WO2018233618A1 - Méthode d'établissement d'un modèle de profil de glycome de glycoprotéine sérique de cirrhose du foie - Google Patents

Méthode d'établissement d'un modèle de profil de glycome de glycoprotéine sérique de cirrhose du foie Download PDF

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WO2018233618A1
WO2018233618A1 PCT/CN2018/091921 CN2018091921W WO2018233618A1 WO 2018233618 A1 WO2018233618 A1 WO 2018233618A1 CN 2018091921 W CN2018091921 W CN 2018091921W WO 2018233618 A1 WO2018233618 A1 WO 2018233618A1
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reagent
serum
cirrhosis
peak
map
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PCT/CN2018/091921
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Chinese (zh)
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陈翠英
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江苏先思达生物科技有限公司
陈翠英
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Publication of WO2018233618A1 publication Critical patent/WO2018233618A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a method for establishing a serum glycoprotein glycoform map model of liver cirrhosis.
  • Cirrhosis is a clinically common chronic progressive liver disease with diffuse liver damage caused by long-term or repeated action of one or more causes.
  • the three most important causes of cirrhosis are alcohol, hepatitis B virus (HBV) and hepatitis C virus (HCV).
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • 57% of cirrhosis is caused by hepatitis B virus (30%) and hepatitis C virus infection (27%).
  • cirrhosis is caused mainly by hepatitis B virus.
  • liver biopsy can provide important information on the stage of liver fibrosis, diffuse liver fibrosis in liver histology with pseudolobular formation. Because liver biopsy has certain risks, and individual liver biopsy specimens may not fully reflect the degree of liver fibrosis, in recent years, a number of non-invasive diagnostic techniques have been developed for the evaluation of liver fibrosis, including images of liver stiffness. Learning techniques, serological signs and various scoring systems.
  • the direct indicators of serum biochemistry mainly include hyaluronic acid (HA), laminin (LN), type III procollagen peptide and type IV collagen.
  • Indirect indicators mainly include platelet count (PLT), r-globulin and prothrombin time. (PT), alanine aminotransferase, bilirubin and apolipoprotein A1, etc., but the above indicators are lack of specificity for diagnosing cirrhosis.
  • Various commonly used imaging methods such as B-mode ultrasound, CT, magnetic resonance imaging (MRI), etc.
  • the liver capsule is thickened, the liver surface contour is irregular, the echogenic heterogeneity of the liver parenchyma is enhanced, or the CT value is increased or a nodule Symptoms of cirrhosis and portal hypertension, such as changes in the proportion of leaves, changes in the thickness of the spleen, and enlargement of the diameter of the portal vein and splenic vein.
  • Color Doppler ultrasonography or radionuclide scanning can measure blood flow and functional portal shunt in hepatic artery and portal vein.
  • Liver elasticity measurements mainly include transient elastic wave scanning (Fibroscan) and magnetic resonance elastography (magnetic resonance elastography).
  • the advantage of this kind of technology is that it can measure the elasticity of a larger range or even the whole liver, and to some extent, it can make up for the insufficient sampling error of liver biopsy; its disadvantage is that liver fat deposition, ascites, abdominal cavity and the like can affect The effect was measured.
  • Such techniques are not yet able to distinguish adjacent liver fiber staging, but have certain clinical guiding significance for the diagnosis of compensatory cirrhosis.
  • Glycoproteins are a class of binding proteins formed by post-translational modification of proteins, ie, glycosylation. Glycosylation of proteins is one of the most common post-translational modifications of proteins. It is the process of transferring sugars to specific amino acid residues on proteins and proteins to form glycosidic bonds under the action of glycosyltransferases. Most glycoproteins are secreted proteins that are widely found in cell membranes, interstitial cells, plasma, and mucus. Glycoproteins have a variety of biological functions. Because of the importance of sugar chains in glycoproteins to maintain the biological functions of the body, changes in sugar chains help to elucidate the molecular mechanisms of abnormal biological behavior such as inflammation, invasion and metastasis of surrounding cells by surrounding cells. At present, changes in N-glycans have been found in a variety of tumors.
  • the structure of the sugar chain is very complicated and has microscopic heterogeneity.
  • the analysis methods of the sugar chain structure mainly include: (1) High performance liquid chromatography (HPLC): the method has high resolution, high detection speed and high repeatability, high-performance liquid phase. Columns can be used repeatedly, but the column efficiency will decrease with the increase in the number of uses, and the mobile phase is toxic. Equipment operation requires highly trained professionals, and the equipment is relatively expensive, and the solvent needs to be strictly purified.
  • Mass spectrometry has the advantages of high sensitivity, various structural information and suitable for analysis of mixtures, but the mass spectrometer is precise, the equipment operation is complicated, and the mass spectrometer is expensive, which is not suitable for popularization and promotion in clinical practice;
  • Capillary electrophoresis Capillary electrophoresis has low cost, high column efficiency, high sensitivity, high speed, low injection volume, and simple operation, but the repeatability is not high and the stability is not as good as HPLC.
  • G-Test is a capillary micro-electrophoresis technique (DSA-FACE) based on DNA sequencer.
  • DSA-FACE capillary micro-electrophoresis technique
  • the N-glycan of glycoprotein in serum samples is fluorescently labeled and separated by capillary microelectrophoresis.
  • the content of the N-oligosaccharide chain obtained by measuring the fluorescence signal is a fingerprint (referred to as G-Test map).
