血清學(xué)指標在非酒精性脂肪性肝病診斷中的意義

萬 艷， 常劍波， 白艷霞， 李倩楠， 戴光榮

(延安大學(xué)附屬醫(yī)院， 陜西 延安 716000)

血清學(xué)指標在非酒精性脂肪性肝病診斷中的意義

萬 艷， 常劍波， 白艷霞， 李倩楠， 戴光榮

(延安大學(xué)附屬醫(yī)院， 陜西 延安 716000)

近年來，脂肪性肝病發(fā)病率呈明顯上升趨勢，肝臟活組織檢查是診斷脂肪肝的金標準，但有其不可避免的缺點。目前尚缺乏一種方便、價廉、準確、適用于臨床的無創(chuàng)診斷方法。主要回顧了多項血清學(xué)指標在非酒精性脂肪性肝病(NAFLD)診斷中的意義，認為血清學(xué)指標中肝酶、AST與ALT比值、血清鐵蛋白、血清硒、尿酸、高空腹胰島素濃度、視黃醇結(jié)合蛋白4、細胞角蛋白18、PNPLA3基因、TM6SF2基因在NAFLD的診斷中有較大的價值，而IL-28B對NAFLD的診斷價值存在較大爭議，尚需更多臨床試驗證實。相信在不久的將來，多項血清指標組合會成為較準確，且適用于臨床的早期診斷NAFLD、肝脂肪變性程度及肝纖維化程度的無創(chuàng)方法。

脂肪肝； 生物學(xué)標記； 診斷； 綜述

近年來，脂肪肝發(fā)病率呈明顯上升趨勢，已成為我國第一大非感染性慢性肝病，其中，以非酒精性脂肪性肝病(NAFLD)最多見，其疾病譜包括單純性非酒精性脂肪肝(NAFL)、非酒精性脂肪性肝炎(NASH)及相關(guān)肝硬化和肝癌[1]。有研究表明NAFLD主要與肥胖、飲食習(xí)慣及基因有關(guān)[2]。據(jù)統(tǒng)計，25%的脂肪肝患者的肝臟逐漸纖維化，6%的患者最終進展為肝硬化，生命受到威脅[3]。NAFLD是可逆性疾病，盡早干預(yù)和治療可完全恢復(fù)。因此，NAFLD患者的早期診斷是關(guān)鍵。雖然，肝臟活組織檢查是診斷NAFLD的金標準，但肝臟穿刺為有創(chuàng)操作，重復(fù)性差，且存在取樣誤差；影像學(xué)檢查價格昂貴，不便于隨訪；而血清學(xué)檢查簡便無創(chuàng)，對NAFLD的診斷意義備受矚目。

1 實驗室檢查

1.1 肝臟酶學(xué) 肝臟酶學(xué)中血清ALT、AST、ALP、GGT等常用于普通人群肝病的篩查。一項來自英國的橫斷面研究[4]顯示，NAFLD為體檢人群肝酶異常的主要原因。我國一項研究[5]表明，肥胖成人的血清ALT水平與TG含量獨立相關(guān)，可作為NAFLD程度的預(yù)測指標。Maximos等[6]研究也表明，NAFLD患者的血漿轉(zhuǎn)氨酶升高主要取決于胰島素抵抗程度和TG含量，且ALT升高者肝細胞脂肪變性程度更嚴重。然而，Cruz等[7]以腹部B超檢查為對照，證實AST、ALT、GGT、胰島素抵抗內(nèi)穩(wěn)態(tài)模型聯(lián)合B超可用于肝臟脂肪變性程度的評估，且在顯示脂肪變性程度方面，AST比ALT更有價值。已知GGT早已納入脂肪肝參數(shù)系統(tǒng)[8]。最新一項研究[9]納入35例NAFLD患者，通過肝活組織檢查復(fù)查肝臟病變情況顯示，血清AST和GGT水平升高與NAFLD活動度評分惡化高度相關(guān)。Kawamura等[10]建立了一個同時納入AST和ALT等指標的評分系統(tǒng)，可準確預(yù)測NASH患者的肝纖維化分期，且預(yù)測進展期纖維化(≥3期)的受試者工作特征曲線下面積(AUC)為0.909，優(yōu)于其他幾種纖維化評分系統(tǒng)。

1.2 AST與ALT比值(AAR) 早有報道稱AAR參與了肝脂肪變性指數(shù)系統(tǒng)[11]、肝脂評分系統(tǒng)[12]、肝纖維化評分系統(tǒng)[13]。最近，F(xiàn)allatah等[14]通過評估肝纖維化用的FibroScan檢查，也證實了AAR與FibroScan之間呈高度正相關(guān)。

