WO2019037106A1 - 用于评价乙肝肝病nk细胞功能的实用性模型的构建方法 - Google Patents

用于评价乙肝肝病nk细胞功能的实用性模型的构建方法 Download PDF

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WO2019037106A1
WO2019037106A1 PCT/CN2017/099120 CN2017099120W WO2019037106A1 WO 2019037106 A1 WO2019037106 A1 WO 2019037106A1 CN 2017099120 W CN2017099120 W CN 2017099120W WO 2019037106 A1 WO2019037106 A1 WO 2019037106A1
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cells
indicators
hepatitis
clinical
cell function
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黄月华
曾首杰
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黄月华
璞晞（广州）生物免疫技术有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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  • the invention belongs to the field of biomedicine, and particularly relates to a practical model for evaluating the function of NK cells of hepatitis B liver disease and a construction method thereof.
  • NK cells Natural killer cells are important immune cells in the body. Unlike T cells, their killing activity is MHC-free, independent of antibodies, so it is called natural killing active cells; NK cells are fighting tumors and anti-virus infections. And immune regulation plays an important role, is the first line of defense against the invasion of pathogens and cell mutations in the body. According to the density of CD56 on the surface of NK cells, they can be divided into two subtypes, CD56bright and CD56dim.
  • the functions of NK cells include: (a) natural killing activity: by non-specific recognition of target cells and release of killing mediators such as perforin, NK cytotoxic factor, tumor necrosis factor (TNF- ⁇ ) and the like. NK cell activation is controlled by the balance between inhibitory and activating receptors. (b) Secretion of cytokines, activated NK cells can synthesize and secrete a variety of cytokines to exert immunomodulatory effects.
  • NK cells are abundant in normal liver, accounting for about 31% of intrahepatic lymphocytes. NK cells are mediated by cytokines in the intrahepatic microenvironment, including IL-2, IL-12, IFN- ⁇ ; NK cells act as effector cells to induce hepatocyte apoptosis via FasL-Fas system, and cytoplasmic granules contain perforin And granzymes, and secrete the cytokines IFN- ⁇ and TNF- ⁇ to inhibit pathogen infection or tumors.
  • cytokines in the intrahepatic microenvironment including IL-2, IL-12, IFN- ⁇
  • NK cells act as effector cells to induce hepatocyte apoptosis via FasL-Fas system
  • cytoplasmic granules contain perforin And granzymes, and secrete the cytokines IFN- ⁇ and TNF- ⁇ to inhibit pathogen infection or tumors.
  • NK cells play an important role in clearing tumor cells, virus-infected cells, certain self-organized cells (such as blood cells), parasites, etc., and are one of the main lines of defense against the tumors and anti-infective immune factors, especially in In the advanced stage of chronic inflammation, tumor and other serious diseases, the T cell function in the body collapses, and the role of NK cells is more important. Therefore, objective evaluation of NK cell function is essential for disease status assessment, treatment medication guidance, and prognosis.
  • the liver is the body's largest immune organ.
  • the diseased liver is full of and infiltrated with a large number of immune cells.
  • the clinical outcome of liver disease is closely related to the repair and improvement of immune function in the body.
  • the relevant technologies of the immune system have been continuously improved. From the determination of immunoglobulins and complements in the 1970s, the immune status of the body can be estimated roughly and indirectly. It has been gradually replaced by the detection of lymphocyte subsets and cytokines.
  • Representative methods include: (1) biological methods based on DNA detection, such as PCR, Northern blotting, in situ hybridization, etc.
  • Single immune cytokines are detected and their regulatory pathways are studied, but they are not suitable for discriminating clinically complex cases; (2) biological activity detection methods such as ELISA, ELISPOT, etc., such methods are only for single cells or single immune Detection of cell secretory factors, rather than the average cytokine levels of a group of immune cells, is also not conducive to the identification of clinically complex cases; (3) flow cytometry and liquid chip detection: this is currently more accurate The detection technology can detect multiple cytokines. The clinician estimates the immune status of the patients according to different cytokine levels, but does not involve the evaluation of the overall function of the immune cells.
