屈曉晶 路遙 曹衛(wèi)華 張英 冉崇平 齊天林 胡蕾蘋 郝紅曉 張璐 李明慧 謝堯

100015 北京，首都醫(yī)科大學(xué)附屬北京地壇醫(yī)院肝病中心肝病二科

·病毒病診斷與治療·

慢性乙型肝炎患者NK細(xì)胞功能與干擾素療效相關(guān)性研究

屈曉晶 路遙 曹衛(wèi)華 張英 冉崇平 齊天林 胡蕾蘋 郝紅曉 張璐 李明慧 謝堯

100015 北京，首都醫(yī)科大學(xué)附屬北京地壇醫(yī)院肝病中心肝病二科

目的探討慢性乙型肝炎(Chronic hepatitis B, CHB)患者外周血NK細(xì)胞頻數(shù)及功能分子與干擾素α(Interferon-alpha, IFNα)療效的相關(guān)性。方法采集乙型肝炎病毒(Hepatitis B virus, HBV)慢性感染免疫清除(Immune clearance, IC)期經(jīng)聚乙二醇干擾素α-2a(Peg-IFNα-2a)治療患者在基線及治療12和24周靜脈全血，采用流式細(xì)胞儀檢測(cè)外周血CD3-CD56+NK細(xì)胞、CD56brightNK細(xì)胞和CD56dimNK細(xì)胞頻數(shù)及NK細(xì)胞表面受體IFNAR2和NKp46的表達(dá)。血漿HBV DNA、HBsAg、HBeAg含量及肝功能由北京地壇醫(yī)院檢驗(yàn)科提供。結(jié)果本實(shí)驗(yàn)共入組41例IC期經(jīng)Peg-IFNα-2a治療患者，包括21例應(yīng)答不佳 (Poor response, PG)患者和20例應(yīng)答(Good response, GR)患者，結(jié)果顯示：與基線比較，GR組CD3-CD56+NK細(xì)胞頻數(shù)在治療24周升高(11.74, 5.69%～18.15% vs 13.7, 9.36%～20.18%,P>0.05)，在PR組同樣升高(8.94, 6.26%～14.15% vs 12.5, 7.64%～16.55%,P>0.05)。GR組CD56brightNK細(xì)胞頻數(shù)在治療24周顯著升高(9.49, 6.2%～12.48% vs 12.98, 7.75%～20.93%,P>0.05)，在PR組也同樣顯著升高(11.45, 8.27%～19.13% vs 17.52, 12.3%～22.42%,P=0.0239)。GR組NK細(xì)胞NKp46表達(dá)水平在治療24周顯著升高(90.55, 83.8～94.78 vs 93.8, 92.28～96.4,P=0.0263)，而PR組卻未升高(95, 90.6～96.15 vs 94.3, 92.1～95.6,P>0.05)。GR組NK細(xì)胞NKp46high表達(dá)水平在治療24周顯著升高(12.4, 8.58～19.08 vs 39.3, 23.15-49.3,P=0.0011)，其升高幅度顯著高于PR組(14.2, 9.78～17.65 vs 27.58, 19.13～36.56,P=0.006)。結(jié)論慢性乙型肝炎患者經(jīng) Peg-IFN-α-2a治療后外周血CD3-CD56+NK細(xì)胞和CD56brightNK細(xì)胞頻數(shù)升高，對(duì)Peg-IFN-α-2a治療應(yīng)答患者NK細(xì)胞上NKp46表達(dá)上調(diào)，而且Peg-IFN-α-2a治療使NKp46high表達(dá)顯著上調(diào)，尤其在應(yīng)答患者。

Fundprogram: Bejing Municipal Administration of Hospitals Clinical and Technical Innovation Project (XMLX201706)