1.3 血清鐵蛋白 2011年Manousou等[15]研究認為血清鐵蛋白是NASH的獨立危險因素。后來，Kowdley等[16]證實當血清鐵蛋白>1.5倍正常值上限(ULN)可用于診斷NASH，并可預(yù)測NAFLD相關(guān)肝纖維化。近期，馬會民等[17]選取243例NAFLD患者的研究結(jié)果顯示血清鐵蛋白水平升高，認為其可能通過影響肝臟脂質(zhì)代謝參與NAFLD進展。與此同時，馮紅萍等[18]納入250例NAFLD患者，并根據(jù)患者首診時CT檢查結(jié)果將其分為輕、中、重3組，對3組患者的血清鐵蛋白值進行比較分析，結(jié)果提示血清鐵蛋白在輕度脂肪性肝病時即可較早地反映肝功能損傷，比ALT、AST更靈敏，并認為血清鐵蛋白可作為NAFLD病情進展程度的獨立預(yù)測指標。Goh等[19]應(yīng)用AST水平、血清鐵蛋白水平、BMI、血小板計數(shù)、2型糖尿病和高血壓等變量建立的評分系統(tǒng)用于預(yù)測NAFLD患者發(fā)生NASH風(fēng)險的AUC為0.81。

1.4 血清硒 硒是人類必不可少的微量元素，主要來自空氣、食物和水。一些動物研究[20-22]表明硒暴露能引起血清肝酶水平增加及Kupffer細胞激活，其肝組織胰島素抵抗和甘油三酯濃度均高于對照組。隨后，Stranges等[23]進行流行病學(xué)研究發(fā)現(xiàn)，高硒暴露可能導(dǎo)致代謝異常，包括血脂異常、2型糖尿病或胰島素抵抗。最近，在中國上海，一項納入8550例研究對象的橫斷面研究[24]顯示血漿硒水平與NAFLD患病率呈正相關(guān)，且血漿硒水平升高者的低密度脂蛋白膽固醇、空腹血糖、糖化血紅蛋白、ALT、AST和GGT水平均較健康人群升高(P值均< 0.05)。由此可見，硒暴露可能會導(dǎo)致NAFLD發(fā)生的風(fēng)險增加。

1.5 尿酸 尿酸是嘌呤在肝臟代謝的最終產(chǎn)物，其生成和排泄決定了血清尿酸的水平。目前有關(guān)尿酸在NAFLD中的發(fā)生機制的主要假說是“二次打擊學(xué)說“，大量研究顯示高血清尿酸與NAFLD的發(fā)生與發(fā)展密切相關(guān)。Sirota等[25]也認為尿酸濃度升高是NAFLD的獨立危險因素，且NAFLD損傷嚴重程度隨尿酸的增高而加重。Yuan等[26]證實了血清尿酸水平每升高1 mg/dl，導(dǎo)致 NAFLD的風(fēng)險增加21%。李韶豐等[27]指出控制尿酸有望成為NAFLD綜合治療手段之一，早期對高尿酸血癥合并NAFLD患者綜合干預(yù)至關(guān)重要。一項82 608例成人參與的研究[28]表明，血清尿酸和ALT水平呈顯著正相關(guān)，具有量效關(guān)系。1.6 空腹胰島素濃度 大量研究證實胰島素抵抗及2型糖尿病可促進NAFLD的發(fā)展，空腹胰島素濃度已參與肝脂評分系統(tǒng)[12]。但是，在發(fā)展成2型糖尿病之前，葡萄糖代謝和NAFLD患者肝組織病變之間的關(guān)系并不為眾所周知。Masuda等[29]納入103例NAFLD患者研究在糖尿病前期，葡萄糖代謝與肝纖維化是否有相關(guān)性，結(jié)果證實纖維化3期組的女性數(shù)量、年齡、AAR、空腹胰島素濃度、糖化血紅蛋白、透明質(zhì)酸和Ⅳ型膠原蛋白7s比纖維化0～2期組明顯更高，認為在NAFLD發(fā)展至2型糖尿病之前，高空腹胰島素濃度是預(yù)測肝纖維化嚴重程度的關(guān)鍵。

