Circulating CD33+HLA-DR-MDSCs cells in HCC patients

3討論

世界癌癥報(bào)告顯示，全世界肝細(xì)胞癌(HCC)每年新發(fā)病例50萬～100萬，總體病死率95%，肝癌已經(jīng)成為當(dāng)前社會主要的健康負(fù)擔(dān)[8]。腫瘤細(xì)胞可以通過形成多種不同的機(jī)制來逃避宿主的免疫系統(tǒng)對其免疫監(jiān)視和免疫攻擊，創(chuàng)造出一個(gè)免疫抑制性的微環(huán)境，從而促進(jìn)腫瘤的增殖、轉(zhuǎn)移和影響腫瘤治療的效果[9,10]。T 淋巴細(xì)胞介導(dǎo)的細(xì)胞免疫在機(jī)體預(yù)防和排斥腫瘤方面起重要作用。本研究通過對肝細(xì)胞癌患者外周血免疫功能的分析發(fā)現(xiàn)，肝細(xì)胞癌患者外周血總T細(xì)胞比例為(56.15±6.27)%，顯著低于正常健康組(70.21±6.69)%；CD3+CD4+T細(xì)胞在調(diào)控或“輔助”其他淋巴細(xì)胞發(fā)揮著重要的調(diào)控作用，是調(diào)節(jié)CD8+T細(xì)胞殺傷能力的主要因素,而Th1、Th2平衡是其效應(yīng)之一，尤其是其向Th1和Th2的分化情況更能夠體現(xiàn)機(jī)體的免疫功能狀態(tài)[11,12]。我們通過檢測發(fā)現(xiàn)，肝細(xì)胞癌患者Th細(xì)胞向Th2漂移，Th1細(xì)胞為(9.65±4.12)%，Th2細(xì)胞為(7.49±2.18)%，Th1/Th2比值為(1.28±0.86)，顯著低于正常健康人群P<0.05；進(jìn)一步對HCC的BCLC分期中各期Th1/Th2比值分析發(fā)現(xiàn)，隨著病情的進(jìn)展，Th細(xì)胞向Th2漂移更為顯著，C期和D期Th1/Th2比值分別為(1.06±0.68)和 (0.88±0.54)，顯著高于A期和B期。

MDSCs是來源于髓系祖細(xì)胞和未成熟髓細(xì)胞，是機(jī)體免疫反應(yīng)的一群獨(dú)特的細(xì)胞群體，腫瘤細(xì)胞自分泌的一些因子如IL-6和VEGF細(xì)胞因子等被認(rèn)為是刺激MDSCs擴(kuò)增和激活的重要因素，MDSCs可通過促進(jìn)血管生成和抑制免疫應(yīng)答促進(jìn)腫瘤生長[13,14]。研究發(fā)現(xiàn)，MDSCs通過生成的活性氧族和活性氮族成分影響T細(xì)胞功能，同時(shí)通過耗竭微環(huán)境中精氨酸和L-半胱氨酸抑制T細(xì)胞激活與增殖[15,16]。我們的研究發(fā)現(xiàn)，肝癌患者外周血CD33+-HLA-DR-MDSCs細(xì)胞在單個(gè)核細(xì)胞中所占的比例為(2.25±0.41)%，明顯高于健康志愿者(1.38±0.29)%，P<0.05；在腫瘤分期上，隨著分期級別的升高，MDSCs的比例與呈上升趨勢，尤其表現(xiàn)為C和D期患者外周血CD33+HLA-DR-MDSCs細(xì)胞在單個(gè)核細(xì)胞中所占的比例分別為(2.43±0.39)%和(2.50±0.48)%，顯著高于A和B期患者外周血CD33+HLA-DR-MDSCs細(xì)胞在單個(gè)核細(xì)胞中所占的比例(1.82±0.21)%和(2.01±0.28)%，P<0.05，表明隨著MDSCs的升高，免疫系統(tǒng)受抑制程度加重，這與文獻(xiàn)報(bào)道中MDSCs與腫瘤患者腫瘤負(fù)荷、有無轉(zhuǎn)移及臨床分期的有關(guān)結(jié)論相一致。結(jié)合我們對肝細(xì)胞癌患者T細(xì)胞和Th細(xì)胞的檢測我們發(fā)現(xiàn)，MDSCs比例變化趨勢與肝癌患者外周血總T細(xì)胞比例變化密切相關(guān)，說明MDSCs可能是影響肝癌患者T細(xì)胞免疫功能的重要原因，MDSCs還可以直接影響CD4+T細(xì)胞的功能狀態(tài)，這可能是導(dǎo)致肝細(xì)胞癌患者Th2細(xì)胞占優(yōu)勢的原因之一。

綜上所述，MDSC在HCC的外周血大量擴(kuò)增，其通過各種途徑抑制免疫系統(tǒng)的活性，促進(jìn)肝癌細(xì)胞的免疫逃逸。進(jìn)一步探索和研究MDSCs生物學(xué)特性和免疫調(diào)控機(jī)制，將有助于通過監(jiān)測MDSCs的數(shù)量和功能來評價(jià)機(jī)體的免疫功能和逆轉(zhuǎn)MDSC的免疫抑制作用，增強(qiáng)腫瘤免疫治療療效。

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[收稿2015-07-23修回2015-08-18]

(編輯張曉舟)