乙型肝炎病毒(Hepatitis B virus, HBV)感染是引發(fā)慢性肝炎、肝硬化及肝癌的重要原因之一[1-2]。在急性HBV感染中，適應(yīng)性免疫的病毒特異性CD8+T細(xì)胞在清除病毒過程中起非常重要作用，但在慢性HBV感染，病毒特異性CD8+T細(xì)胞反應(yīng)微弱并呈現(xiàn)功能性耗竭，這可能是HBV長(zhǎng)期存在的原因[3]。固有免疫重要組成的NK細(xì)胞對(duì)病毒特異性CD8+T細(xì)胞的耗竭起重要作用[4]。對(duì)NK細(xì)胞的研究有助于了解HBV與人體免疫的相關(guān)作用。

急性HBV感染研究顯示CD3-CD56+NK細(xì)胞早期被快速激活并發(fā)揮抗病毒作用，而且能及時(shí)觸發(fā)適應(yīng)性免疫反應(yīng)[5]。然而在慢性HBV感染NK細(xì)胞似乎受到抑制，研究發(fā)現(xiàn)慢性HBV感染患者NK細(xì)胞上抑制性受體NKG2A表達(dá)上調(diào)，而且調(diào)節(jié)性CD4+CD25+T細(xì)胞分泌的抑制性細(xì)胞因子IL-10增加[6]，雖然外周血NK細(xì)胞的數(shù)量不變，細(xì)胞毒活性保持，但其分泌細(xì)胞因子如干擾素γ(Interferon-gamma, IFNγ)和腫瘤壞死因子α(Tumor necrosis factor-alpha，TNFα)等的能力受損[7]。干擾素α(Interferon-alpha, IFNα)不僅具有直接抗病毒、調(diào)節(jié)免疫作用，而且還有抗腫瘤等作用，目前被廣泛應(yīng)于慢性乙型肝炎患者的治療[8]。研究顯示，在CHB患者治療過程中IFNα對(duì)NK細(xì)胞及樹突狀細(xì)胞(Dendritic cell,DCs)等多種免疫細(xì)胞有調(diào)節(jié)作用[9]。因此，研究慢性乙型肝炎(Chronic hepatitis B, CHB)患者經(jīng)IFNα治療過程中NK細(xì)胞與IFNα的相互作用有助于了解IFNα的具體作用機(jī)制，而且有助于免疫治療新策略的研發(fā)。

1 材料與方法

1.1研究對(duì)象本實(shí)驗(yàn)入選了2014年9月至2016年1月到首都醫(yī)科大學(xué)附屬北京市地壇醫(yī)院就診的慢性HBV感染免疫清除(Immune clearance, IC)期接受聚乙二醇干擾素α-2a(Peg-IFNα-2a)治療患者。為了探討NK細(xì)胞與Peg-IFNα-2a療效的相關(guān)性，我們以Peg-IFN-α-2a治療24周時(shí)HBsAg下降程度為依據(jù)分組，HBsAg下降<1 log的為應(yīng)答不佳(Poor response, PG)組，HBsAg下降≥1 log的應(yīng)答(Good response，GR)組，受試者特征如表1。本研究經(jīng)過首都醫(yī)科大學(xué)附屬北京市地壇醫(yī)院倫理委員會(huì)通過，所有患者簽署知情書。

1.2細(xì)胞表面染色為了檢測(cè)CD3-CD56+NK細(xì)胞，在100 μl靜脈全血中加入3 μl PerCP Mouse anti-Human CD3，3 μl APC Mouse anti-Human CD56，5uL PE anti-Human CD335(NKp46)，5 μl FITC Anti-Human IFNAR2 Antibody，避光孵育15～20 min，染色后的細(xì)胞用FACS Lysing Solution裂解，磷酸鹽緩沖液(PBS)洗滌，流式細(xì)胞儀檢測(cè)和FlowJo7.6分析。

