王 玲,蔚梅芳,武希潤(rùn)(山西省榮軍醫(yī)院內(nèi)科,太原 000;洛陽(yáng)市中心醫(yī)院消化科;山西醫(yī)科大學(xué)第二醫(yī)院消化科;通訊作者,E-mail:xirunwu66@6.com)

乙肝肝硬化患者外周血25-(OH)D3、Th17細(xì)胞、CD4+Treg細(xì)胞的變化及意義

王 玲1,蔚梅芳2,武希潤(rùn)3*
(1山西省榮軍醫(yī)院內(nèi)科,太原 030031;2洛陽(yáng)市中心醫(yī)院消化科;3山西醫(yī)科大學(xué)第二醫(yī)院消化科;*通訊作者,E-mail:xirunwu66@163.com)

目的 檢測(cè)乙肝肝硬化患者外周血中25-(OH)D3、Th17、CD4+Treg細(xì)胞變化,為維生素D治療乙肝肝硬化提供依據(jù)。 方法 選取乙肝肝硬化患者21例,正常對(duì)照者15例,采用電化學(xué)發(fā)光免疫測(cè)定法檢測(cè)25-(OH)D3水平,采用流式細(xì)胞儀檢測(cè)Th17細(xì)胞、CD4+Treg細(xì)胞水平,并計(jì)算Th17/Treg比率。 結(jié)果 健康對(duì)照組外周血25-(OH)D3為(31.87±5.97)ng/ml,乙肝肝硬化組25-(OH)D3明顯降低,為(11.89±6.04)ng/ml,兩者相比差異具有統(tǒng)計(jì)學(xué)意義(t=9.510,P=0.001)。與健康對(duì)照組相比,乙肝肝硬化組外周血Th17細(xì)胞百分比(0.913%±0.216%vs2.498%±2.583%)增高,CD4+Treg細(xì)胞百分比降低(3.964%±1.116%vs2.333%±1.714%),Th17/Treg比率(1.199±0.973vs0.256±0.138)增高,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。 結(jié)論 乙肝肝硬化患者外周血25-(OH)D3水平顯著降低,Th17增高,CD4+Treg降低,存在CD4+Treg、Th17、Treg/Th17免疫平衡紊亂,增加外周血25-(OH)D3水平有可能調(diào)節(jié)乙肝肝硬化患者體內(nèi)CD4+Treg、Th17、Treg/Th17變化,改變其免疫狀態(tài)。

乙肝肝硬化; 25-羥維生素D3; CD4+Treg細(xì)胞; Th17細(xì)胞

肝硬化是慢性肝病進(jìn)行性發(fā)展的結(jié)果,在我國(guó)以乙肝肝硬化最多見(jiàn),占肝硬化病因66%。正常情況下,CD4+T細(xì)胞亞群,CD4+CD25+調(diào)節(jié)性T細(xì)胞(regulatory T cells,Treg)和輔助T細(xì)胞17(T helper type 17 cell,Th17)在維持機(jī)體免疫平衡方面發(fā)揮重要作用,Thl7/Treg和Thl/Th2處于相對(duì)穩(wěn)定的免疫平衡狀態(tài)中。Thl/Th2免疫平衡失調(diào)導(dǎo)致HBV不能被宿主完全清除是病情遷延不愈、出現(xiàn)慢性肝損傷的主要原因,Th17/Treg的免疫平衡失調(diào)與HBV相關(guān)急慢性肝衰竭的免疫損傷和HBV再感染有關(guān)[1]。維生素D(VitD)是重要的雙向免疫調(diào)節(jié)劑,具有免疫調(diào)節(jié)、抗炎及抗纖維化等作用。25-(OH)D3能夠調(diào)節(jié)CD4+、CD8+細(xì)胞數(shù)量及比例,上調(diào)Th2細(xì)胞和Treg細(xì)胞的活性,抑制Th1和Th17細(xì)胞的活性,促進(jìn)CD4+T細(xì)胞分化時(shí)偏向Th2[2]。然而,VitD、Th17、CD4+Treg細(xì)胞在乙肝肝硬化發(fā)病中的作用及其相互關(guān)系研究少有報(bào)道。本研究著重檢測(cè)乙肝肝硬化患者的外周血25-(OH)D3、Th17、Treg細(xì)胞水平變化,旨在為25-(OH)D3臨床應(yīng)用于乙肝肝硬化治療提供依據(jù)。