  • the object of the present invention is to provide a simple and convenient method for constructing a practical model for evaluating the function of NK cells of hepatitis B liver disease for the above technical problems to be solved, and the evaluation model constructed by the method can accurately evaluate hepatitis B liver disease NK cells.
  • the overall function is to provide a simple and convenient method for constructing a practical model for evaluating the function of NK cells of hepatitis B liver disease for the above technical problems to be solved, and the evaluation model constructed by the method can accurately evaluate hepatitis B liver disease NK cells.
  • the present invention provides a method for constructing a practical model for evaluating NK cell function of hepatitis B liver disease, comprising:
  • NK cell function and NK cell function were used as feedback variables to screen out the indicators related to the functional status of NK cells from 14 easily available clinical-virological indicators.
  • the screening method is a stepwise variable screening method, and the standard of screening is AIC.
  • the Youden Index is used to determine the optimal cutoff value of the probability that the patient's immune function is an active group, and the patient is divided into a NK cell functional active group and a NK cell dysfunctional group;
  • Age is age
  • Fibriscan is liver hardness value
  • HBeAg is hepatitis B E antigen
  • HBcAb is hepatitis B core antibody
  • ALB is albumin.
  • the construction method of the present invention further comprises a verification step.
  • the construction method of the present invention externally validates the model using a new patient cohort.
  • the clinical application equation for evaluating NK cell function is:
  • the model constructed by the method of the invention is practical, convenient and simple, can effectively evaluate the overall immune status of each NK cell, and provides an indirect reference for clinical disease evaluation, immunotherapy and anti-viral treatment, and promotes the diagnosis and treatment of liver diseases. Further precision.
  • Figure 1 shows the frequency distribution of NK cells and their subtypes.
  • Figure 2 shows the analysis of NK cells and their subpopulations secreting active cytokines IFN- ⁇ and TNF- ⁇ .
  • Figure 3 shows the correlation between cytokines secreted by NK cells and their subtypes and clinical virological indicators.
  • Figure 4 shows a ROC curve analysis diagram.
  • Figure 5 shows a nomogram
  • Figure 6 shows a roadmap for constructing a NK cell function application model.
  • the inventive idea of the present invention is to pass the activation cytokines secreted by NK cells and their subgroups of liver diseases patients, mainly IFN- ⁇ and TNF- ⁇ ; Monoclonal antibodies for multi-parameter, rapid quantitative analysis of individual cells or other biological particles; The overall immune status of NK cells in patients with natural disease duration was newly evaluated, and a clinical application scoring model capable of assessing the overall immune status of NK cells was established.
  • the aim is to use this simple and practical model to individually evaluate the overall immune status of patients with NK cells, and provide reference for clinical disease assessment, immunotherapy and antiviral therapy, and promote the diagnosis and treatment of liver diseases to further precision.
  • the method of the present invention involves the following:
  • Study cohort 800 patients with hepatitis B disease were included. The diagnostic criteria were based on the 2016 US Liver Disease Research Guide.
  • Validation cohort 300 patients with liver disease.
  • CA chronic hepatitis active patients
  • CAN chronic hepatitis inactive patients
  • use flow cytometry to detect the frequency of peripheral blood NK cell subsets ( Total NK cells, NK dim cells and NK bright cells) and secreted cytokines (IFN- ⁇ and TNF- ⁇ ) were compared between the two groups.
  • IFN- ⁇ and TNF- ⁇ secreted cytokines
  • FIG 1 it is the frequency distribution of NK cells and their subtypes.
  • a in Figure 1 shows a representative NK cell flow detection technique
  • B, C, and D show the levels of total NK cells and their subpopulations (NK dim and NK bright ) in two groups of liver diseases, respectively.
  • CA is a subject of chronic hepatitis active period
  • CAN chronic hepatitis inactive subject
  • HC is a healthy control.
  • Flow cytometry was used to detect the levels of cytokines secreted by peripheral blood NK cells and their subpopulations, and comparison between the two groups was performed.
  • NK cells and their subpopulations were secreted for the analysis of active cytokines IFN- ⁇ and TNF- ⁇ .