1.3QuantiBRITEPE 微球定量功能分子 QuantiBRITE微球表面標(biāo)記的熒光素與NK細(xì)胞表面結(jié)合的抗體上的熒光素相同,均為PE標(biāo)記，使其有匹配的熒光光譜。每個(gè)BD QuantiBRITE PE管里有一個(gè)凍干的顆粒，顆粒由4種已知的標(biāo)有不同PE分子數(shù)量的微球混合而成，用流式細(xì)胞儀測(cè)定時(shí)制作標(biāo)準(zhǔn)曲線。把細(xì)胞表面CD分子的PE熒光強(qiáng)度值轉(zhuǎn)換為每個(gè)細(xì)胞表面CD分子的絕對(duì)值。

表1 PR組和GR組受試者特征

注：SD，標(biāo)準(zhǔn)差；ALT，丙氨酸氨基轉(zhuǎn)移酶

Notes: SD, standard deviation; ALT, alanine aminotransferase

圖1 IC期患者經(jīng)Peg-IFNα-2a治療PR組和GR組NK細(xì)胞及其亞群頻數(shù)的變化aP<0.05，bP<0.01， cP<0.001，P<0.05表示差別有統(tǒng)計(jì)學(xué)意義Fig.1 The change of the frequency of NK cells and their subgroups in PR and GR groups from patients in IC phase treated with Peg-IFNα-2aaP<0.05, bP<0.01, cP<0.001, P<0.05 was considered to be statistically significan

1.4統(tǒng)計(jì)學(xué)方法所有數(shù)據(jù)以中位數(shù)(median)及四分位數(shù)(Q1，Q3)表示，應(yīng)用GraphPad Prism 5軟件進(jìn)行統(tǒng)計(jì)分析。不同組間多重比較采用Kruskal-Wallis H分析完成，組間兩兩比較采用Mann-Whitney非參數(shù) U檢驗(yàn)方法，連續(xù)變量的前后比較采用wilcoxon秩和檢驗(yàn)。aP<0.05，bP<0.01,cP<0.001，P<0.05表示差別有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1Peg-IFNα-2a治療使外周血CD3-CD56+NK和CD56brightNK細(xì)胞頻數(shù)升高與基線比較，GR組CD3-CD56+NK細(xì)胞頻數(shù)在治療24周升高(11.74, 5.69～18.15% vs 13.7, 9.36%～20.18%,P>0.05)，在PR組同樣升高(8.94, 6.26%～14.15% vs 12.5, 7.64%～16.55%,P>0.05)(圖1 A)。GR組CD56brightNK細(xì)胞頻數(shù)在治療24周顯著升高(9.49, 6.2%～12.48% vs 12.98, 7.75%～20.93%,P>0.05)，在PR組同樣顯著升高(11.45, 8.27%～19.13% vs 17.52, 12.3%～22.42%,P=0.0239)(圖1B)。GR組CD56dimNK細(xì)胞頻數(shù)在治療24周降低(90.03, 86.95%～93.46% vs 86.3, 77.95%～92.13%,P>0.05)，在PR組也降低(88.6, 80%～91.63% vs 81.6, 77.14%～87.25%,P=0.0123)(圖1C)。

圖2 IC期患者經(jīng)Peg-IFNα-2a治療PR組和GR組NK細(xì)胞表面受體IFNAR2和NKp46的變化FMI，平均熒光強(qiáng)度；ABC，抗體結(jié)合量；*P<0.05，**P<0.01, ***P<0.001，P<0.05表示差別有統(tǒng)計(jì)學(xué)意義Fig.2 The change of the expression of IFNAR2 and NKp46 on NK cells in PR and GR groups from patients in IC phase treated with Peg-IFNα-2a. FMI, mean fluorescent intensity, ABC, antibody binding capacity*P<0.05, **P<0.01, ***P<0.001, P<0.05 was considered to be statistically significant.

