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WO2017011929A1 - 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 - Google Patents

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 Download PDF

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WO2017011929A1
WO2017011929A1 PCT/CN2015/000515 CN2015000515W WO2017011929A1 WO 2017011929 A1 WO2017011929 A1 WO 2017011929A1 CN 2015000515 W CN2015000515 W CN 2015000515W WO 2017011929 A1 WO2017011929 A1 WO 2017011929A1
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liver
protein
content
detecting
degree
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French (fr)
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王贵强
邓永琼
赵鸿
李俊
王艳
洪源
周继远
贾伟
方雨晴
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Peking University First Hospital
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Peking University First Hospital
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the use of a substance for detecting serum angiopoietin-like protein 2 in the field of biomedicine for the preparation of a product for detecting liver inflammation and fibrosis.
  • HBV chronic hepatitis B virus
  • liver biopsy is an invasive examination, there are errors in interpretation and sampling, and there is a risk of bleeding, bile leakage, etc., and it is not applicable to all patients.
  • the existing non-invasive methods are only suitable for the judgment of the degree of liver fibrosis, and the study population only focuses on patients with chronic hepatitis B, and is not suitable for the judgment of treatment decisions for all patients with chronic HBV infection. Therefore, there is an urgent need to develop non-invasive methods to assist in the formulation of clinical treatment decisions.
  • Angiopoetin-like protein 2 (angptl2) is a member of the angiopoietin-like white family, which regulates the deposition and degradation of extracellular matrix by increasing the expression and activity of metalloproteinases.
  • the functional receptor integrin of angiopoietin-like protein is widely expressed on macrophages, vascular endothelial cells, and fine surface of fat.
  • the technical problem to be solved by the present invention is how to detect the degree of inflammation and degree of fibrosis in the liver of patients with chronic hepatitis B virus (HBV) infection, and to determine the treatment decision of the patient.
  • HBV chronic hepatitis B virus
  • the present invention first provides any of the following uses 1)-8):
  • serum angiopoietin-like protein 2 as a marker for detecting the degree of human liver inflammation and the degree of fibrosis in the preparation of screening or assisted screening for the application of liver puncture liver disease patients;
  • serum angiopoietin-like protein 2 as a marker for detecting the degree of human liver inflammation and/or the substance used in the method of fibrosis for detecting the degree of inflammation and/or the degree of fibrosis in human liver;
  • M3 the use of a substance for detecting the degree of human liver inflammation and/or the degree of fibrosis using serum angiopoietin-like protein 2 as a marker for screening or assisting screening for the need for liver puncture liver disease patients;
  • the present invention also provides the following products of P1 or P2:
  • P1 a product for detecting a degree of liver inflammation and/or a degree of fibrosis, which is the substance for detecting a serum angiogenic factor 2 protein content;
  • a product for detecting a degree of liver inflammation and/or a degree of fibrosis which is a substance for detecting serum angiopoietin-like protein 2 content, hepatitis B e antigen, HBV DNA content, and ALT content.
  • the substance for detecting serum angiopoietin-like protein 2 content may include an agent and/or an instrument for detecting serum angiopoietin-like protein 2 content, such as detecting serum angiopoietin-like protein 2 content by enzyme-linked immunosorbent assay.
  • Required reagents and instruments may include serum angiopoietin-like protein 2, serum angiopoietin-like protein 2 antibody, and other reagents and instruments required for performing an enzyme-linked immunosorbent assay (ELISA).
  • the above-mentioned substance for detecting serum angiopoietin-like protein 2 may be composed only of serum angiopoietin-like protein 2 and serum angiopoietin-like protein 2 antibody, or may be composed only of serum angiopoietin-like protein 2, It can be composed only of serum angiopoietin-like protein 2 antibody.
  • the serum angiopoietin-like protein 2, the serum angiopoietin-like protein 2 antibody, and other reagents required for performing an enzyme-linked immunosorbent assay (ELISA) can be individually packaged.
  • the substance for detecting serum angiopoietin-like protein 2 content, hepatitis B e antigen, HBV DNA content and ALT content may be detected by a substance for detecting serum angiopoietin-like protein 2 content, and detecting hepatitis B e antigen.
  • a substance for detecting serum angiopoietin-like protein 2 content, and detecting hepatitis B e antigen substance that detects HBV DNA content
  • substance that detects ALT content substance that detects ALT content.
  • the substance for detecting the serum angiopoietin-like protein 2 content, the substance for detecting the hepatitis B e antigen, the substance for detecting the HBV DNA content, and the substance for detecting the ALT content may each include a reagent and/or a kit and/or an instrument.
  • the serum angiopoietin-like protein 2 antibody may be a serum angiopoietin-like protein 2 monoclonal antibody and/or a serum angiopoietin-like protein 2 polyclonal antibody.
  • the serum angiopoietin-like protein 2 may be a marker-labeled serum angiopoietin-like protein 2.
  • the serum angiopoietin-like protein 2 may specifically be human serum angiopoietin-like protein 2.
  • the serum angiopoietin-like protein 2 may be a protein whose amino acid sequence is the sequence 1 in the sequence listing.
  • the serum angiopoietin-like protein 2 antibody may be a biotin-labeled serum angiopoietin-like protein 2 antibody.
  • the serum angiopoietin-like protein 2 antibody recognizes a protein whose amino acid sequence is SEQ ID No. 1 in the Sequence Listing.
  • the substance for detecting the serum angiopoietin-like protein 2 content may be the serum angiopoietin-like protein 2 and/or the serum angiopoietin-like protein 2 antibody.
  • the substance for detecting the serum angiopoietin-like protein 2 content may be a reagent and an apparatus required for detecting the serum angiopoietin-like protein 2 content by an enzyme-linked immunoreactivity reaction.
  • the human may specifically be a liver disease patient infected with chronic hepatitis B virus (HBV).
  • HBV chronic hepatitis B virus
  • the liver disease patient may be a chronic hepatitis B virus (HBV) infected person.
  • the serum angiopoietin-like protein 2 content may specifically be a serum angiopoietin-like protein 2 concentration in human serum or plasma;
  • the HBV DNA content may specifically be a HBV DNA concentration in human serum or plasma;
  • the ALT content may specifically be the ALT concentration in human serum or plasma.
