CN110954700A - 血清gdnf在预测肝纤维化程度中的应用 - Google Patents
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Abstract
本发明涉及血清GDNF在预测肝纤维化程度中的应用。本发明研究了血清GDNF预测肝纤维化程度的效果，从肝组织的病理分期，病理染色阳性面积进行相关性分析，并与公认的肝纤维化无创性诊断指标对血清GDNF预测肝纤维化的准确性加以验证，发现GDNF可作为预测肝纤维化严重性的标志物，具有较高的准确性，在预测F4 vs F0‑3阶段、F4 vs F3阶段显著优于公认的肝纤维化无创性诊断指标，具有重要临床价值。
Description
技术领域
本发明涉及生物医学技术领域，具体地说，涉及血清GDNF在预测肝纤维化程度中的应用。
背景技术
胶质细胞源性神经营养因子(glial cell linederived neuro trophic factor,GDNF)于1993年由Lin等从大鼠神经胶质细胞系B49的培养液中首先纯化并命名。其在神经系统的发育、生长、损伤及修复等过程中发挥重要的神经营养作用，神经营养活性较高，不仅可以保护多巴胺(DA)运动神经元，对于中枢及外周的运动神经元、感觉神经元及交感神经元等多种神经元具有同等的营养作用，在神经系统变性类疾病及缺血性脑血管疾病等治疗中发挥重要作用。还有研究表明GDNF可以通过促进已破坏肠上皮细胞增殖、加强肠上皮间的细胞连接和抗肠上皮细胞凋亡等途径保护肠上皮屏障的完整性。亦有部分证据表明GDNF可以通过干预增殖平滑肌与ENS修复的平衡体系抑制肠道炎症的进展。
肝纤维化(hepatic fibrosis,HF)指的是慢性肝脏疾病发展的过程中，肝细胞被反复破坏后再生，胶原、糖蛋白、蛋白多糖等细胞外基质在肝脏中弥漫型过度沉积与异常分布，是肝脏对慢性损伤的病理性修复反应，是各种慢性肝病向肝硬化发展过程中的关键步骤和影响慢性肝病预后的重要环节。肝纤维化经过早期、有效的治疗可以逆转，明确诊断其改变程度，对其预后判断、选择用药及疗效判定都有重要的作用。目前诊断肝纤维化的手段有：肝脏穿刺活检术、弹性超声、肝纤维四项等血清学指标。血清学指标判定肝纤维化是最方便实用的诊断方法，因此受到重视。血清学指标包括细胞外间质成份(ECM)、胶原酶类和细胞因子三大类。目前已知的与肝纤维化有一定关系的血清学指标有III型前胶原氨基酸前肽、IV型胶原、VI型胶原、纤维连接素、层粘蛋白、脯氨酸羟化酶、脯氨酸肽酶、腺苷脱氨酶、单胺氧化酶、TGF-β、TNF-a、PDGF等，种类庞多。然而尚缺乏灵敏度、特异性良好，准确度高的标志物。
发明内容
本发明的目的是针对现有技术中的不足，提供一种新的、准确度高的预测肝纤维化程度的血清标志物。
第一方面，本发明提供了血清GDNF在制备预测或诊断肝纤维化程度的试剂或试剂盒中的应用。
优选地，所述预测或诊断肝纤维化程度是指预测或诊断个体处于肝纤维化分期的哪个阶段。
第二方面，本发明提供了检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断肝纤维化程度的试剂或试剂盒中的应用。
优选地，所述预测或诊断肝纤维化程度是指预测或诊断个体处于肝纤维化分期的哪个阶段。
第三方面，本发明提供了血清GDNF在制备预测或诊断肝纤维化程度为F0-3还是F4的试剂或试剂盒中的应用。
第四方面，本发明提供了检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断肝纤维化程度为F0-3还是F4的试剂或试剂盒中的应用。
第五方面，本发明提供了血清GDNF在制备预测或诊断肝纤维化程度为F3还是F4的试剂或试剂盒中的应用。
第六方面，本发明提供了检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断肝纤维化程度为F3还是F4的试剂或试剂盒中的应用。
第七方面，本发明提供了血清GDNF在制备预测或诊断个体是否存在肝纤维化的试剂或试剂盒中的应用。
优选地，所述预测或诊断个体是否存在肝纤维化是指预测或诊断个体处于非肝纤维化分期还是肝纤维化期。