  • a in Figure 2 shows the levels of activating factors secreted by NK cells and subpopulations in chronic liver disease; B shows the levels of IFN- ⁇ and TNF- ⁇ secreted by NK cells and their subpopulations in both groups. Distribution map.
  • NK cell function (NK-H) and NK cell function (NK-L) as feedback variables
  • the screening method is a stepwise variable screening method, and the standard of screening is AI C.
  • the Youden Index the best cutoff value for "probability of patient immune function as High group" was determined, and patients were divided into high risk group and low risk group.
  • Figure 4 it is a ROC curve analysis chart: a significant clinical indicator screened out in chronic liver disease as an indirect assessment of the specificity and sensitivity analysis of NK cell function.
  • the AUC area of all subjects was 0.818, and the AUC area of the non-hepatitis active patient group was 0.7608.
  • NK cell function The clinical application equation used to evaluate NK cell function is:
  • Example 1 Regular clinical biochemical tests were performed on 12 patients with chronic hepatitis B to understand their NK cell immune function:
  • the NK cell function detection values of the 12 cases are as follows:
  • the cell function status is as follows:
  • Example 2 Hepatitis B patients, male, 35 years old, review the clinical indicators within six months of surgery, and observe the sequence of virology and biochemical indicators.
  • the time nodes are: 0 weeks, 12 weeks, 24 weeks.
  • the Vnk value at different time points that is, the NK cell immune function test results are as follows:
  • the cell function status is as follows:
  • the NK cell applicability evaluation model of the present invention can not only judge a single instant The overall immune function status of NK cells; further, the trend of the overall immune function of NK cells during non-treatment or treatment can be monitored; since the present invention is based on advanced flow cytometry and flow sorting single cells