  • the product for detecting the degree of human liver inflammation and/or the degree of fibrosis may be various reagents or kits for detecting the degree of inflammation and/or degree of fibrosis in human liver.
  • the diagnostic or auxiliary diagnosis of a patient with liver disease requires a liver puncture test product may be various reagents or kits for diagnosing or assisting diagnosis of whether a liver disease patient needs a liver puncture test.
  • the screening or assisted screening of whether the liver puncture liver disease patient product is available may be a variety of reagents or kits for screening or assisting screening for patients requiring liver puncture liver disease.
  • substances that detect serum angiopoietin-like protein 2 levels and substances that detect other indicators can be combined to prepare products for detecting the degree of inflammation and/or fibrosis of human liver, and whether diagnosis or assisted diagnosis of liver disease patients is required.
  • HBV DNA content in human serum or plasma alanine aminotransferase (ALT) content, hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg), platelet (PLT) Count, aspartate aminotransferase (AST) content, glutamyl transpeptidase (GGT) content, albumin (Abumin) content, total bilirubin (Tbil) content, prothrombin time (PT), can also be a fibrosis model Such as APRI, FIB-4 and AAR.
  • the substance for detecting other indicators may be a reagent and/or an instrument used for detection by a conventional method.
  • the product can be a reagent or kit, and can also be a system consisting of reagents or kits and instruments.
  • the method for determining a treatment plan for a patient with liver disease according to the content of serum angiopoietin-like protein 2 is as follows: detecting serum angiopoietin-like protein 2 content in a patient with chronic HBV infection, and if the serum angiopoietin-like protein 2 content is ⁇ in a patient with chronic HBV infection ⁇ 7.36 ng / ml, the patient needs immediate antiviral treatment, if the serum angiopoietin-like protein 2 content of patients with liver disease ⁇ 3.95 ng / ml, the patient does not need treatment, if the serum angiogenic protein 2 content of patients with liver disease is greater than 3.95 The ng/ml is less than 7.36 ng/ml, and the patient needs to undergo liver penetration to determine the treatment plan.
  • HBV DNA content can also be detected first, if the patient does not meet the Asia Pacific and American Liver Disease Association for treatment of chronic hepatitis B virus (HBV) infection.
  • ALT alanine aminotransferase
  • HBeAg status can also be detected first, if the patient does not meet the Asia Pacific and American Liver Disease Association for treatment of chronic hepatitis B virus (HBV) infection.
  • the immediate treatment criteria and follow-up criteria in the decision-making guidelines determine the treatment options for patients with liver disease based on the method of determining the therapeutic profile of patients with liver disease based on serum angiopoietin-like protein 2 levels.
  • the immediate treatment criteria for the Asia-Pacific and American Hepatology Society guidelines for chronic hepatitis B are HBeAg-positive, HBV DNA ⁇ 20000 IU/mL, ALT ⁇ 2 ULN or HBeAg-negative, HBV DNA ⁇ 2000 IU/mL, ALT ⁇ 2 ULN;
  • the liver penetrating is for pathological examination of liver tissue by liver puncture.
  • the degree of inflammation of the liver may be mild and/or moderately severe; the hepatic activity index (HAI) of the mild inflammation is 0-4 (ie 0 ⁇ HAI ⁇ 4), liver disease with mild inflammation of the liver
  • HAI hepatic activity index
  • the serum angiopoietin-like protein 2 content of the patient is ⁇ 3.95 ng/ml, and the liver activity index of the moderate to severe inflammation is greater than or equal to 5 (ie, HAI ⁇ 5), and the serum angiopoietin-like protein 2 of the liver disease in the liver is severely inflammatory.
  • the content is ⁇ 7.36 ng / ml.
  • the degree of liver fibrosis may be marked fibrosis and/or non-significant fibrosis;
  • the apparent fibrosis is a serum angiopoietin-like protein of a liver disease patient with Fibrosis of 3 or more (ie, F ⁇ 3) and liver fibrosis 2 content ⁇ 7.36 ng / ml
  • the non-significant fibrosis is Fibrosis 0-2 (ie 0 ⁇ F ⁇ 2)
  • the serum angiopoietin-like protein 2 content of liver disease patients with liver non-significant fibrosis ⁇ 3.95 ng / Ml.
  • Figure 1 shows the relationship between the content of angptl2 in serum and the degree of inflammation in the liver and the degree of fibrosis.
  • A is the relationship between angptl2 content and liver inflammation degree
  • B is the relationship between angptl2 content and liver fibrosis degree.
  • Figure 2 is a single index angptl2 diagnosis of liver transfusion to determine the ROC curve of moderate to severe inflammation or significant fibrosis in patients in the treatment decision group. Among them, 1 indicates a cutoff value of 3.95 ng/ml, and 2 indicates a cutoff value of 7.36 ng/ml.
  • Figure 3 is a flow chart of treatment decisions for ash patients who did not meet the criteria for the immediate treatment of chronic hepatitis B guidelines and follow-up criteria for the IA Society of Hepatology.
  • Receiver operating characteristic reflects the balance between sensitivity and specificity.
  • the area under the ROC curve is an important test accuracy index. The larger the area under the ROC curve, the greater the diagnostic value of the test.
  • Sensitivity true positive rate: The actual disease is correctly judged as the percentage of disease according to the test standard. The greater the sensitivity, the better, and the ideal sensitivity is 100%.
  • Example 1 angptl2 can be used to determine treatment decisions in patients with chronic HBV infection
  • Study subjects (1) Case source: 309 patients with chronic HBV (hepatitis B virus) infection. (2) Inclusion criteria: age 18-65 years old; HBsAg positive history for half a year or more (quantitative detection of HBsAg in serum using the US Roche kit (Roche HBsAg II assay)). (3) Exclusion criteria: patients with chronic hepatitis C and AIDS, combined with hereditary liver disease, drug liver, non-alcoholic fatty liver and other basic patients; primary liver cancer, cirrhosis patients and pregnant women. Informed consent was signed before all subjects were enrolled.
  • angptl2 can be used to determine treatment decisions in patients with chronic HBV infection
  • liver puncture 309 patients with chronic HBV infection were under color Doppler guidance according to standard liver pathological puncture criteria.