第八方面，本发明提供了检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断个体是否存在肝纤维化的试剂或试剂盒中的应用。
优选地，所述预测或诊断个体是否存在肝纤维化是指预测或诊断个体处于非肝纤维化分期还是肝纤维化期。
第九方面，本发明提供了血清GDNF在制备预测或诊断个体是否存在肝硬化的试剂或试剂盒中的应用。
优选地，所述预测或诊断个体是否存在肝硬化是指预测或诊断个体处于非肝硬化分期还是肝硬化期。
第十方面，本发明提供了检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断个体是否存在肝硬化的试剂或试剂盒中的应用。
优选地，所述预测或诊断个体是否存在肝硬化是指预测或诊断个体处于非肝硬化分期还是肝硬化期。
本文中，所述GDNF蛋白和基因可参考NCBI ID:2668。
本文中，肝纤维化具体分级标准参考文献：Histological grading and stagingof chronic hepatitis,Journal of Hepatology 1995；22:696-699。在病理分期中包括炎症分级inflammation stage(简称G，是指肝脏病变的炎症程度)和纤维化分期fibrosisstage(国内简称S分期，国际上简称F stage，是指肝脏病变的纤维化程度)，两者综合在一起判断肝脏的病变程度。
本发明优点在于：
本发明研究了血清GDNF预测肝纤维化程度的效果，从肝组织的病理分期，病理染色阳性面积进行相关性分析，并与公认的肝纤维化无创性诊断指标对血清GDNF预测肝纤维化的准确性加以验证，发现GDNF可作为预测肝纤维化严重性的标志物，具有较高的准确性，在预测F4 vs F0-3阶段、F4 vs F3阶段显著优于公认的肝纤维化无创性诊断指标，具有重要临床价值。
附图说明
图1：正常人与HBV患者基本信息。
图2：患者血清GDNF水平与病理分期的相关性。
图3：血清GDNF在肝脏各病理分级中的表达。A、344例患者根据组织学分级和纤维化分期测定血清GDNF含量。B、血清GDNF含量在HBV患者肝脏病理染色的阳性面积。
图4：患者肝脏GDNF水平与病理分期的相关性。
图5：肝脏GDNF在肝脏各病理分级中的表达。A、mGDNF在HBV患者病理分期中的表达。B、mGDNF的表达与病理染色阳性面积。
图6：GDNF预测肝纤维化严重性。
图7：ROC分析血清GDNF对患者肝纤维化和肝硬化的预测。
图8：慢性HBV患者血清GDNF的总体风险指数。
图9：血清GDNF与无创性肝纤维化诊断APRI、FIB4、Fibrometer、Fornindex和Hepascore对肝纤维化分期预测的比较。
图10：血清GDNF对APRI、FIB4、Fibrotest、Forn指数、Hepascore、经Delong检验的AUROC。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
1、研究方案
本研究通过对350例正常人(2017年12月至2019年7月)的血清和385例慢性乙肝患者的肝活检标本和血清(包括两部分：一为带血清的普陀医院344例；二为上海市公共卫生临床中心41例无血清仅有肝脏活检组织)GDNF含量、肝活检组织GDNF mRNA相对表达量及肝功能(ALT、Alb等)、肝纤维化病理学分期(炎症及纤维化分期)、分析GDNF作为预测肝纤维化指标的科学依据。
2、研究对象
本研究方案经上海中医药大学普陀医院临床伦理委员会和复旦大学上海市公共卫生临床中心批准，符合赫尔辛基宣言的规定。
收集2011年6月至2019年7月在上海中医药大学普陀医院感染科首次就诊的慢性乙肝患者，行肝活检术，记录患者基本信息资料，收集血清样本，测定GDNF含量。
收集2013年11月至2016年3月在复旦大学上海市公共卫生临床中心中西医结合部首次就诊的慢性乙肝患者，行肝穿刺活检术，记录患者基本信息，收集肝穿刺组织41例。
(1)入选标准