1 材料與方法

1.1 臨床資料

病例選擇2015-04～2015-09在山西醫(yī)科大學(xué)第二醫(yī)院明確診斷為乙肝肝硬化病人(肝硬化組)21例,其中男13例,女8例,年齡29-74歲,中位年齡59歲。健康對(duì)照組15例來(lái)自2015-06～2015-12山西醫(yī)科大學(xué)第二醫(yī)院體檢中心,其中男7例,女8例,年齡39-72歲,中位年齡58歲。

1.2 方法

抽取空腹靜脈血6-8 ml,加入肝素抗凝,取3-3.5 ml人外周血淋巴細(xì)胞分離液加入10 ml的滅菌離心管中,分離外周血單個(gè)核細(xì)胞(PBMC),調(diào)整細(xì)胞數(shù)為1×106/ml。淋巴細(xì)胞表面染色、洗滌、破膜、胞內(nèi)染色,進(jìn)行Treg細(xì)胞、Th17細(xì)胞標(biāo)記,流式細(xì)胞儀檢測(cè)。

1.3 統(tǒng)計(jì)學(xué)處理

采用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,組間比較采用LSD-t檢驗(yàn),P<0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 乙肝肝硬化患者外周血25-(OH)D3變化

乙肝肝硬化患者外周血25-(OH)D3水平較對(duì)照組明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見(jiàn)表1)。

組別n25?(OH)D3tp健康對(duì)照組1531 87±5 979 5100 001肝硬化組 2111 89±6 04

2.2 乙肝肝硬化患者外周血Th17細(xì)胞、CD4+Treg細(xì)胞、Th17/Treg細(xì)胞變化

乙肝肝硬化患者外周血Th17細(xì)胞增多,CD4+Treg細(xì)胞減少,Thl7/Treg細(xì)胞比值增高,表現(xiàn)為Thl7/Treg細(xì)胞失衡,與健康對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見(jiàn)表2)。

組別nTh17(%)Treg(%)Th17/Treg 健康對(duì)照組150 913±0 2163 964±1 1160 256±1 138 肝硬化組 212 498±2 5832 333±1 7141 199±0 973 t-2 5203 141-3 950 P<0 05<0 05<0 05

3 討論

肝硬化患者體內(nèi)也存在免疫功能紊亂,有研究表明,Th17細(xì)胞的數(shù)量與肝損傷標(biāo)記水平密切相關(guān)[3]。具有強(qiáng)大促炎效應(yīng)的Th17細(xì)胞通過(guò)IL-17/IL-17R信號(hào)途徑直接激活肝星狀細(xì)胞,而激活的肝星狀細(xì)胞分泌大量促炎性細(xì)胞因子加劇肝臟炎癥反應(yīng),加速肝硬化的進(jìn)程。Treg細(xì)胞通過(guò)分泌細(xì)胞因子TGF-β或細(xì)胞間直接接觸作用于肝星狀細(xì)胞[4,5],抑制特異性T細(xì)胞的免疫反應(yīng),維持免疫耐受。國(guó)內(nèi)外許多研究[6-9]表明,慢性乙型肝炎患者外周血中Th17細(xì)胞、Treg細(xì)胞水平均明顯升高,在HBV感染過(guò)程中,Treg細(xì)胞及其高表達(dá)的轉(zhuǎn)錄因子FoxP3共同抑制HBcAg誘導(dǎo)的Th17細(xì)胞亞群的增殖、分化,造成Th17/Treg比例的失衡[10],Th17/Treg免疫平衡被打破會(huì)進(jìn)一步加快肝硬化的進(jìn)程,進(jìn)而加大罹患肝癌的風(fēng)險(xiǎn)[11]。

保持Th17/Treg的免疫平衡可以有效維持免疫應(yīng)答的正常狀態(tài),但Th17/Treg免疫平衡狀態(tài)與乙肝肝硬化發(fā)生發(fā)展的關(guān)系尚不清楚。在4周、8周和12周四氯化碳誘導(dǎo)的肝纖維化小鼠模型體內(nèi),Th17細(xì)胞數(shù)增加,但Treg細(xì)胞數(shù)逐漸減少[12]。Yu等[13]發(fā)現(xiàn),在乙肝肝硬化尤其是失代償期患者體內(nèi),IL-10、IL-23、IL-17、TGF-β都明顯升高,TGF-β/IL-17、Th17/Treg比率升高,提示Th17/Treg免疫平衡失調(diào)可能通過(guò)HSC激活促進(jìn)肝纖維化及肝硬化進(jìn)程。本研究中,乙肝肝硬化組外周血Th17細(xì)胞水平高,Treg細(xì)胞水平低,Th17/Treg比率高,提示乙肝肝硬化存在Th17、Treg、Th17/Treg免疫紊亂,免疫抑制效應(yīng)減弱而免疫炎性效應(yīng)增強(qiáng)誘導(dǎo)了外周血T細(xì)胞亞群的紊亂以及外周免疫耐受的破壞,有可能促進(jìn)肝硬化的發(fā)生、發(fā)展。