  • the liver tissue puncture specimen has a length of 2.0 cm or more and contains at least 11 intact portal areas.
  • the liver puncture specimens were fixed, dehydrated, transparent, waxed, embedded, and sectioned, and subjected to conventional HE staining, special staining (including reticular fibers, Masson trichrome staining) and immunohistochemical staining (HBsAg, HBcAg, and CK7).
  • the pathological specimens were uniformly interpreted by centralization blind method. The degree of liver inflammation and liver fibrosis were graded according to the Ishak criteria.
  • the degree of inflammation in the liver is mild to moderate to severe; the hepatic activity index (HAI) of mild inflammation is 0-4 (ie 0 ⁇ HAI ⁇ 4), and the index of liver activity of moderate to severe inflammation is greater than or equal to 5 (ie HAI) ⁇ 5).
  • the degree of liver fibrosis is markedly fibrotic and non-significant fibrosis; significant fibrosis is Fibrosis greater than or equal to 3 (ie, F ⁇ 3), and non-significant fibrosis is Fibrosis 0-2 (ie, 0 ⁇ F ⁇ 2).
  • liver biopsy liver biopsy
  • liver penetrating tissue less than 2.0 CM, or less than 11 portal areas
  • Hematology and biochemical examinations are based on the results of the patient within 4 weeks of liver puncture.
  • HBV DNA in serum was performed using Roche COBAS AmpliPrep/COBAS TaqMan (detection range: 2.0 x 101 - 1.7 x 108 IU/ml). Determination of hepatitis B e antigen and hepatitis B e antibody in serum using Roche, USA The assays were qualitatively analyzed. Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were quantified using an automated biochemical analyzer.
  • ALT serum alanine aminotransferase
  • AST serum aspartate aminotransferase
  • the clinical treatment decision criteria (see Table 1) were established. Patients who met the immediate treatment criteria, follow-up criteria, and liver penetration criteria were divided into immediate treatment group, follow-up group, and need. Liver wear determines the treatment decision group.
  • liver puncture is required to make treatment decisions.
  • the serum levels of HBV DNA, HBsAg, ALT and AST in patients with treatment decision-making group were lower than those in the follow-up group.
  • 0 ⁇ F ⁇ 2 accounted for 115 cases (66.5%)
  • F ⁇ 3 accounted for 58 cases (33.5%)
  • 0 ⁇ HAI ⁇ 4 accounted for 115 cases (66.5%)
  • HAI ⁇ 5 accounted for 58 cases (33.5%).
  • Serum serum angiopoietin-like protein 2 (the amino acid sequence of serum angiopoietin-like protein 2 is shown in SEQ ID NO: 1 in the sequence listing) was measured in 298 eligible biopsy specimens: ELISA kit - Human ANGPTL2 Assay kit ( Immuno-Biological Laboratories Co., Ltd, Japan) (Human ANGPTL2 Assay kit contains an antibody recognizing serum angiopoietin-like protein 2 and serum angiopoietin-like protein 2 as a standard) was analyzed. In order to avoid repeated freezing and thawing of blood, blood specimens are dispensed after collection and stored in a -80 ° C refrigerator. All tests are completed within 2 weeks.
  • the coefficient of variation between the complex holes is greater than 10% and the standard R square is less than 0.99.
  • the measurement range is from 0.05 ng/ml to 3.5 ng/ml, and the samples are all diluted 10-fold.
  • the relationship between serum angptl2 content and hepatic inflammation and fibrosis was analyzed. The results are shown in Figure 1.
  • the content of angptl2 was 4.55 ⁇ 2.01ng/ml.
  • the content of angptl2 was 5.68 ⁇ 2.37ng/ml; the correlation between serum angptl2 and liver fibrosis grade was better.
  • angptl2 content was 4.52 ⁇ 1.75 ng / ml, in 98 patients with F ⁇ 3, angptl2 content was 6.27 ⁇ 2.69 ng / Ml.
  • the area under the working curve (AUC) was 0.707, while the liver was diagnosed with the previous fibrosis models APRI, FIB-4, and AAR.
  • the AUC of marked fibrosis were 0.672, 0.661, and 0.553, respectively (Table 3), indicating that the use of serum angptl2 as a single indicator for the diagnosis of liver fibrosis is superior to the fibrotic models APRI, FIB-4, and AAR.
  • ALT ⁇ ULN ALT ⁇ ULN
  • HAI ⁇ 5 severe inflammation
  • F ⁇ 3 obvious fibrosis
  • the single-index angptl2 diagnosed moderate to severe inflammation or marked fibrosis with an AUC of 0.691 (Fig. 2). Further leave-one-out cross-validation suggests that a single indicator can correctly determine 64.0% of patients.
  • the moderate and severe inflammation and obvious fibrosis were diagnosed with the concentration of 7.36 ng/ml of the index, the specificity (true negative rate) and the positive predictive rate (the probability of the test was positive, the probability of the patient) were 91.76.
  • the serum angptl2 content ⁇ 7.36 ng / ml can be used as the moderate or severe inflammation or significant fibrosis threshold, the serum angptl2 content ⁇ 3.95 ng / ml as the exclusion of moderate to severe inflammation or significant fibrosis threshold, liver treatment to determine treatment decisions Among the 173 patients in the group, 86 patients (49.7%) did not need to undergo liver puncture to determine the treatment plan (Fig.
  • ALT is normal (ALT ⁇ ULN)
  • the levels of angptl2 in the serum of patients with severe inflammation (HAI ⁇ 5) and obvious fibrosis (F ⁇ 3) are milder than those of mild inflammation (0 ⁇ HAI ⁇ 4).
  • the specificity (true negative rate, ie the probability of being diagnosed as non-patient without actual disease) and the positive predictive rate (test diagnosis) For those who were positive, the probability of being a patient was 91.76% and 72% respectively; when the angptl2 content was 3.95 ng/ml as the cut-off value for the diagnosis of moderate to severe inflammation and obvious fibrosis, the specificity (true negative rate, ie, actual The probability of being diagnosed as a non-patient and the negative predictive rate (the probability of being negative in the test diagnosis) were 78.41% and 70.8%, respectively.
  • the invention determines the treatment decision of chronic HBV infection by checking the content of serum angptl2, and can avoid clinical liver puncture in 49.7% of patients.