①符合《慢性乙型肝炎防治指南(2015年版)》慢性乙型肝炎诊断标准(中华医学会肝病学分会.慢性乙型肝炎防治指南(2015年版).中华实验和临床感染病杂志(电子版).2015；19(5):1-18.)；
②年龄18-70周岁。
(2)排除标准
①14周岁以下或70周岁以上；
②妊娠妇女；
③胸部影像学(胸片或CT)证实支气管炎、肺炎、胸腔积液、间质性病变等；
④有严重的基础病：重度COPD(FEV1/EVC<70％，FEV1占预计值<30％；或出现呼吸衰竭或右心衰竭)、严重肝功能不全(ALT或AST≥正常值3倍)、严重肾功能不全(血清肌酐>2mg/dL)、慢性充血性心力衰竭(心功能NYHAⅢ-Ⅳ级)；
⑤免疫缺陷患者、器官移植或近3月内服用免疫抑制剂或糖皮质激素；
⑥就诊时出现下列情况之一者：
a、呼衰：PaO2<60mmHg伴或不伴PCO2>50mmHg或PaO2/FiO2≤300；
b、循环衰竭：收缩压<90mmHg或需要血管活性药物维持血压；
c、肾衰：有效液体复苏后仍出现少尿或无尿(尿量≤0.5ml/Kg/h)；
d、肝衰：(a)腹胀或腹水。(b)黄疸进行性加深(每天上升17μmol/L)。(c)明显出血倾向，PTA＜40％。(d)肝性脑病。具备四条中任意三条者。
3、检测方法
3.1 血清GDNF的浓度测定：
采用R&D GDNF试剂盒(DY212)及(DY008)，具体步骤为：制备ELISA板稀释捕获抗体向360μg的Capture(捕获抗体管内)加入1ml PBS混匀，稀释至Capture抗体工作液为2μg/ml，向96孔板加入100μl/孔，密封紧密后置于室温过夜。洗板，尽可能吸净Capture，洗板3次。制备反应液，混匀，每孔加入300μL的1*Regent Dilute工作液，37℃烘箱孵育1h，吸净液体。制备标准品，后按照住院号从小到大，加入样本，每个加入样本100μL。封闭置于37℃烘箱孵育2h，洗液洗2-3遍；滴加检测抗体100μl/孔，封闭好，至37℃2h孵育。洗板后加100μlHRP，每孔加入底物液100μl，37℃孵育20min，避光。每孔加终止液50μl，450nm检测吸光度。
3.2 肝功能、肾功能、血脂、血常规、凝血功能、血清HBV-DNA、肝纤维化纤四项检测：
丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、碱性磷酸酶(GGT)、γ-谷氨酰转肽酶(γ-GT)、总胆红素(TBil)、白蛋白(Alb)、触珠蛋白(HPT)、胆碱酯酶(CHE)、肌酐(Cr)、尿素氮(BUN)、甘油三酯(TC)、总胆固醇(TG)、白细胞、红细胞、血红蛋白(Hb)、淋巴细胞、凝血酶原时间(PT)、国际标准化比值(INR)、透明质酸(HA)、IV型胶原(CIV)、层粘连蛋白(LN)和III型前胶原(PCIII)、血清HBV-DNA等由我院检验科检测。
3.3 肝组织病理学检测：
肝脏用16G×20cm一次性针头行穿刺术(肝脏标本长度至少为15毫米)，4％福尔马林固定，石蜡包埋。切片(3毫米厚)用苏木精和伊红(HE)、网状、天狼星红和Masson’s染色。肝穿刺组织病理判定按照Scheuer国际通用肝纤维分期评估方案进行分期(Guido C,MariaG,Aurelio S,Gioacchino L.Impact of liver biopsy size on histologicalevaluation of chronic viral hepatitis:the smaller the sample,the milder thedisease.Journal of Hepatology.2003；39(2):239-244.)，具体：S0，无纤维化；S1，汇管区扩大纤维化，小叶结构完整，无纤维间隔形成；S2，汇管区周围纤维化、纤维间隔形成，小叶结构大部分保留；S3，较多纤维间隔分隔破坏肝小叶，致小叶结构紊乱，但无肝硬化；S4，肝硬化。
3.4 无创性肝纤维化指标计算方法：
APRI：[AST(U/L)/AST(正常值上限)]×100/PLT计数(109/L)；
FIB-4：[年龄×AST(U/L)]/[PLT计数(×109/L)×ALT(U/L)1/2]；