近年來(lái)研究表明,在慢性肝病,尤其是肝硬化患者,存在VitD缺乏[14]。本研究通過(guò)檢測(cè)乙肝肝硬化患者體內(nèi)的25-(OH)D3水平發(fā)現(xiàn),其25-(OH)D3水平較對(duì)照組明顯降低,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

越來(lái)越多的研究表明,VitD是免疫調(diào)節(jié)劑,與免疫調(diào)節(jié)有關(guān)。25-(OH)D3缺乏與丙肝肝硬化嚴(yán)重的肝細(xì)胞壞死及肝纖維化密切相關(guān)[15]。Gal-Tanamy等[16]研究表明VitD通過(guò)誘導(dǎo)IFN-γ和MxA基因表達(dá)抑制人類肝細(xì)胞丙型肝炎病毒(HCV)復(fù)制,并增強(qiáng)VitD受體表達(dá),產(chǎn)生協(xié)同抗病毒治療作用,最終可能延緩或阻止丙肝肝硬化進(jìn)展。Treg細(xì)胞有效抑制了過(guò)強(qiáng)的免疫反應(yīng),及時(shí)阻止肝細(xì)胞的持續(xù)損傷,但同時(shí)也阻止了機(jī)體清除HBV的能力,使乙型肝炎感染慢性化。1,25-(OH)2D3被轉(zhuǎn)運(yùn)到靶組織中結(jié)合VitD受體后進(jìn)行信號(hào)轉(zhuǎn)導(dǎo),直接或間接作用于淋巴細(xì)胞,抑制T淋巴細(xì)胞的增殖、分化,抑制Th17細(xì)胞分泌促炎細(xì)胞因子IL-17[16]。VitD對(duì)T細(xì)胞也有一定的刺激作用,T細(xì)胞受刺激后可以表達(dá)細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原-4(CTLA-4)和FoxP3,從而誘導(dǎo)Treg細(xì)胞產(chǎn)生[17]。本實(shí)驗(yàn)結(jié)果表明,肝硬化患者25-(OH)D3明顯降低,Th17細(xì)胞增加,Treg細(xì)胞降低、Th17/Treg增加,推測(cè)25-(OH)D3缺乏打破了Th17/Treg免疫平衡,參與乙肝肝硬化發(fā)生、發(fā)展。

[1] Lohse AW, Weiler-Normann C, Tiegs G. Immune-mediated liver injury[J]. Hepatology,2010, 52:136-144.

[2] Daniel C, Sartory NA, Zahn N,etal. Immune modulatory treatment of trinitrobenzene sulfonicacid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile[J]. J Pharmacol Exp Ther, 2008, 324(1): 23-33.

[3] Zhang GL, Xie DY, Ye YN,etal. High level of IL-27 positively correlated with Th17 cells may indicate liver injury in patients infected with HBV[J]. Liver Int,2014,34:266-273.

[4] Li J, Qiu SJ, Wang FP,etal. Significance of the balance between regulatory T(Treg)and T helper 17(Th17)cells during hepatitis B virus related liver fibrosis[J]. PLoS One, 2012, 7(6): e39307.

[5] 饒少鋒,游晶,張茹薏.Th17/Treg細(xì)胞與HBV感染相關(guān)肝病關(guān)系的研究進(jìn)展[J].實(shí)用醫(yī)學(xué)雜志,2015,31,(7):1050-1052.

[6] Li J, Shi J, Ren W,etal. Regulatory role of CD4(+)CD25(+)Foxp3(+)regulatory T cells on IL-17-secreting T cells in chronic hepatitis B patients[J]. Dig Dis Sci, 2014, 59(7): 1475-1483.

[7] 薛芝敏,姚冬梅.Th17細(xì)胞與肝臟疾病關(guān)系的研究進(jìn)展[J].世界華人消化雜志,2013,21(13):1185-1190.

[8] 陸曉曉,張占卿.Treg、Th17的發(fā)育調(diào)控及其與慢性乙型肝炎發(fā)病和進(jìn)展的相關(guān)性[J].肝臟,2013,18(11):782-785.

[9] Nan XP, Zhang Y, Yu HT,etal. Inhibition of viral replication downregulates CD4(+)CD25(high)regulatory T cells and programmed death-ligand 1 in chronic hepatitis B[J]. Viral Immunol, 2012, 25(1): 21-28.