  • angptl2 content can be used as a non-invasive single index to determine the degree of liver inflammation and fibrosis.
  • You can use angptl2 content to determine the degree of inflammation and fibrosis in the liver of patients with chronic hepatitis B virus (HBV) infection, or use angptl2 Content and other common clinical indicators of patients with liver disease to determine the degree of inflammation and fibrosis in the liver of patients with chronic hepatitis B virus infection; the preparation of substances for detecting serum angiopoietin-like protein 2 can be used to detect the degree of liver inflammation and/or fiber Level of product, diagnosis or diagnosis of liver disease patients requiring liver puncture testing products or screening or assisted screening for liver puncture liver disease patients, or serum angiopoietin-like protein 2 content In combination with substances that detect common clinical indicators in patients with other liver diseases, prepare products for the detection of the degree of inflammation and/or fibrosis of human liver, diagnose or assist in the diagnosis of liver disease patients for products requiring

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Abstract

公开了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。血清中angptl2含量与慢性乙型肝炎病毒感染患者肝脏的炎症程度及纤维化程度具有相关性，angptl2含量可作为非侵袭性单一指标判断肝脏炎症程度及纤维化程度。检测血清血管生成素样蛋白2含量的物质或其与检测肝病患者的其他常见临床指标的物质联合可用于制备检测人肝脏炎症程度和/或纤维化程度的产品、诊断或辅助诊断肝病患者是否需进行肝穿刺检查的产品、或筛查或辅助筛查是否需进行肝穿刺肝病患者的产品。

Description

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 技术领域
本发明涉及生物医学领域中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。
背景技术
亚太及美国肝病协会关于慢性乙型肝炎病毒(HBV)感染者治疗决策指南推荐：ALT≥2ULN、HBV DNA≥20000IU/mL(HBeAg阳性者)或者HBV DNA≥2000IU/mL(HBeAg阴性者)的慢性HBV感染者需给予抗病毒治疗。而对于不满足上述界值的灰区患者，则需肝脏穿刺活检病理结果的辅助确定治疗方案。肝穿刺病理显示中重度炎症或明显纤维化者需立即进行抗病毒治疗。但肝脏穿刺活检是侵袭性检查，存在判读和取样误差，且有出血、胆漏等风险，不适用于应用于所有患者。而已有的非侵袭性方法仅适用于肝脏纤维化程度的判断，且研究人群仅关注慢性乙型病毒性肝炎患者，不适用于全部慢性HBV感染患者治疗决策的判断。因此，临床亟需开发非侵袭性方法辅助临床治疗决策的制定。
血管生成素样蛋白2(Angiopoetin-like protein 2，angptl2)是血管生成素样白家族成员之一，能通过增加金属蛋白酶的表达及活性从而调节细胞外基质的沉积及降解。血管生成素样蛋白的功能性受体整合素广泛表达于巨噬细胞、血管内皮细胞、脂肪细表面。
发明公开
本发明所要解决的技术问题是如何检测慢性乙型肝炎病毒(HBV)感染患者肝脏的炎症程度及纤维化程度，并确定患者的治疗决策。
为解决上述技术问题，本发明首先提供了下述1)-8)中任一用途：
1)检测血清血管生成素样蛋白2(angptl2)含量的物质在制备检测人肝脏炎症和/或纤维化产品中的应用；
2)检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、HBV DNA含量和丙氨酸转氨酶(ALT)含量的物质在制备检测人肝脏炎症程度和/或纤维化程度产品中的应用；
3)以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在制备检测人肝脏炎症程度和/或纤维化程度产品中的应用；
4)下述Y1-Y3中任一应用：
Y1、检测血清血管生成素样蛋白2含量的物质在制备筛查或辅助筛查是否需进行肝穿刺肝病患者产品中的应用；
Y2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在制备筛查或辅助筛查是否需进行肝穿刺 肝病患者产品中的应用；
Y3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和纤维化程度方法中所用的物质在制备筛查或辅助筛查是否需进行肝穿刺肝病患者产品中的应用；
5)下述Z1-Z3中任一应用：
Z1、检测血清血管生成素样蛋白2含量的物质在制备诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品中的应用；
Z2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在制备诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品中的应用；