Fibrometer：-0.007PLT(G/L)-0.049PI(％)+0.012AST(U/L)+0.005A2M(mg/dL)+0.021HA(μg/L)-0.270urea(mmol/L)+0.027年龄+3.718；
Fornindex：7.811-3.131×ln(PLT计数)+0.781×ln(GGT)+3.467×ln(年龄)-0.014×(总胆固醇(mg/dl))；
Hepascore：
y＝exp[4.185818-(0.0249×年龄)+(0.7464×性别)+(1.0039×α2M)+(0.0302×HA)+(0.0691×BUN)-(0.0012×GGT)]，男＝1，女＝0；
Fibrotest：4.467×log(α2M)-1.357×log HAP+1.017×log GGT+0.0281×年龄+1.737×log TBil-1.184×ApoA-I+0.301×(性别)-5.540，男＝1，女＝0。
3.5 肝组织GDNF、α-SMA、Col1A1等mRNA表达检测：
按照Trizol(Takara,9109)法提取总RNA后溶于DEPC水，核酸定量分析仪测定总RNA的浓度和纯度，A260nm/A280nm值在1.8-2.1之间即可。参照逆转录试剂盒说明书进行逆转录合成cDNA，反应条件：37℃15min，85℃5s，4℃。PCR扩增：取cDNA 3μL加SYBR Green 5μl，上引物0.4μl，下引物0.4μl，d-dH2O 1.2μl，制成10μl反应体系，在实时荧光定量PCR系统中进行扩增反应。PCR扩增条件：95℃30min预变性；95℃5s变性，60℃30s退火，45个循环，和60℃1min延伸。PCR结束后，采用2-△△CT法分析结果，计算GDNF、α-SMA等基因在各组的相对表达量。
4、统计学方法
数据统计及统计分析采用SPSS 23.0统计软件进行，检测样本的正态性和方差齐性；多组数据比较采用ONE-WAY ANOVA检验，两因素相关性分析采用直线回归分析，多因素相关性分析采用多元回归分析。计量结果以均数±标准差表示。P<0.05为有统计学意义。通过线性回归分析GDNF与其他连续变量之间的关系。通过Spearman’s检测GDNF与组织学评分之间的关系。采用多元回归分析临床、生物学和组织学因素与血清GDNF水平或F4分期的相关性。对使用Youden指数确定了每种模式的最佳截值计算AUROC、截止值和以下性能参数：灵敏度、特异性、阳性预测值(PPV)和阴性预测值(NPV)，用于比较血清GDNF与APRI、FIB4、Fibrotest、Hepacore、Fibrometer、Forn index预测F4的值。
5、结果
5.1 患者基本信息
如图1所示，对照组与慢性乙型肝炎患者组的年龄，性别组成无差异，BUN、红细胞无差异，慢性乙型肝炎患者的ALT、AST和Cr水平高于对照组，血小板和白细胞(WBC)水平低于对照组(P＝0.000)。
5.2 血清GDNF含量在HBV肝纤维化患者中表达升高
检测血清GDNF统计结果表明：350例对照组血清GDNF值为11.4(7.2-17.6)，显著低于344例HBV患者(F0＝26，F1＝84，F2＝133，F3＝67，F4＝34)的血清GDNF值28.3(26.2-31.3)(P＝0.000)(图1)。将344例慢性乙肝患者平均分为低GDNF组(sGDNF-low)和高GDNF组(sGDNF-high)，统计结果表明：G0和G1期患者的血清GDNF水平(平均28.88pg/ml)低于G2(平均32.05pg/ml)和G3-G4(平均35.41pg/ml)(P＝0.040)。与血清GDNF低的HBV患者相比，血清GDNF高的HBV患者的纤维化分期更高(P<0.000)，统计肝穿刺Masson染色和网状纤维染色阳性面积结果表明，在高血清GDNF组中Masson染色和网状纤维染色阳性面积也更大(P＝0.0037)(P＝0.0187)(图2和图3)。
我们将293例HBV患者肝脏GDNF mRNA分为低GDNF组(mGDNF-low)和高GDNF组(mGDNF-high)，统计结果表明：mGDNF高组的表达量在比mGDNF低组的表达量在炎症分期(P＝0.004)、纤维化阶段(P＝0.014)和网状纤维阳性面积(P＝0.045)更高。因此我们得出结论：肝内GDNF mRNA水平在很大程度上与患者血清中sGDNF一致(图4和图5)。