2 生物化學(xué)標志物

2.1 視黃醇結(jié)合蛋白(retinol-binding protein，RBP)4 RBP-4是視黃醇類結(jié)合蛋白家族中的一員，主要由肝臟分泌，脂肪組織也少量分泌，主要與維生素A的儲存、代謝和轉(zhuǎn)運有關(guān)。研究認為RBP-4與代謝綜合征、胰島素抵抗、2型糖尿病、慢性肝病、慢性腎病、心腦血管疾病均有關(guān)，而這些大多為NAFLD的高危因素，故認為RBP-4與NAFLD發(fā)病有關(guān)。早在2006年，Graham等[30]證實了血清RBP-4水平與胰島素抵抗程度呈正相關(guān)。我國孫立山等[31]也認為血清RBP-4是NAFLD發(fā)病相關(guān)的獨立危險因素。后來，Chang等[32]研究認為RBP-4可作為腹部肥胖的一個標志物，且隨著肝臟脂肪含量的增加，BMI、腰圍、腰臀比、TG、ALT、AST、GGT也逐漸升高，高密度脂蛋白逐漸下降；而總膽固醇、低密度脂蛋白、ALP無顯著變化。Liu等[33]通過動物實驗證實RBP-4 mRNA在NAFLD模型中異常升高，與肝TG積累呈正相關(guān)，并認為可能與線粒體含量減少和線粒體脂肪酸β-oxidation受損有關(guān)。

2.2 細胞角蛋白(cytokeratin,CK)18 CK-18與細胞凋亡相關(guān)，當細胞壞死時，血液中CK-18濃度隨之升高，而NAFLD的進展與肝實質(zhì)細胞脂肪變性、壞死和凋亡密切相關(guān)。Maliken等[34]研究表明血清CK-18及其裂解物的水平隨著肝細胞壞死、凋亡程度增加而升高，且與肝細胞壞死水平呈正相關(guān)。Castera等[35]也表明CK-18的主要價值在于可提示NASH的存在，并能評價炎癥程度及纖維化的發(fā)生。Chalasani等[36]總結(jié)了一些后續(xù)研究和薈萃分析，證實血清CK-18評估NASH的敏感度和特異度分別為78%和87%，其AUC為0.82。Kazankov等[37]聯(lián)合sCD163和CK-18，認為NAFLD/NASH患者細胞凋亡可能與巨噬細胞活化有關(guān)。

NAFLD具有遺傳傾向，F(xiàn)eldstein等[38]報道CK-18有望成為NASH患兒非侵入性診斷標志物之一。隨后，李娜等[39]研究也證實了這一觀點。然而，由于不同實驗研究所所用的特定臨界值不同，缺乏統(tǒng)一標準，美國肝病學(xué)會指南并未將CK-18作為臨床常用的檢測指標。近期，Zwolak等[40]同時檢測了RBP-4和CK-18，結(jié)果認為RBP-4與肥胖的NAFLD 患者密切相關(guān)，CK-18與非肥胖的NAFLD患者密切相關(guān)。

大量研究證實，炎癥反應(yīng)在NAFLD的發(fā)展中起關(guān)鍵作用，炎性標志物有IL-6、超敏C反應(yīng)蛋白(hs-CRP)、脂聯(lián)素、TNFα，其中脂聯(lián)素具有抗炎作用[41]，其水平下降可使NAFLD發(fā)展至NASH[42]。TNFα則通過參與氧化應(yīng)激和脂質(zhì)氧化介導(dǎo)肝臟炎癥反應(yīng)，從而導(dǎo)致肝損傷。藍常明等[43]將98例輕、中、重度NAFLD患者和38例作為對照組的健康體檢者進行血清TNFα檢測，Spearman相關(guān)分析結(jié)果顯示血清TNFα水平與NAFLD病情嚴重等級呈正相關(guān)(r=0.516，P<0.05)，且在NAFLD發(fā)展、NASH及肝纖維化中起重要作用。

3 遺傳基因

3.1 PNPLA3基因 PNPLA3基因位于人類第22號染色體的長臂上，可翻譯出一條包含481個氨基酸的跨膜多肽鏈，在脂肪組織和肝臟中高表達，其表達主要受營養(yǎng)狀況的影響，基因的多態(tài)性影響了PNPLA3的表型，從而易引起能量代謝的紊亂。PNPLA3 rs738409 G可降低脂聯(lián)素水平，已在多個國家被證明與NAFLD及脂肪變性程度密切相關(guān)[44]。最近，一項基于肝活組織病理基礎(chǔ)的多中心研究[45]顯示，PNPLA3 rs738409基因多態(tài)性可使肝臟脂肪含量增加及肝纖維化加重。同時，一項針對日本的經(jīng)肝活組織檢查證實為NAFLD患者的研究[46]認為，嚴重的肝纖維化和PNPLA3 rs738409基因多態(tài)性是NAFLD患者發(fā)生肝細胞癌的獨立危險因素。由此可見，PNPLA3與NAFLD患者的肝脂肪含量、脂肪變性程度、肝纖維化，甚至肝癌的發(fā)生均有密切關(guān)系。