Z3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在制备诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品中的应用；
6)下述L1-L3中任一应用：
L1、检测血清血管生成素样蛋白2含量的物质在检测人肝脏炎症程度和/或纤维化程度中的应用；
L2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在检测人肝脏炎症程度和/或纤维化程度中的应用；
L3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化方法中所用的物质在检测人肝脏炎症程度和/或纤维化程度中的应用；
7)下述M1-M3中任一应用：
M1、检测血清血管生成素样蛋白2含量的物质在筛查或辅助筛查是否需进行肝穿刺肝病患者中的应用；
M2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在筛查或辅助筛查是否需进行肝穿刺肝病患者中的应用；
M3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在筛查或辅助筛查是否需进行肝穿刺肝病患者中的应用；
8)下述N1-N3中任一应用：
N1、检测血清血管生成素样蛋白2含量的物质在诊断或辅助诊断肝病患者是否需进行肝穿刺检测中的应用；
N2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在诊断或辅助诊断肝病患者是否需进行肝穿刺检测中的应用；
N3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤 维化程度方法中所用的物质在诊断或辅助诊断肝病患者是否需进行肝穿刺检测中的应用。
为解决上述技术问题，本发明还提供了下述P1或P2的产品：
P1、检测人肝脏炎症程度和/或纤维化程度产品，为所述检测血清血管生成素样蛋白2含量的物质；
P2、检测人肝脏炎症程度和/或纤维化程度产品，为所述检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、HBV DNA含量和ALT含量的物质。
本发明中，所述检测血清血管生成素样蛋白2含量的物质可包括检测血清血管生成素样蛋白2含量的试剂和/或仪器，如通过酶联免疫反应检测血清血管生成素样蛋白2含量所需的试剂和仪器。具体来说，检测血清血管生成素样蛋白2含量的物质可包括血清血管生成素样蛋白2、血清血管生成素样蛋白2抗体和进行酶联免疫吸附实验(ELISA)所需要的其它试剂和仪器，当然，上述检测血清血管生成素样蛋白2含量的物质可只由血清血管生成素样蛋白2和血清血管生成素样蛋白2抗体组成，也可只由血清血管生成素样蛋白2组成，也可只由血清血管生成素样蛋白2抗体组成。所述血清血管生成素样蛋白2、所述血清血管生成素样蛋白2抗体和进行酶联免疫吸附实验(ELISA)所需要的其它试剂均可独立包装。
本发明中，所述检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、HBV DNA含量和ALT含量的物质可由检测血清血管生成素样蛋白2含量的物质、检测乙型肝炎e抗原的物质、检测HBV DNA含量的物质和检测ALT含量的物质组成。检测血清血管生成素样蛋白2含量的物质、检测乙型肝炎e抗原的物质、检测HBV DNA含量的物质和检测ALT含量的物质均可包括试剂和/或试剂盒和/或仪器。
本发明中，所述血清血管生成素样蛋白2抗体可为血清血管生成素样蛋白2单克隆抗体和/或血清血管生成素样蛋白2多克隆抗体。
本发明中，所述血清血管生成素样蛋白2可为标记物标记的血清血管生成素样蛋白2。
本发明中，所述血清血管生成素样蛋白2具体可为人血清血管生成素样蛋白2。
本发明中，所述血清血管生成素样蛋白2可为氨基酸序列是序列表中序列1的蛋白质。
本发明中，所述血清血管生成素样蛋白2抗体可为用生物素标记的血清血管生成素样蛋白2抗体。
本发明中，所述血清血管生成素样蛋白2抗体可识别氨基酸序列是序列表中SEQ ID No.1的蛋白质。
本发明中，所述检测血清血管生成素样蛋白2含量的物质可为所述血清血管生成素样蛋白2和/或所述血清血管生成素样蛋白2抗体。
本发明中，所述检测血清血管生成素样蛋白2含量的物质可为通过酶联免疫反应检测所述血清血管生成素样蛋白2含量所需的试剂和仪器。
本发明中，所述人具体可为慢性乙型肝炎病毒(HBV)感染的肝病患者。所述肝病患者可为慢性乙型肝炎病毒(HBV)感染者。
本发明中，所述血清血管生成素样蛋白2含量具体可为人血清或血浆中的血清血管生成素样蛋白2浓度；所述HBV DNA含量具体可为人血清或血浆中的HBV DNA浓度；所述ALT含量具体可为人血清或血浆中的ALT浓度。
本发明中，所述检测人肝脏炎症程度和/或纤维化程度产品可为用于检测人肝脏炎症程度和/或纤维化程度的各种试剂或试剂盒。所述诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品可为用于诊断或辅助诊断肝病患者是否需进行肝穿刺检测的各种试剂或试剂盒。所述筛查或辅助筛查是否需进行肝穿刺肝病患者产品可为用于筛查或辅助筛查是否需进行肝穿刺肝病患者的各种试剂或试剂盒。
在实际应用中，可将检测血清血管生成素样蛋白2含量的物质与检测其他指标的物质联合在一起制备检测人肝脏炎症程度和/或纤维化程度的产品、诊断或辅助诊断肝病患者是否需进行肝穿刺检测的产品或筛查或辅助筛查是否需进行肝穿刺肝病患者的产品。其他指标可为肝病患者的常见临床指标，如人血清或血浆中HBV DNA含量、丙氨酸转氨酶(ALT)含量、乙型肝炎e抗原(HBeAg)、乙肝表面抗原(HBsAg)、血小板(PLT)计数、谷草转氨酶(AST)含量、谷氨酰转肽酶(GGT)含量、白蛋白(Abumin)含量、总胆红素(Tbil)含量、凝血酶原时间(PT)，也可为纤维化模型，如APRI、FIB-4和AAR。
检测其他指标的物质可为利用常规方法检测时用到的试剂和/或仪器。所述产品可为试剂或试剂盒，还可为由试剂或试剂盒与仪器组成的系统。
本发明中，根据血清血管生成素样蛋白2含量确定肝病患者治疗方案的方法如下：检测慢性HBV感染患者血清血管生成素样蛋白2含量，如果慢性HBV感染患者血清血管生成素样蛋白2含量≥7.36ng/ml，该患者需立即进行抗病毒治疗，如果肝病患者血清血管生成素样蛋白2含量≤3.95ng/ml，该患者不需治疗，如果肝病患者血清血管生成素样蛋白2含量大于3.95ng/ml小于7.36ng/ml，该患者需进行肝穿来确定治疗方案。