  • the detection technology the value is real and reliable; re-integration of biochemical, virological indicators, the use of biometric big data calculation method, the construction of the model, convenient, practical and accurate.
  • the method of the present invention aims to construct a model for detecting and assessing the immune status of NK lymphocytes, with the sole purpose of obtaining intermediate results that are not directly related to the diagnosis or health status of the disease.
  • a comprehensive evaluation of a variety of parameters including not limited to hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody, pre-S1 antigen level and other serological responses and biochemical responses,
  • the NK lymphocyte immune status known only by the model involved in the present invention is not capable of directly detecting and/or diagnosing hepatitis B disease.

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Abstract

提供了一种用于评价乙肝肝病NK细胞功能的实用性模型的构建方法，包括：(1)将乙肝肝病患者分为慢性肝炎活动期患者和慢性肝炎非活动期患者，利用流式细胞技术检测外周血NK细胞及其亚群的频数分布，并进行比较；(2)利用流式细胞技术检测外周血NK细胞及其亚群分泌的细胞因子水平，并进行比较；(3)收集患者的临床指标，将临床指标与NK细胞免疫评价指标进行相关性分析；(4)以NK细胞功能活跃及NK细胞功能低下为反馈变量，利用多变量logistic回归模型筛选出与患者NK细胞功能状态有关的指标；(5)建立评估模型；(6)构建列线图。

Description

用于评价乙肝肝病NK细胞功能的实用性模型的构建方法 技术领域
本发明属于生物医学领域，具体涉及一种用于评价乙肝肝病NK细胞功能的实用性模型及其构建方法。
背景技术
自然杀伤(natural killer，NK)细胞是机体重要的免疫细胞，与T细胞不同的是其杀伤活性无MHC限制，不依赖抗体，因此称为自然杀伤活性细胞；NK细胞在抗击肿瘤、抗病毒感染和免疫调节发挥重要作用，是人体防御病原体入侵和体内细胞变异发生的第一道防线。根据NK细胞表面CD56密度的不同，可将其分为CD56bright和CD56dim两种亚型。NK细胞的功能包括：(a)自然杀伤活性：通过非特异性识别靶细胞并释放杀伤介质，如穿孔素(Perforin)、NK细胞毒因子、肿瘤坏死因子(TNF-α)等。NK细胞活化由抑制性和活化性受体间的平衡所控制。(b)分泌细胞因子，活化的NK细胞可以合成和分泌多种细胞因子，发挥免疫调节作用。
NK细胞在正常肝脏中很丰富，约占肝内淋巴细胞的31％。NK细胞在肝内微环境中的细胞因子介导，包括IL-2、IL-12、IFN-γ；NK细胞作为效应细胞经FasL-Fas系统引起肝细胞凋亡，胞浆颗粒中含有穿孔素和颗粒酶，并分泌细胞因子IFN-γ和TNF-α抑制病原体感染或肿瘤。因此，NK细胞在清除肿瘤细胞、病毒感染细胞、某些自身组织细胞(如血细胞)、寄生虫等扮演着重要角色，是机体抗肿瘤、抗感染的重要免疫因素的主要防线之一，尤其在慢性炎症、肿瘤等严重疾病进展期，体内的T细胞功能塌陷，NK细胞的作用更为重要。因此，客观评价NK细胞功能对疾病状态评估、治疗用药指导、预后判断至关重要。
然而，目前缺乏用于评价肝病NK细胞总体功能的临床检测模型。
肝脏是人体最大的免疫器官，病变肝脏充满和浸润着大量免疫细胞，肝病临床结局与体内免疫功能的修复和改善息息相关。然而，在许多慢性肝病和肝癌患者体内，随着生命组学的发展，免疫系统的相关技术不断进步，从70年代开始的免疫球蛋白、补体等测定，能粗略和间接地估计机体的免疫状态，已经逐步被淋巴细胞亚群、细胞因子的检测替代，代表性的检测手段包括：(1)基于DNA检测的生物学方法，如PCR、RNA印迹、原位杂交等技术，这类方法能够对单一免疫细胞因子进行检测，并研究其调控通路，但不宜作为临床复杂病例的判别；(2)生物活性检测方法，如ELISA、ELISPOT等，这类方法只对单一细胞或者单一免疫细 胞分泌因子进行检测，而非对某一群免疫细胞的平均细胞因子水平进行检测，同样，也不利于作为临床复杂病例的判别；(3)流式细胞技术和液态芯片检测：这是目前较为精准的检测技术，可对多个细胞因子进行检测，临床医生根据不同细胞因子水平对患者的免疫状态进行预估，但并未涉及免疫细胞总体功能评价。