3.2 TM6SF2基因 TM6SF2基因位于第19號染色體上，編碼一段由351個氨基酸構(gòu)成的蛋白，主要在腦組織、腎臟、肝臟及小腸表達，其中在小腸組織表達最高。2014年Kozlitina等[47]首次報道了TM6SF2 rs58542926與NAFLD易感的相關(guān)性，TM6SF2 rs58542926在p.E167K位點上將谷氨基酸轉(zhuǎn)換成賴氨基酸，且AST和ALT與這種改變呈高度正相關(guān)。近日，一項針對中國漢族人群關(guān)于PNPLA3 rs738409、rs2294918、NCAN rs2228603、GCKR rs780094、LYPLAL1 rs12137855和TM6SF2 rs58542926的全基因組關(guān)聯(lián)研究證實了TM6SF2和PNPLA3一樣，是中國NAFLD患者最重要的危險等位基因[48]。最新研究[45]認為TM6SF2 rs58542926主要與NAFLD患者肝脂肪堆積有關(guān)，與肝纖維化聯(lián)系不大。

Krawczyk等[49]聯(lián)合檢測PNPLA3 rs738409和TM6SF2 rs58542926，結(jié)果證實在含有PNPLA3 rs738409的患者中，TM6SF2變體的存在會加重血清轉(zhuǎn)氨酶升高，并進一步加重肝臟脂肪變性。

3.3 IL-28B IFN家族包括Ⅰ型(IFNα、IFNβ等)、Ⅱ型(IFNγ)和Ⅲ型(IFNλs，包括IFNλ1、IFNλ2和IFNλ3，又分別稱為IL-29、IL-28A和IL-28B)。IL-28B即IFNλ3，其基因的編碼位于人類第19號染色體上(19q13.13)，其單核苷酸多態(tài)性可以影響基因轉(zhuǎn)錄及翻譯，進而影響IFNλ3的合成。大量研究也證實了IL-28B與病毒性肝炎及其相關(guān)肝硬化、肝癌的發(fā)生發(fā)展、抗病毒治療的應(yīng)答反應(yīng)及預(yù)后密切相關(guān)。近年來，隨著NAFLD患者的增多，越來越多的研究者開始探索IL-28B與NAFLD的相關(guān)性。有研究發(fā)現(xiàn)IL-28B基因多態(tài)性與肝臟脂肪變性[50]、炎癥反應(yīng)[51]和肝纖維化[52]的嚴重程度有關(guān)。Petta等[53]認為在NAFLD患者中，IL-28B rs12979860 CC基因型和PNPLA3 rs738409 GG與肝損傷的嚴重程度相關(guān)。有研究[54]表明，在非肥胖的NAFLD患者中，攜帶IL-28B rs12979860 CC基因型的患者肝小葉炎性反應(yīng)和F2～F4期肝纖維化患病率比攜帶TT/TC基因型的患者高(28/46和9/48，P<0.001)，而在肥胖的NAFLD患者中卻未發(fā)現(xiàn)這種差異[55]，并證實IL-28B rs12979860位點與NAFLD無相關(guān)性。Hashemi等[56]針對伊朗人的一項研究認為IL-28B rs8099917位點也不是NAFLD的危險因素。李江文[57]收集190例NAFLD患者和183例正常人的血液標本，采用多重高溫連接酶檢測反應(yīng)技術(shù)對IL-28B rs12979860、IL-28B rs8099917兩位點進行基因檢測，結(jié)果也證實IL-28B rs12979860、IL-28B rs8099917兩位點與NAFLD無明顯相關(guān)性。目前，關(guān)于IL-28B與NAFLD相關(guān)性的研究相對較少，尚需大量研究加以證實。