确定慢性HBV感染患者治疗方案也可先检测肝病患者HBV DNA含量、丙氨酸转氨酶(ALT)含量以及HBeAg状态，如果患者不满足亚太及美国肝病协会关于慢性乙型肝炎病毒(HBV)感染者治疗决策指南中的立即治疗标准和随访标准，根据血清血管生成素样蛋白2含量确定肝病患者治疗方案的方法确定肝病患者治疗方案。亚太及美国肝病学会关于慢性乙型病毒性肝炎指南中的立即治疗标准为HBeAg为阳性、HBV DNA≥20000IU/mL、ALT≥2ULN或HbeAg为阴性、HBV DNA≥2000IU/mL、ALT≥2ULN；亚太及美国肝病协会关于慢性乙型肝炎病毒(HBV)感染者治疗决策指南中的随访标准为HBeAg为阳性、HBV DNA≥2×107IU/mL、 ALT<ULN或HBeAg为阴性、HBV DNA<2000IU/mL、ALT<ULN。
所述肝穿为将肝脏穿刺取肝脏组织做病理学检查。
所述肝脏炎症程度和所述肝脏纤维化程度均按照Ishak标准进行分级。所述肝脏炎症程度可为轻度和/或中重度；所述轻度炎症的肝脏活动指数(hepatic activity index，HAI)为0-4(即0≤HAI≤4)，肝脏轻度炎症的肝病患者的血清血管生成素样蛋白2含量≤3.95ng/ml，所述中重度炎症的肝脏活动指数大于等于5(即HAI≥5)，肝脏中重度炎症的肝病患者的血清血管生成素样蛋白2含量≥7.36ng/ml。所述肝脏纤维化程度可为明显纤维化和/或非明显纤维化；所述明显纤维化为Fibrosis大于等于3(即F≥3)，肝脏明显纤维化的肝病患者的血清血管生成素样蛋白2含量≥7.36ng/ml，所述非明显纤维化为Fibrosis为0-2(即0≤F≤2)，肝脏非明显纤维化的肝病患者的血清血管生成素样蛋白2含量≤3.95ng/ml。
附图说明
图1为血清中angptl2含量与肝脏炎症程度及纤维化程度的关系。其中，A为angptl2含量与肝脏炎症程度的关系；B为angptl2含量与肝脏纤维化程度的关系。
图2为单一指标angptl2诊断需肝穿确定治疗决策组患者的中重度炎症或明显纤维化的ROC曲线。其中，1表示界值3.95ng/ml，2表示界值7.36ng/ml。
图3为173例为不符合亚太及美国肝病学会关于慢性乙型病毒性肝炎指南立即治疗标准和随访标准的灰区患者的治疗决策流程图。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所涉及的名词解释：
受试者工作特征(receiver operating characteristic,ROC)反映了灵敏度与特异度间的平衡，ROC曲线下面积是重要的试验准确度指标，ROC曲线下面积越大，试验的诊断价值越大。
灵敏度(真阳性率)：实际有病而按试验标准被正确判断为有病的百分率，灵敏度越大越好，理想灵敏度为100％。
特异度(真阴性率)：实际无病而按试验标准被正确判断为无病的百分率，特异度越大越好，理想特异度为100％。
下述实施例中的统计分析均采用SPSS17.0软件进行。计量资料用均数±标准差表示，采用Kruskal-Wallis and Mann-Whitney U-tests进行分析；分类资料采用Chi-squared test进行分析。变量与病理分级之间的相关性采用Spearman’s rank test进行分析。二元logistic回归用于确定独立影响因素。 变量对炎症纤维化的诊断性能用ROC分析进行评价，表示为曲线下面积(area under the curve，AUC)，敏感性，特异性，阳性预测率(试验诊断为阳性者，确为患者的概率)，阴性预测率(试验诊断为阴性者，确为非患者的概率)，准确率。Leave-one-out交叉验证法对变量的诊断稳健性进行验证。
实施例1、angptl2可以用来确定慢性HBV感染患者的治疗决策
1.研究对象：(1)病例来源：309例慢性HBV(hepatitis B virus，乙型肝炎病毒)感染患者。(2)纳入标准：年龄18-65岁；HBsAg阳性史半年及以上(血清中HBsAg的定量检测采用用美国罗氏公司试剂盒(Roche
Figure PCTCN2015000515-appb-000001
HBsAg II assay))。(3)排除标准：合并慢性丙型病毒性肝炎、艾滋病感染者，合并遗传性肝病、药物肝、非酒精性脂肪肝等基本的患者；原发性肝癌、肝硬化患者及孕妇。所有研究对象入组前均签署知情同意书。
2、angptl2可以用来确定慢性HBV感染患者的治疗决策
肝穿刺组织病理学检查：将309例慢性HBV感染患者在彩色多普勒引导下按照标准的肝脏病理穿刺标准进行。肝组织穿刺标本的长度2.0cm以上，包含至少11个完整的汇管区。肝穿刺标本经固定、脱水、透明、浸蜡、包埋、切片后，进行常规HE染色、特殊染色(包括网状纤维、Masson三色染色)和免疫组化染色(HBsAg、HBcAg及CK7)。病理标本统一进行中心化盲法判读。肝脏炎症程度和肝脏纤维化程度均按照Ishak标准进行分级。肝脏炎症程度为轻度和中重度；轻度炎症的肝脏活动指数(hepatic activity index，HAI)为0-4(即0≤HAI≤4)，中重度炎症的肝脏活动指数大于等于5(即HAI≥5)。肝脏纤维化程度为明显纤维化和非明显纤维化；明显纤维化为Fibrosis大于等于3(即F≥3)，非明显纤维化为Fibrosis为0-2(即0≤F≤2)。
309例慢性HBV感染患者中有11例患者的肝穿刺活组织检查(肝活检)不合格(肝穿组织小于2.0CM，或少于11个汇管区)，其余298例患者肝活检合格。
血液学及生化检查均以病人在肝穿刺4周内的结果为准。
血清中HBV DNA的定量检测采用美国罗氏公司COBAS AmpliPrep/COBAS TaqMan进行分析(检测范围：2.0×101–1.7×108IU/ml)。血清中乙型肝炎e抗原及乙型肝炎e抗体测定采用美国Roche
Figure PCTCN2015000515-appb-000002
assays进行定性分析。血清中丙氨酸转氨酶(ALT)及血清中谷草转氨酶(AST)的定量均采用全自动生化分析仪进行检测。
根据亚太及美国肝病学会关于慢性乙型病毒性肝炎指南制定临床治疗决策标准(见表1)，将分别符合立即治疗标准、随访标准和肝穿标准的患者分为立即治疗组、随访组和需肝穿确定治疗决策组。
在这298例活检标本合格的患者中，101例符合立即治疗标准(HBeAg(+)、HBVDNA≥20000、ALT≥2ULN或HBeAg(-)、HBV DNA≥2000、ALT≥2ULN)，属于 立即治疗组，该组的患者应立即进行抗病毒治疗；24例符合随访标准(HBeAg(+)、HBVDNA≥2×107、ALT<ULN或HBeAg(-)、HBV DNA<2000、ALT<ULN)，属于随访组，该组的患者不需治疗；其余173例为不符合亚太及美国肝病学会关于慢性乙型病毒性肝炎指南立即治疗标准和随访标准的灰区患者，属于需肝穿确定治疗决策组，按照亚太及美国肝病学会关于慢性乙型病毒性肝炎指南需采用肝脏穿刺制定治疗决策。需肝穿确定治疗决策组患者血清中HBV DNA、HBsAg、ALT和AST的含量较随访组高较立即治疗组低。需肝穿确定治疗决策组的173例患者中0≤F≤2占115例(66.5％)，F≥3占58例(33.5％),0≤HAI≤4占115例(66.5％),HAI≥5占58例(33.5％)。