5.3 GDNF可预测肝纤维化
通过检测350例正常人血清和344例(F0＝26，F1＝84，F2＝133，F3＝67，F4＝34)HBV患者血清GDNF含量结果做ROC曲线分析，发现血清GDNF可显著区分正常人与HBV患者[ROC＝0.77(0.71-0.83，P<0.001)]，同时对无肝纤维化患者(F0期)与肝纤维化(F1-4期)也可显著区分[ROC＝0.72(0.66-0.76)，P<0.001)]，进一步统计发现，血清GDNF可将F2期与F3-4期[ROC＝0.64(0.57-0.70)]显著分开，并且血清GDNF可以显著预测F4期肝硬化[ROC＝0.79，(0.71-0.88)，P<0.001]，这些结果提示GDNF可作为预测肝纤维化严重性的标志物，具有重要临床价值(图6)。
5.4 血清GDNF对预测肝纤维化的准确性
为了进一步研究血清GDNF水平对肝纤维化的诊断准确性，我们首先将350例正常人和344例慢性乙肝患者中分为非肝纤维化和肝纤维化期，即正常人和F0期为非肝纤维化患者，F1,F2,F3,F4期为肝纤维化患者，计算ROC值，诊断率为0.83(95％CI，0.80-0.87)。再将350例正常人和344例慢性乙肝患者中分为非肝硬化期和肝硬化期，即正常人和F0,F1,F2,F3期为非肝硬化患者，F4期为肝纤维化患者，计算ROC值，诊断率为0.84(95％CI，0.79-0.89)(图7)。结果表明血清GDNF对区分非肝纤维化和肝纤维化及区分非肝硬化和肝硬化具有较高的准确性，作为预测肝纤维化的血清标志物具有较高的准确性。
5.5 HBV肝纤维化患者中影响GDNF的因素
为了评估患者的什么临床指标可能会影响血清GDNF水平，我们对HBV患者的临床指标进行分析发现，血清GDNF的水平与ALT、Alb和肝纤维化分期密切相关，风险指数分别为ALT([RR]1.002，95％[CI]，1.000-1.003)，Alb([RR]0.935，95％[CI]，0.881-0.992)，纤维化分期([RR]1.468，95％[CI]，1.134-1.902)。进一步多重回归分析结果表明，血清GDNF水平与肝纤维化分期([RR]1.609、95％[CI]，1.128-2.294)显著相关，以上结果进一步证实了血清GDNF可作为预测肝纤维化的指标(图8)。
5.6 血清GDNF含量与无创性肝纤维化诊断APRI、FIB4、Fibrometer、Fornindex和Hepascore的比较
我们将血清GDNF与APRI、FIB-4、Fibrometer、Hepascore、Fornindex和Fibrotest的ROC曲线下面积进行了比较，结果表明：在预测F≥2和F≥3的病理分期中血清GDNF的ROC曲线下面积低于APRI、FIB-4、Fibrometer、Hepascore、Fornindex和Fibrotest，血清GDNF在预测F4阶段和F0-3阶段则显著优于APRI、FIB-4、Fibrometer、Hepascore、Fornindex和Fibrotest。血清GDNF水平与APRI、FIB-4、Fibrometer、Hepascore、Fornindex相比，在诊断F4与F3纤维化分期相比，ROC曲线下面积为0.74(95％CI，0.65-0.83；P＝0.000)，明显高于APRI(0.55；95％CI，0.44-0.65；P＝0.0124)、FIB-4(0.55；95％CI，0.44-0.65；P＝0.0181)、Fibrometer(0.53；95％CI，0.43-0.64)；P＝0.0133)、Forn index(0.57；95％CI，0.47-0.67；P＝0368)和Hepascore(0.55；95％CI，0.45-0.65；P＝0.0211)。表明血清GDNF对F4期CHB患者的预测优于APRI、FIB4、Fibrometer、Fornindex和Hepascore评分(图9和图10)。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.血清GDNF在制备预测或诊断肝纤维化程度的试剂或试剂盒中的应用。