2.2NKp46在GR組表達(dá)上調(diào)與基線比較，GR組NK細(xì)胞IFNAR2表達(dá)水平在治療24周升高(3.3, 2.3～4.7 vs 4.46, 2.7-5.92,P>0.05)，其升高幅度與PR組差異無(wú)統(tǒng)計(jì)學(xué)意義(4.86, 3.53～6.81 vs 5.94, 2.77～7.85,P>0.05)(圖2 A)。GR組NK細(xì)胞NKp46表達(dá)水平在治療24周顯著升高(90.55, 83.8～94.78 vs 93.8, 92.28～96.4,P=0.0263)，而PR組卻未升高(95, 90.6～96.15 vs 94.3, 92.1～95.6,P>0.05)(圖2 C)。GR組NK細(xì)胞NKp46ABC在治療24周顯著升高(8405.67, 6762.03～13090.4 vs 14974.3, 8489.77～26243,P=0.0016)，其升高幅度與PR組差異無(wú)統(tǒng)計(jì)學(xué)意義(8496.88, 6786.24～13202.1 vs 14952.1, 8253.18～23975.1,P=0.0074)(圖2 E)。

2.3經(jīng)Peg-IFNα-2a治療，NKp46high表達(dá)顯著上調(diào)，尤其在GR組與基線比較，GR組NK細(xì)胞NKp46high表達(dá)水平在治療24周顯著升高(12.4, 8.58～19.08 vs 39.3, 23.15～49.3,P=0.0011)，其升高幅度顯著高于PR組(14.2, 9.78～17.65 vs 27.58, 19.13～36.56,P=0.006)(圖2 F)。GR組NK細(xì)胞NKp46dim表達(dá)水平在治療24周顯著降低(76, 59.33～85.13, vs 58.2, 41.05～71.2,P=0.016)，其降低幅度顯著高于PR組(80.4, 74.2～84.95 vs 70.7, 59.6-80.07,P=0.0091)(圖2 G)。

2.4NK細(xì)胞功能與Peg-IFN-α-2a療效相關(guān)性分析為了探討CD3-CD56+NK細(xì)胞與Peg-IFNα-2a療效是否具有相關(guān)性，我們把所有IC期經(jīng)Peg-IFNα-2a治療患者的血漿HBsAg和HBV DNA的下降程度分別作自變量，CD3-CD56+NK細(xì)胞、CD56brightNK細(xì)胞和CD56dimNK細(xì)胞頻數(shù)及IFNAR2MFI、 NKp46MFI、NKp46ABC以及NK細(xì)胞上IFNAR2、NKp46、NKp46high和NKp46dim表達(dá)水平的變化作因變量進(jìn)行Spearman等級(jí)相關(guān)檢驗(yàn)，結(jié)果未顯示其有相關(guān)性(數(shù)據(jù)未呈現(xiàn))。

3 討論

IFNα可以通過不同的機(jī)制發(fā)揮直接抗病毒的作用，比如阻止包含RNA的核心顆粒形成，加速核心顆粒衰減和降解前基因組RNA(Pregenomic RNA)[10-11]，也有報(bào)道顯示IFNα可直接促使共價(jià)閉合環(huán)狀DNA(Covalently closed circular DNA, cccDNA)降解[12]。而且部分患者經(jīng)過IFNα治療停藥后可獲得長(zhǎng)期抑制病毒復(fù)制的免疫，這些研究支持IFNα的直接抗病毒活性在控制HBV感染方面的重要性，然而關(guān)于IFNα在調(diào)節(jié)免疫反應(yīng)尤其是調(diào)節(jié)固有免疫方面作用機(jī)制的研究結(jié)果不盡相同。