可见，以上的所有检测技术都没有涉及免疫细胞总体功能评价的方法，同时无法对多个细胞因子进行检测，以便临床医生根据不同细胞因子水平对患者的免疫状态进行预估，且带有较强的主观性、相当昂贵、操作繁琐，无法广泛应用。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种简单、方便的用于评价乙肝肝病NK细胞功能的实用性模型的构建方法，通过该方法构建的评估模型能够准确评价乙肝肝病NK细胞的总体功能。
为了实现以上发明目的，本发明提供了一种用于评价乙肝肝病NK细胞功能的实用性模型的构建方法，其包括：
(1)将乙肝肝病患者分为慢性肝炎活动期患者(CA)和慢性肝炎非活动期患者(CAN)，利用流式细胞技术检测外周血NK细胞及其亚群的频数分布，并进行两组间的比较；
(2)利用流式细胞技术检测外周血NK细胞及其亚群分泌的细胞因子水平，并进行两组间的比较；
(3)收集患者的临床指标人口统计学、病毒学、肝功能、肝硬度数值，将临床指标与NK细胞免疫评价指标进行相关性分析，找出有密切相关性的临床指标；
(4)以NK细胞功能活跃及NK细胞功能低下为反馈变量，利用多变量logistic回归模型从14个易获得的临床-病毒学指标中筛选出与患者NK细胞功能状态有关的指标。优选地，筛选的方法是逐步变量筛选法，筛选的标准是AIC。在另一个优选的实施例中，利用约登指数(Youden Index)，确定患者免疫功能为活跃组的概率的最佳cutoff值，把患者划分为NK细胞功能活跃组与NK细胞功能低下组；
(5)以Age、Fibriscan、HBeAg、HBcAb、ALB临床-病毒学指标建立评估模型：
  Estimate Std.Error Pr(>|z|)
(Intercept) 8.079761402 4.561524966 0.076513297
age 0.025878032 0.028016963 0.355665519
fibriscan 0.372221664 0.159550049 0.019650975
HBeAg -0.000459896 0.000281939 0.102850506
HBcAb -2.41486034 0.585470381 3.71E-05
ALB -0.184567872 0.089708896 0.039646921
其中，Age为年龄；Fibriscan为肝硬度值；HBeAg为乙肝E抗原；HBcAb为乙肝核心抗体；ALB为白蛋白。
(6)构建列线图，得到用于评价NK细胞功能的临床应用型方程，使用常规临床指标间接提示NK细胞的总体功能状态。
优选地，本发明的构建方法还包括验证步骤。
更优选地，本发明的构建方法利用新的患者队列对模型进行外部验证。
优选地，用于评价NK细胞功能的临床应用型方程为：
Figure PCTCN2017099120-appb-000001
通过计算，所获得的数值与cutoff值(0.0361)比较，>＝0.0361表示N细胞功能活跃；低于0.0361为NK细胞功能低下。
与现有技术相比，本发明的方法构建的模型实用、方便、简单，能够有效评价各NK细胞整体免疫状态，为临床病情评估、免疫治疗及抗病毒治疗等提供间接参考，推动肝病的诊疗更进一步精准化。
附图说明
图1示出了为NK细胞及其亚型的频数分布。
图2示出了NK细胞及其亚群分泌活性细胞因子IFN-γ和TNF-α分析。
图3表明了NK细胞及其亚型分泌各个细胞因子与临床病毒学指标间的相关性。
图4示出了ROC曲线分析图。
图5示出了列线图。
图6示出了NK细胞功能应用型模型的构建技术路线图。
具体实施方式
下面结合具体实施例和附图，对本发明的技术方案作进一步的说明，但本发明不限于以下实施例。如未特别指出，本发明所涉及的检测技术，如流式细胞术，为本领域已知的技术。
鉴于目前免疫功能检测手段的不足，本发明的发明思路是：通过对肝病患者NK细胞及其亚群分泌的活化性细胞因子，主要是IFN-γ和TNF-α；进行在细胞分子水平上通过单克隆抗体对单个细胞或其他生物粒子进行多参数、快速的定量分析检测；然后经统计分析，重 新评价各自然病程时期患者的NK细胞整体免疫状态，并转化建立了能够评估NK细胞整体免疫状态的临床应用评分模型。旨在利用此简便、实用性模型，个体化评价患者NK细胞整体免疫状态，为临床病情评估、免疫治疗及抗病毒治疗等提供参考，推动肝病的诊疗朝精准化更进一步。
概括地讲，本发明的方法涉及如下：
1、研究队列：纳入800例乙肝肝病患者，诊断标准参照2016年美国肝病研究指南。