2.检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断肝纤维化程度的试剂或试剂盒中的应用。
3.血清GDNF在制备预测或诊断肝纤维化程度为F0-3还是F4的试剂或试剂盒中的应用。
4.检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断肝纤维化程度为F0-3还是F4的试剂或试剂盒中的应用。
5.血清GDNF在制备预测或诊断肝纤维化程度为F3还是F4的试剂或试剂盒中的应用。
6.检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断肝纤维化程度为F3还是F4的试剂或试剂盒中的应用。
7.血清GDNF在制备预测或诊断个体是否存在肝纤维化的试剂或试剂盒中的应用。
8.检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断个体是否存在肝纤维化的试剂或试剂盒中的应用。
9.血清GDNF在制备预测或诊断个体是否存在肝硬化的试剂或试剂盒中的应用。
10.检测血清中GDNF蛋白或基因含量的试剂在制备预测或诊断个体是否存在肝硬化的试剂或试剂盒中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102654502A (zh) * | 2011-03-02 | 2012-09-05 | 上海交通大学附属第一人民医院 | 慢性乙型肝炎肝纤维化的蛋白标记物及其检测方法 |
| US20140370521A1 (en) * | 2011-12-15 | 2014-12-18 | The USA , as Represented by the Secretary, Department of Health & Human Services | Identification of two novel biomarkers for niemann-pick disease type c |
| WO2017011929A1 (zh) * | 2015-07-17 | 2017-01-26 | 北京大学第一医院 | 检测血清血管生成素样蛋白2含量的物质在制备检测肝脏炎症和纤维化程度产品中的应用 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102654502A (zh) * | 2011-03-02 | 2012-09-05 | 上海交通大学附属第一人民医院 | 慢性乙型肝炎肝纤维化的蛋白标记物及其检测方法 |
| US20140370521A1 (en) * | 2011-12-15 | 2014-12-18 | The USA , as Represented by the Secretary, Department of Health & Human Services | Identification of two novel biomarkers for niemann-pick disease type c |
| WO2017011929A1 (zh) * | 2015-07-17 | 2017-01-26 | 北京大学第一医院 | 检测血清血管生成素样蛋白2含量的物质在制备检测肝脏炎症和纤维化程度产品中的应用 |
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| LE TAO ETAL.: "Glial cell line-derived neurotrophic factor (GDNF) mediates hepatic stellate cell activation via ALK5/Smad signalling" * |
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