Bruder等[9]研究發(fā)現(xiàn)Peg-IFNα-2a可以使CD56brightNK細(xì)胞顯著擴(kuò)增，而且促進(jìn)CD56dimNK細(xì)胞的激活并加強(qiáng)其功能，CD56brightNK的絕對(duì)分子計(jì)數(shù)與HBsAg滴度呈負(fù)相關(guān)。我們前期研究發(fā)現(xiàn)慢性HBV感染免疫清除期患者外周血CD3-CD56+NK頻數(shù)降低[13]，這可能是肝炎發(fā)生時(shí)NK細(xì)胞功能耗竭所致，隨后經(jīng)過Peg-IFNα-2a治療無(wú)論應(yīng)答組還是應(yīng)答不佳組CD3-CD56+NK細(xì)胞頻數(shù)均升高，且這種升高伴隨著ALT的降低，間接提示NK細(xì)胞與肝臟損傷相關(guān)，隨著炎癥減輕NK細(xì)胞數(shù)量也在恢復(fù)。正如Zheng等研究所得，在HBV感染炎癥發(fā)生時(shí)外周血NK細(xì)胞數(shù)量降低，激活的NK細(xì)胞富集在肝臟組織，且CD69+NK細(xì)胞與ALT成正相關(guān)[14]，遺憾的是我們并沒有得出ALT與CD3-CD56+NK細(xì)胞、CD56brightNK細(xì)胞和CD56dimNK細(xì)胞頻數(shù)及IFNAR2MFI、NKp46MFI和NKp46ABC以及NK細(xì)胞上IFNAR2、NKp46、NKp46high和NKp46dim的表達(dá)具有相關(guān)性。

我們的研究發(fā)現(xiàn)經(jīng)過Peg-IFNα-2a治療的免疫清除期患者CD56brightNK細(xì)胞的頻數(shù)升高，而且有報(bào)道顯示在丙型肝炎和丁型肝炎患者IFNα同樣可以使CD56brightNK細(xì)胞頻數(shù)升高[15、16]。Bruder等[9]發(fā)現(xiàn)經(jīng)Peg-IFNα-2a治療的慢乙肝患者CD56brightNK細(xì)胞絕對(duì)數(shù)與HBsAg滴度呈負(fù)相關(guān)，但我們并沒有發(fā)現(xiàn)CD56brightNK細(xì)胞頻數(shù)與HBsAg含量相關(guān)。有報(bào)道顯示恩替卡韋(entecavir, ETV)抗病毒治療雖然可以增加CD56brightNK細(xì)胞的激活，但更主要的會(huì)增強(qiáng)CD56dimNK細(xì)胞分泌IFN-γ的能力[17]。我們發(fā)現(xiàn)應(yīng)答組CD56brightNK細(xì)胞頻數(shù)在前12周擴(kuò)增顯著，我們猜測(cè)Peg-IFNα-2a治療擴(kuò)增CD56brightNK細(xì)胞發(fā)揮抗病毒作用主要發(fā)生在治療前期。

我們的研究發(fā)現(xiàn)Peg-IFNα-2a治療無(wú)論應(yīng)答組還是應(yīng)答不佳組NK細(xì)胞上NKp46分子量均明顯增加，然而Peg-IFNα-2a應(yīng)答不佳組NK細(xì)胞上NKp46表達(dá)水平較基線無(wú)明顯改變，治療應(yīng)答組NK細(xì)胞NKp46表達(dá)水平卻上調(diào)，提示Peg-IFNα-2a治療促進(jìn)NK細(xì)胞的激活，而且激活的NK細(xì)胞與Peg-IFNα-2a療效相關(guān)。

HCV感染患者研究NKp46highNK細(xì)胞與抑制病毒復(fù)制和肝臟損傷密切相關(guān)，在HBV感染的研究還較少。我們發(fā)現(xiàn)免疫清除期患者Peg-IFNα-2a治療NKp46high表達(dá)升高，而且在應(yīng)答組顯著升高。因此，我們推測(cè)激活的NKp46highNK細(xì)胞與Peg-IFNα-2a療效密切相關(guān)。

本研究結(jié)果顯示慢性乙型肝炎患者經(jīng) Peg-IFNα-2a治療外周血CD3-CD56+NK細(xì)胞和CD56brightNK細(xì)胞頻數(shù)升高，對(duì)Peg-IFNα-2a治療應(yīng)答患者NK細(xì)胞上NKp46表達(dá)上調(diào)，而且Peg-IFNα-2a治療使NKp46high表達(dá)顯著上調(diào)，尤其在應(yīng)答患者。本研究初步探討了Peg-IFNα-2a在治療慢性乙型肝炎患者過程中NK細(xì)胞頻數(shù)和功能變化的基本規(guī)律，而激活的NK細(xì)胞發(fā)揮作用的具體機(jī)制仍需進(jìn)一步研究。