Severson等[44]認為遺傳因素及基因多態(tài)性導(dǎo)致NAFLD及其最終結(jié)果，這些基因是未來提高診斷及管理水平的關(guān)鍵。

4 目前已存在的血清指標組合在NAFLD中的應(yīng)用

診斷脂肪變性的參數(shù)系統(tǒng)有：脂肪變性測試系統(tǒng)[58]，包括6個變量(肝硬度、FibroTest指數(shù)、BMI、TC、TG和血糖)；脂肪肝參數(shù)[8]，包括4個變量(BMI、腰圍、TG和GGT)；脂質(zhì)積累量[59]，包括3個變量(腰圍、TG及性別)；肝脂肪變性指數(shù)系統(tǒng)[11]，包括3個變量(AAR、BMI和2型糖尿病)；NAFLD肝脂評分系統(tǒng)[12]，包括5個變量(代謝綜合征、2型糖尿病、空腹胰島素水平、AST及AAR)，可預(yù)測肝臟脂肪的百分比。

用于肝纖維化評價的系統(tǒng)包括：NAFLD肝纖維化評分、BARD評分、NIKEI、NASH-CRN回歸得分、AST與PLT比值指數(shù)、FIB-4指數(shù)、King′s評分、GUCI、Lok指數(shù)、Forns分數(shù)和肝纖維化血清學(xué)指標。Lykiardopoulos等[60]納入158例NAFLD患者，其中38例處于肝纖維化早期階段，以肝活組織檢查結(jié)果為金標準，研究表明FIB-4(包括血小板、ALT、AST和年齡)和King′s評分(年齡、AST、血小板)診斷肝纖維化的AUC分別為0.84和0.83；并用創(chuàng)建的包括年齡、空腹血糖、透明質(zhì)酸等指標的預(yù)測早期肝纖維化的模型(LINKI-1)和包括這些指標合并血小板計數(shù)，且用數(shù)學(xué)方法夸大其反面影響的替代模型(LINKI-2)證實，在總?cè)褐蠰INKI-1和LINKI-2模型的AUC高達0.91和0.89。同時有研究[61]表明，通過多項血清指標組合的無創(chuàng)模型雖然診斷NAFLD的效能差，但排除肝硬化的準確性>90%。

5 展望

綜上所述，血清學(xué)指標中肝酶、AAR、血清鐵蛋白、血清硒、尿酸、高空腹胰島素濃度、PNPLA3、TM6SF2、RBP-4、CK-18對NAFLD的診斷價值值得肯定，而IL-28B對NAFLD的診斷價值存在較大爭議，尚需更多臨床試驗證實。并且多項血清指標組合在評價肝脂肪變性、肝纖維化及肝硬化方面的研究還處于初期階段，尚不能有效的對NAFLD進行全面的評估。相信通過大量的研究，更全面的血清指標組合將會問世，其診斷NAFLD的價值或許有望取代肝臟活組織病理檢查。

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引證本文：WAN Y, CHANG JB, BAI YX, et al. Significance of serological markers in diagnosis of nonalcoholic fatty liver disease[J]. J Clin Hepatol, 2017, 33(5): 963-968. (in Chinese) 萬艷， 常劍波， 白艷霞， 等. 血清學(xué)指標在非酒精性脂肪性肝病診斷中的意義[J]. 臨床肝膽病雜志, 2017, 33(5): 963-968.

(本文編輯：朱 晶)

Significance of serological markers in diagnosis of nonalcoholic fatty liver disease

WANYan,CHANGJianbo,BAIYanxia,etal.

(Yan′anUniversityAffiliatedHospital,Yan′an，Shaanxi716000,China)

In recent years, the incidence rate of fatty liver tends to increase markedly, and liver biopsy is the gold standard for the diagnosis of fatty liver, but it has some inevitable shortcomings. At present, there lacks a convenient, cheap, and accurate noninvasive diagnostic method for clinical practice. This article reviews the significance of various serological markers in the diagnosis of nonalcoholic fatty liver disease (NAFLD) and points out that liver enzyme, aspartate aminotransferase/alanine aminotransferase ratio, serum ferritin, serum selenium, uric acid, high fasting insulin level, retinol-binding protein-4, cytokeratin-18, PNPLA3 gene, and TM6SF2 gene have great significance in the diagnosis of NAFLD. There are still controversies over the value of interleukin-28B in the diagnosis of NAFLD, and more clinical trials are needed. We believe that in the near future, a combination of various serum markers may become an accurate noninvasive method for the diagnosis of NAFLD and the assessment of the degree of liver fatty degeneration and fibrosis.

fatty liver； biological markers； diagnosis； review

10.3969/j.issn.1001-5256.2017.05.037

2016-11-24；

2016-12-29。

萬艷(1990-)，女，主要從事慢性肝病的診療研究。

戴光榮，電子信箱：daiguangrong6810@sina.cn。

R575.5

A

1001-5256(2017)05-0963-06