[10] Li J, Wu W, Peng G,etal. HBcAg induces interleukin-10 production, inhibiting HBcAg-specific Th17responses in chronic hepatitis B patients[J]. Immunol Cell Biol, 2010, 88:834-841.

[11] Li J, Qiu S J, She WM,etal. Significance of the balance between regulatory T(Treg) and Th17 cells during hepatitis B virus related liver fibrosis[J]. PLoS One, 2012, 7: e39307.

[12] Sun XF, Gu L, Deng WS,etal. Impaired balance of T helper 17/T regulatory cells in carbon tetrachloride-induced liver fibrosis in mice[J]. World J Gastroenterol,2014, 20(8): 2062-2070.

[13] Yu X, Guo R, Ming D,etal. Ratios of regulatory T cells/T-helper 17 cells and transforming growthfactor-beta1/interleukin-17 to be associated with the development of hepatitis B virus-associated liver cirrhosis[J]. Gastroenterol Hepatol, 2014, 29 (5): 1065-1072.

[14] Guo GY, Shi YQ, Wang L,etal. Serum vitamin D level is associated with disease severity and response to ursodeoxycholic acid in primary biliary cirrhosis[J]. Aliment Pharmacol Ther, 2015, 42(2):221-230.

[15] Petta S, CammC, Scazzone C,etal. Low vitamin D serum level is related to severe fibrosis and low responsiveness tointerferon-based therapy in genotype 1 chronic hepatitis C[J]. Hepatology,2010,51: 1158-1167.

[16] Gal-Tanamy M, Bachmetov L, Ravid A,etal. Vitamin D: an innate antiviral agent suppressing hepatitis C virus in human hepatocytes[J]. Hepatology, 2011, 54: 1570-1579.

[17] Kang SW, Kim SH, Lee N,etal. 1,25-Dihyroxyvitam in D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved noncoding sequence region[J]. J Immuno1, 2012, 188(11): 5276-5282.

Alterationsof25-hydroxyvitaminD3,Th17cellsandCD4+TregulatorycellsinperipheralbloodinpatientswithhepatitisBcirrhosis

WANG Ling1,YU Meifang2,WU Xirun3*
(1DepartmentofInternalMedicine,ShanxiRongjunHospital,Taiyuan030031,China;2LuoyangCentreHospital,HenanProvince;3DepartmentofGastroenterology,SecondHospitalofShanxiMedicalUniversity;*Correspondingauthor,E-mail:xirunwu66@163.com)

ObjectiveTo explore the alterations of 25-hydroxyvitamin D3,Th17 cells and CD4+T regulatory cells in peripheral blood of patients with hepatitis B cirrhosis.MethodsTwenty-one patients with hepatitis B cirrhosis and 15 healthy volunteers were enrolled in the study. Electrochemical luminescence immunoassay was used to detect the level of 25-hydroxyvitamin D3.The Th17 cell and CD4+Treg cells in the peripheral blood mononuclear cell were detected by flow cytometry.ResultsCompared with normal controls [(31.87±5.97)ng/ml], the peripheral blood 25-hydroxyvitamin D3 concentrations decreased significantly in patients with hepatitis B cirrhosis[(11.89±6.04)ng/ml,t=9.510,P=0.001]. The percentage of Th17 cells was higher in patients with hepatitis B cirrhosis than that of normal controls(2.498%±2.583%vs0.913%±0.216%). In hepatitis B cirrhosis patients, the percentage of CD4+Treg cells was lower than that of healthy controls(2.333%±1.714%vs3.964%±1.116%). Compared with normal controls,the ratio of Th17/Treg increased in patients with hepatitis B cirrhosis(0.256±0.138vs1.199±0.973,P<0.05).ConclusionIn patients with hepatitis B cirrhosis,the peripheral blood 25-hydroxyvitamin D3 concentration decreases, Th17 cells increases, CD4+Treg cells decreases and the ratio of Th17/Treg increases. There are the immune disorders of CD4+Treg cells, Th17 cells and the ratio of Treg/Th17 in hepatitis B cirrhosis patients. Increased peripheral blood level of 25-hydroxyvitamin D may adjust immune status of hepatitis B cirrhosis.

liver cirrhosis; 25-hydroxyvitamin D3; CD4+Treg cells; Th17 cells

王玲,女,1968-12生,學(xué)士，副主任醫(yī)師,E-mail:983095232@qq.com

2017-11-19

R512.6

A

1007-6611(2017)12-1233-03

10.13753/j.issn.1007-6611.2017.12.007