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FunctionsofNKcellsinchronichepatitisBpatientstreatedwithinterferon

QuXiaojing,LuYao,CaoWeihua,ZhangYing,RanChongping,QiTianlin,HuLeiping,HaoHongxiao,ZhangLu,LiMinghui,XieYao
SecondDivisionofLiverCenter,BeijingDitanHospital,CapitalMedicalUniversityBeijing100015,China

XieYao,Email:xieyao00120184@sina.com;LiMinghui,Email:wuhm2000@sina.com

ObjectiveTo elucidate the functions of peripheral blood NK cells in chronic hepatitis B patients treated with interferon.MethodsVenous whole blood samples were obtained from patients in the immune clearance (IC) phase treated with peg-interferon-alpha-2a (Peg-IFNα-2a) at baseline (t=0), 12 weeks (t=12) and 24 weeks (t=24). The frequencyies of peripheral blood CD3-CD56+NK cells, CD56brightNK cells and CD56dimNK cells, the expression level of IFNAR2 and NKp46 on NK cells were detected by flow cytometry. The levels of serum HBV DNA, HBsAg, HBeAg and ALT were detected by Ditan Hospital clinical laboratory.ResultsForty-one patients in the IC phase treated with Peg-IFNα-2a, including 21 poor response (PR) patients and 20 good response (GR) patients, were recruited for this study. Theresult were as follows: The frequency of peripheral blood CD3-CD56+NK cells was increased at week 24 in GR compared with the baseline(11.74, 5.69%-18.15% vs 13.7, 9.36-20.18%,P>0.05), and it also was increased in PR(8.94, 6.26%-14.15% vs 12.5, 7.64-16.55%,P>0.05). The frequency of peripheral blood CD56brightNK cells was increased at week 24 in GR compared with the baseline(9.49, 6.2%-12.48% vs 12.98, 7.75%-20.93%,P>0.05), and it also was increased in PR(11.45, 8.27%-19.13% vs 17.52, 12.3%-22.42%,P=0.0239). The expression level of NKp46 on NK cells was significantly increased at week 24 in GR compared with the baseline(90.55, 83.8-94.78 vs 93.8, 92.28-96.4,P=0.0263), but it was not increased in PR(95, 90.6-96.15 vs 94.3, 92.1-95.6,P>0.05). The expression level of NKp46highon NK cells was significantly increased at week 24 in GR compared with the baseline(12.4, 8.58-19.08 vs 39.3, 23.15-49.3,P=0.0011), at that range of the increase was significantly higher than PR(14.2, 9.78-17.65 vs 27.58, 19.13-36.56,P=0.006).ConclusionsThe frequencies of peripheral blood CD3-CD56+NK cells and CD56brightNK cells were increased from patients in the IC phase treated with Peg-IFNα-2a. The expression level of NKp46 on NK cells was increased in GR patients treated with Peg-IFNα-2a. The expression level of NKp46highon NK cells was significantly increased, especially in GR patients treated with Peg-IFNα-2a.

Hepatitis B virus; NK cells; Immunopathogenesis; Interferon-alpha

肝炎病毒,乙型；自然殺傷細(xì)胞；免疫病理；干擾素α

2017-06-12)

(本文編輯：陳培莉)

屈曉晶、路遙對(duì)本文同等貢獻(xiàn)

謝堯，Emai:xieyao00120184@sina.com；李明慧，Email:wuhm2000@sina.com

10.3760/cma.j.issn.1003-9279.2017.04.013

北京市醫(yī)院管理局臨床技術(shù)創(chuàng)新項(xiàng)目(XMLX201706)

QuXiaojingandLuYaoarethefirstauthorswhocontributedequallytothearticle