表1、根据亚太及美国肝病学会关于慢性乙型病毒性肝炎指南制定的治疗决策标准
Figure PCTCN2015000515-appb-000003
测定298例活检标本合格的患者血液中血清血管生成素样蛋白2(血清血管生成素样蛋白2的氨基酸序列如序列表中序列1所示)的含量：采用ELISA试剂盒——Human ANGPTL2Assay kit(Immuno-Biological Laboratories Co.,Ltd,Japan)(Human ANGPTL2Assay kit中含有识别血清血管生成素样蛋白2的抗体和作为标准品的血清血管生成素样蛋白2)进行分析。为了避免血液反复冻融，血液标本在采集后即被分装好，并存储于-80℃冰箱。所有检测2周内完成。为了保障检测值的可信度，复孔间变异系数大于10％及标准曲R方小于0.99的均需重新测量。测量范围0.05ng/ml至3.5ng/ml，样本均做10倍稀释。
各组患者的临床特征见表2。
表2、患者的临床特征
Figure PCTCN2015000515-appb-000004
Figure PCTCN2015000515-appb-000005
分析血清中angptl2含量与肝脏炎症及纤维化的关系，结果如图1所示。血清中angptl2含量随肝脏炎症及纤维化水平的增高而增高(P＝0.000)，并且血清中angptl2含量与肝脏炎症分级的相关性较好，p＝0.000,spearman rho＝0.350，在155例0≤HAI≤4的患者中，angptl2含量4.55±2.01ng/ml，在143例HAI≥5的患者中，angptl2含量为5.68±2.37ng/ml；血清中angptl2含量与肝脏纤维化分级的相关性较好，P＝0.000,spearman rho＝0.352，在200例0≤F≤2的患者中，angptl2含量为4.52±1.75ng/ml，在98例F≥3的患者中，angptl2含量为6.27±2.69ng/ml。仅用血清中angptl2含量作为单一指标诊断肝脏明显纤维化(即F≥3)的受试者工作曲线下面积(AUC)为0.707，而用既往的纤维化模型APRI、FIB-4和AAR诊断肝脏明显纤维化的AUC分别为0.672、0.661和0.553(表3)，表明用血清中angptl2含量作为单一指标诊断肝脏明显纤维化要优于纤维化模型APRI、FIB-4和AAR。
表3、Angptl2及其它模型诊断肝脏明显纤维化的比较
Figure PCTCN2015000515-appb-000006
Figure PCTCN2015000515-appb-000007
ALT分层分析显示：ALT正常(ALT＜ULN)时，肝脏中重度炎症(HAI≥5)及明显纤维化(F≥3)患者血清中angptl2的含量分别较轻度炎症(0≤HAI≤4)及非明显纤维化(0≤F≤2)患者高(P＝0.025，P＝0.024)。ULN≤ALT≤2ULN及ALT≥2ULN时，患者血清中angptl2的含量也分别随肝脏炎症及纤维化严重程度而升高(见表4)。
表4、在ALT不同分层时，angptl2与肝脏炎症纤维化的关系
Figure PCTCN2015000515-appb-000008
在需肝穿确定治疗决策组的173例患者中，单因素分析显示肝脏中重度炎症(HAI≥5)及明显纤维化(F≥3)患者血清中angptl2含量、AST含量、GGT含量和PT分别明显高于轻度炎症(0≤HAI≤4)及非明显纤维化(0≤F≤2)患者血清中angptl2、AST、GGT和PT，而其HBsAg、Albumin含量明显较低(表5)，表明血清中angptl2含量、AST含量、GGT含量、PT、HBsAg、Albumin含量与肝脏炎症和纤维化程度具有相关性。对血清中angptl2含量、AST含量、GGT含量、PT、HBsAg、Albumin含量以及临床普遍认为有影响的因素(年龄大于40岁、性别、体重指数大于24))进行多因素分析发现，高水平angptl2能独立预测肝脏中重度炎症(HAI≥5)或肝脏明显纤维化(F≥3)，P＝0.015，OR＝0.788(95％CI：0.650,0.955)。
表5、需肝穿确定治疗决策组中，angptl2与肝脏炎症纤维化的单因素及多因素分析
Figure PCTCN2015000515-appb-000009
Figure PCTCN2015000515-appb-000010
针对需肝穿确定治疗决策组的173例患者，单一指标angptl2诊断中重度炎症或明显纤维化的AUC为0.691(图2)。进一步leave-one-out(留一)交叉验证法提示，单一指标能正确判断出64.0％的患者。当以该指标的浓度7.36ng/ml为界值诊断中重度炎症及明显纤维化时，特异性(真阴性率)及阳性预测率(试验诊断为阳性者，确为患者的概率)分别为91.76％、72％；以3.95ng/ml为界值排除中重度炎症及明显纤维化诊断时，特异性(真阴性率)及阴性预测率(试验诊断为阴性者，确为非患者的概率)分别为78.41％、70.8％。可以将血清中angptl2含量≥7.36ng/ml作为中重度炎症或明显纤维化界值，将血清中angptl2含量≤3.95ng/ml作为排除中重度炎症或明显纤维化界值，需肝穿确定治疗决策组的173例患者中有86例患者(49.7％)的患者不需要做肝穿刺即可确定治疗方案(图3)，其中血清中angptl2含量≥7.36ng/ml的21例患者应立即进行抗病毒治疗；血清中angptl2含量≤3.95ng/ml的65例患者不需治疗；血清中血清血管生成素样蛋白2含量大于3.95ng/ml小于7.36ng/ml的87患者需进行进一步的肝穿刺来确定治疗方案。本发明通过检查血清angptl2的含量来制定慢性HBV感染者的治疗决策，可避免49.7％的患者进行临床肝穿刺。
工业应用
实验证明，血清中angptl2含量与肝脏炎症程度及纤维化程度均相关：血清中angptl2含量随肝脏炎症程度及纤维化程度的增高而增高(P＝0.000)，并且血清中angptl2含量与肝脏炎症分级的相关性较好，p＝0.000,spearman rho＝0.352；血清中angptl2含量与肝脏纤维化分级的相关性较好，P＝0.000,spearman rho＝0.389。ALT正常(ALT＜ULN)时，肝脏中重度炎症(HAI≥5)及明显纤维化(F≥3)患者血清中angptl2的含量分别较轻度炎症(0≤HAI≤4) 及非明显纤维化(0≤F≤2)患者高(P＝0.025，P＝0.024)。ULN≤ALT≤2ULN及ALT≥2ULN时，患者血清中angptl2的含量也分别随肝脏炎症及纤维化严重程度而升高。
实验证明，用血清中angptl2含量作为单一指标诊断肝脏明显纤维化要优于纤维化模型APRI、FIB-4和AAR：针对慢性乙型肝炎病毒感染患者，仅用血清中angptl2含量作为单一指标诊断肝脏明显纤维化的受试者工作曲线下面积(AUC)为0.707，而用纤维化模型APRI、FIB-4和AAR诊断肝脏明显纤维化的AUC分别为0.672、0.661和0.553。angptl2能独立预测肝脏中重度炎症(HAI≥5)或肝脏纤维化(F≥3)，P＝0.015，OR＝0.788(95％CI：0.650,0.955)。