验证队列：300例肝病患者。
2、按照上述指南把研究人群进行分组：慢性肝炎活动期患者(CA，400例)和慢性肝炎非活动期患者(CAN，400例)；利用流式细胞技术检测外周血NK细胞亚群频数(total NK cells,NKdim cells and NKbright cells)及分泌的细胞因子(IFN-γand TNF-α)，并进行两组间的比较。如图1所示，为NK细胞及其亚型的频数分布。其中，图1中的A示出了代表性的NK细胞流式检测技术；B、C、D分别示出了总体NK细胞及其亚群(NKdim和NKbright)在两组肝病中的水平比较。CA为慢性肝炎活动期受试对象，CAN为慢性肝炎非活动期受试对象，HC为健康对照。
3、利用流式细胞技术检测外周血NK细胞及其亚群分泌的细胞因子水平，并进行两组间的比较。如图2所示，为NK细胞及其亚群分泌活性细胞因子IFN-γ和TNF-α分析。其中，图2中的A示出了比较慢性肝病中NK细胞和亚群分泌的活化性因子水平；B则示出了两组病例NK细胞及其亚群分泌IFN-γ和TNF-α的水平的分布图。
4、收集研究队列中各个病例的人口统计学、病毒学、肝功能、肝硬度数值等资料，将临床指标与NK细胞免疫评价指标进行相关性分析，找出有密切相关性的临床指标。如图3所示，为NK细胞及其亚群分泌细胞因子(活化性细胞因子、抑制性细胞因子)和表达的表面受体(活化性受体、抑制性受体)与临床指标的相关性分析。其中深色阴影表明两种存在相关性，相关性越强颜色越深；空白则表示不存在相关性。
5、以NK细胞功能活跃(high NK activity，NK-H)及NK细胞功能低下(low NK activity，NK-L)为反馈变量，利用多变量logistic回归模型从14个易获得的临床-病毒学指标中筛选出与患者NK细胞功能状态有关的指标。筛选的方法是逐步变量筛选法，筛选的标准是AI C。利用约登指数(Youden Index)，确定“患者免疫功能为High group的概率”的最佳cutoff值，把患者划分为高风险组与低风险组。如图4所示，为ROC曲线分析图：慢性肝病中筛出的具有显著意义的临床指标，作为间接评估NK细胞功能的特异性和敏感性分析。所有研究对象的AUC面积为0.818，非肝炎活动患者群的AUC面积为0.7608。
6、最终得到4个重要的临床-病毒学指标(Fibrosis value、HBsAg、HBcAb、ALB)：建 立评估模型：
  Estimate Std.Error Pr(>|z|)
(Intercept) 8.079761402 4.561524966 0.076513297
age 0.025878032 0.028016963 0.355665519
fibriscan 0.372221664 0.159550049 0.019650975
HBeAg -0.000459896 0.000281939 0.102850506
HBcAb -2.41486034 0.585470381 3.71E-05
ALB -0.184567872 0.089708896 0.039646921
7、构建列线图(nomograms)，得到用于评价NK细胞功能的临床应用型方程，使用常规临床指标间接提示NK细胞的总体功能状态。
列线图如图5所示。
用于评价NK细胞功能的临床应用型方程为：
Figure PCTCN2017099120-appb-000002
通过计算，所获得的数值与cutoff值(0.0361)比较，>＝0.0361表示N细胞功能活跃；低于0.0361为NK细胞功能低下。
8、利用新的患者队列对评估模型进行外部验证。
应用实施例
实例一：对12例慢性乙肝患者进行常规临床生化检测，了解其NK细胞免疫功能：
该12例病例的部分临床指标如下：
病例/指标 Age HBeAg HBcAb ALB Fibrosis
1 33 34 1.6 43 5.4
2 23 2 10.7 44 5.2
3 25 46 32.5 43 6.2
4 45 35 0.007 39 7.8
5 34 36 4.5 51 6.3
6 38 0.34 0.3 48 6.8
7 54 0.45 0.67 41 4.7
8 62 0.63 0.76 43 3.5
9 25 47 21.2 49 8.5
10 41 0.32 0.056 41 12.3
11 67 0.32 0.045 40 9.8
12 45 0.12 0.021 39 6.9
(2)利用以上本发明的评估模型(Vnk模型)，该12例病例的NK细胞功能检测值如下：
病例 1 2 3 4 5 6 7 8 9 10 11 12
Vnk 0.2944 8.6199 -28.1683 0.9927 0.0001 0.8818 0.8850 0.7712 4.6581 0.9976 0.9975 0.9887
(3)根据Vnk模型的cutoff值为0.0361，Vnk>＝0.0361表示NK细胞功能正常或活跃(NK-H)；Vnk<0.0361表示NK细胞功能低下(NK-L)，以此判断上述病例的NK细胞功能状态如下：