实验证明，针对不满足亚太及美国肝病协会关于慢性乙型肝炎病毒(HBV)感染者治疗决策指南中的立即治疗标准和随访标准的慢性乙型肝炎病毒感染患者，单一指标angptl2诊断中重度炎症或明显纤维化的AUC为0.691，“交叉验证留一法”验证angptl2诊断中重度炎症或明显纤维化的稳健性，证实单一指标能正确判断出64.0％的患者。当以angptl2含量是7.36ng/ml为界值诊断中重度炎症及明显纤维化时，特异性(真阴性率，即实际未患病而被诊断为非患者的概率)及阳性预测率(试验诊断为阳性者，确为患者的概率)分别为91.76％、72％；以angptl2含量是3.95ng/ml为界值排除中重度炎症及明显纤维化诊断时，特异性(真阴性率，即实际未患病而被诊断为非患者的概率)及阴性预测率(试验诊断为阴性者，确为阴性的概率)分别为78.41％、70.8％。
本发明通过检查血清angptl2的含量来制定慢性HBV感染者的治疗决策，可避免49.7％的患者进行临床肝穿刺。
实验证明，angptl2含量可作为非侵袭性单一指标判断肝脏炎症程度及纤维化程度，可以用angptl2含量来判断慢性乙型肝炎病毒(HBV)感染患者肝脏的炎症程度及纤维化程度，也可以用angptl2含量与其他肝病患者的常见临床指标来判断慢性乙型肝炎病毒感染患者肝脏的炎症程度及纤维化程度；可以将检测血清血管生成素样蛋白2含量的物质制备检测人肝脏炎症程度和/或纤维化程度的产品、诊断或辅助诊断肝病患者是否需进行肝穿刺检测的产品或筛查或辅助筛查是否需进行肝穿刺肝病患者的产品，也可以将检测血清血管生成素样蛋白2含量的物质与检测其他肝病患者的常见临床指标的物质联合在一起制备制备检测人肝脏炎症程度和/或纤维化程度的产品、诊断或辅助诊断肝病患者是否需进行肝穿刺检测的产品或筛查或辅助筛查是否需进行肝穿刺肝病患者的产品，并进一步确定患者的治疗决策。

Claims (11)

  1. 检测血清血管生成素样蛋白2含量的物质在制备检测人肝脏炎症程度和/或纤维化程度产品中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述检测血清血管生成素样蛋白2含量的物质为血清血管生成素样蛋白2和/或血清血管生成素样蛋白2抗体。
  3. 根据权利要求1所述的应用，其特征在于：所述检测血清血管生成素样蛋白2含量的物质为通过酶联免疫反应检测血清血管生成素样蛋白2含量所需的试剂和仪器。
  4. 检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在制备检测人肝脏炎症程度和/或纤维化程度产品中的应用。
  5. 以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在制备检测人肝脏炎症程度和/或纤维化程度产品中的应用。
  6. 下述Y1-Y3中任一应用：
    Y1、检测血清血管生成素样蛋白2含量的物质在制备筛查或辅助筛查是否需进行肝穿刺肝病患者产品中的应用；
    Y2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在制备筛查或辅助筛查是否需进行肝穿刺肝病患者产品中的应用；
    Y3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在制备筛查或辅助筛查是否需进行肝穿刺肝病患者产品中的应用。
  7. 下述Z1-Z3中任一应用：
    Z1、检测血清血管生成素样蛋白2含量的物质在制备诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品中的应用；
    Z2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在制备诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品中的应用；
    Z3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在制备诊断或辅助诊断肝病患者是否需进行肝穿刺检测产品中的应用。
  8. 下述L1-L3中任一应用：
    L1、检测血清血管生成素样蛋白2含量的物质在检测人肝脏炎症程度和/或纤维化程度中的应用；
    L2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在检测人肝脏炎症程度和/或纤维化程度中的应用；
    L3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化方法中所用的物质在检测人肝脏炎症程度和/或纤维化程度中的应用。
  9. 下述M1-M3中任一应用：
    M1、检测血清血管生成素样蛋白2含量的物质在筛查或辅助筛查是否需进行肝穿刺肝病患者中的应用；
    M2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在筛查或辅助筛查是否需进行肝穿刺肝病患者中的应用；
    M3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在筛查或辅助筛查是否需进行肝穿刺肝病患者中的应用。
  10. 下述N1-N3中任一应用：
    N1、检测血清血管生成素样蛋白2含量的物质在诊断或辅助诊断肝病患者是否需进行肝穿刺检测中的应用；
    N2、检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质在诊断或辅助诊断肝病患者是否需进行肝穿刺检测中的应用；
    N3、以血清血管生成素样蛋白2作为标志物的检测人肝脏炎症程度和/或纤维化程度方法中所用的物质在诊断或辅助诊断肝病患者是否需进行肝穿刺检测中的应用。
  11. 下述P1或P2：
    P1、检测人肝脏炎症程度和/或纤维化程度产品，为权利要求1-3中任一所述检测血清血管生成素样蛋白2含量的物质；
    P2、检测人肝脏炎症程度和/或纤维化程度产品，为权利要求4所述检测血清血管生成素样蛋白2含量、乙型肝炎e抗原、乙型肝炎病毒DNA含量和丙氨酸转氨酶含量的物质。
PCT/CN2015/000515 2015-07-17 2015-07-17 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 Ceased WO2017011929A1 (zh)

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