Figure PCTCN2017099120-appb-000003
实例二：乙肝肝癌患者，男性，35岁，手术治疗半年内复查临床指标，进行病毒学和生化指标的序列观察，时间节点是：0周、12周、24周。
(1)临床指标如下：
Figure PCTCN2017099120-appb-000004
(2)根据本发明的NK细胞功能评价模型，在不同时间点的Vnk值，即NK细胞免疫功能检测结果如下：
治疗时间点 0周 12周 24周
Vnk 0.9255 0.9426 0.9871
(3)根据Vnk模型的cutoff值为0.0361，Vnk>＝0.0361表示NK细胞功能正常或活跃(NK-H)；Vnk<0.0361表示NK细胞功能低下(NK-L)，以此判断上述病例的NK细胞功能状态如下：
Figure PCTCN2017099120-appb-000005
因此，从以上实例可以证实，本发明的NK细胞应用性评估模型不仅可以判断单次的瞬 时的NK细胞整体的免疫功能状态；更进一步地，可以监控在非治疗或者治疗过程中NK细胞整体免疫功能的变化趋势；由于本发明建基于先进的流式细胞技术和流式分选单个细胞的检测技术，数值真实可靠；再融合生化、病毒学指标，利用生物统计大数据的计算方法，进行模型的构建，方便、实用、准确。
值得注意的是，本发明的方法旨在构建用于检测和评估NK淋巴细胞免疫状态的模型，直接目的仅仅在于获得中间结果，该中间结果与疾病的诊断或健康状况并无直接必然联系。对于乙肝疾病的治疗和诊断，需要综合评价众多参数，包括并不限于乙肝病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体、前S1抗原水平等多项血清学应答以及生化学应答，而仅仅经由本发明所涉及的模型所获知的NK淋巴细胞免疫状态并不能够直接检测和/或诊断乙肝疾病。
综上所述以上实施例不过是本发明的最佳实施方案，不可理解为对本发明保护范围的限定，对于该领域的技术工作人员根据本发明的实施例所做的不超出本发明技术方案的调整和改动，应认为落在本发明的保护范围内。

Claims (6)

  1. 一种用于评价乙肝肝病NK细胞功能的实用性模型的构建方法，其特征在于包括：
    (1)将乙肝肝病患者分为慢性肝炎活动期患者和慢性肝炎非活动期患者，利用流式细胞技术检测外周血NK细胞及其亚群的频数分布，并进行两组间的比较；
    (2)利用流式细胞技术检测外周血NK细胞及其亚群分泌的细胞因子水平，并进行两组间的比较；
    (3)收集患者的临床指标人口统计学、病毒学、肝功能、肝硬度数值，将临床指标与NK细胞免疫评价指标进行相关性分析，找出有密切相关性的临床指标；
    (4)以NK细胞功能活跃及NK细胞功能低下为反馈变量，利用多变量logistic回归模型从多个易获得的临床-病毒学指标中筛选出与患者NK细胞功能状态有关的指标；
    (5)以Age、Fibrosis检测值、HBeAg、HBcAb、ALB临床-病毒学指标建立评估模型：
      Estimate Std.Error Pr(>|z|) (Intercept) 8.079761402 4.561524966 0.076513297 age 0.025878032 0.028016963 0.355665519 fibriscan 0.372221664 0.159550049 0.019650975 HBeAg -0.000459896 0.000281939 0.102850506 HBcAb -2.41486034 0.585470381 3.71E-05 ALB -0.184567872 0.089708896 0.039646921
    其中，Age为年龄；Fibriscan为肝硬度值；HBeAg为乙肝E抗原；HBcAb为乙肝核心抗体；ALB为白蛋白；
    (6)构建列线图，得到用于评价NK细胞功能的临床应用型方程，使用常规临床指标间接提示NK细胞的总体功能状态。
  2. 根据权利要求1所述的方法，其特征在于还包括验证步骤。
  3. 根据权利要求2所述的方法，其特征在于：利用新的患者队列对模型进行外部验证。
  4. 根据权利要求1所述的方法，其特征在于：用于评价NK细胞功能的临床应用型方程为：
    Figure PCTCN2017099120-appb-100001
  5. 根据权利要求1所述的方法，其特征在于：所述步骤(4)中，筛选的方法是逐步变量筛选法，筛选的标准是AIC。
  6. 根据权利要求1所述的方法，其特征在于：所述步骤(4)中，利用约登指数确定患者免疫功能为活跃组的概率的最佳cutoff值，该临界值为0.0361，把患者划分为NK细胞功能活跃组与NK细胞功能低下组。
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