  • the detection technology has the advantages of high sensitivity, simple operation, trace (2 ⁇ l serum), high reproducibility, good stability, high-throughput (96-well plate) and other sugar chain analysis techniques, which is suitable for general laboratory. It is expected to be used for clinical promotion.
  • the serological marker detection lacks specificity, the imaging test is susceptible to the detection environment, and the specificity and accuracy are not high.
  • the present invention provides a serum glycoprotein glycopeptide map of liver cirrhosis.
  • NA3 three antennas was selected as a specific marker by the model, which can be used for the diagnosis of cirrhosis.
  • Step 1 Collect serum from patients with cirrhosis and normal controls;
  • Step 2 preparing a solution containing 5% SDS (sodium dodecyl sulfate) at a concentration of 10 mM, a pH of 8.3, NH 4 HCO 3 as reagent A, and reagent B from 2.2 U/ ⁇ L of PNGaseF and 3.33% of NP-40.
  • SDS sodium dodecyl sulfate
  • reagent B 2.2 U/ ⁇ L of PNGaseF and 3.33% of NP-40.
  • reagent C was prepared by mixing 20 mM APTS (8-aminoindole-1,3,6-trisulphonic acid) and 1 M NaCNBH 3 in equal volumes.
  • Reagent D consisted of 100 mM NH 4 AC, 2 mU/ ⁇ L of sialidase and hydrogen peroxide are mixed in a volume ratio of 5:1:14;
  • Step 3 preparation of oligosaccharide chain: adding half volume of reagent A to the diluted serum, denaturation at 95 ° C for 5 min, then adding reagent B with the same volume of serum, reacting at 37 ° C for 3 h and then drying;
  • Step 4 labeling of the oligosaccharide chain: adding the reagent C of the same volume as the reagent A in the liquid of the step 3, reacting at 65 ° C for 3 h for fluorescent labeling, and then adding water to terminate the labeling reaction;
  • Step 5 desialic acid treatment: take an equal volume of step 4 fluorescently labeled liquid and reagent D at 45 ° C for 3 h, then add water to terminate the reaction;
  • Step 6 Oligosaccharide chain separation analysis: the liquid after the sialic acid treatment in step 5 is taken, and the fragment analysis is performed by a DNA sequencer to obtain a sugar group map;
  • step 7 the obtained sugar group map is subjected to peak quantification, and the peak height value of each peak is divided by the sum of the heights of all the peaks, and the relative content of each peak is quantitatively calculated, and then the quantified cirrhosis group and The peak value of NA3 in the glycoform map of the normal control group was compared and statistically analyzed.
  • step 1 the serum is inactivated.
  • step 4 the fluorescent labeling time of the serum sample is 3h to ensure the success rate of the label. Under the general experimental conditions, the test requirement can be met within 2.5 hours.
  • step 5 the reaction time for removing the terminal sialic acid is 3 hours, and in order to ensure sufficient contact reaction of the enzyme, the reaction can be made more complete by extending to 4 hours.
  • step 7 the cut-off value of the relative content of NA3 is 6.18.
  • the present invention has the following advantages:
  • the method of the invention adopts the G-Test detection method with high sensitivity, simple operation, only a small amount of sample, high repeatability, good stability and high throughput, and establishes a sugar group map with significant difference between liver cirrhosis patients and normal control personnel.
  • the model was screened for a significant difference in NA3 between the cirrhosis group and the normal control group.
  • the degree of cirrhosis of the test subject can be detected by the peak of the single peak NA3 in the map model established by the method in the glycoform map of the serum of the test subject, compared with the prior art, With higher specificity and accuracy, the sensitivity and specificity for detection of cirrhosis reached 84.3% and 85.0%, respectively.
  • the sugar group map model constructed by the method of the invention can enable many patients with liver cirrhosis to receive routine and non-invasive tests, and help doctors and patients to timely monitor the occurrence and progression of liver cirrhosis, and is expected to be promoted in clinical use.
  • Figure 1 is a graph of serum glycoprotein glycoforms in the normal control group (A) and the liver cirrhosis group (B).
  • Figure 2 is a ROC curve for the differential diagnosis of cirrhosis by NA3.
  • the serum samples of 130 cases of liver cirrhosis and control group were treated by G-Test. Among them, 60 cases of cirrhosis caused by hepatitis B virus and 60 cases of normal control group without hepatitis B virus.
  • the glycan profiles obtained from the G-Test detection technique were statistically analyzed.
  • Reagent A SDS was dissolved in 10 mM NH 4 HCO 3 to prepare a NH 4 HCO 3 solution containing 5% SDS and a pH of 8.3.
  • Reagent B 2.2 U/ ⁇ L of PNGaseF and 3.33% of NP-40 were mixed at a volume ratio of 1:20;
  • Reagent C mixing equal volumes of 20 mM APTS and 1 M NaCNBH 3 ;
  • Reagent D 100 mM NH4AC, sialidase (2 mU/ ⁇ L) and hydrogen peroxide were mixed at a volume ratio of 5:1:14.
  • Step 1 Preparation of oligosaccharide chain: 2 ⁇ L of reagent A was added to 4 ⁇ L of diluted serum, and denatured at 95 ° C for 5 minutes, then an equivalent volume (4 ⁇ L) of reagent B was added, and reacted at 37 ° C for 3 hours and then dried;
  • Step 2 labeling of the oligosaccharide chain: adding 2 ⁇ L of reagent C to the liquid of step 1, reacting at 65 ° C for 3 hours for fluorescent labeling, and then adding 200 ⁇ L of water to terminate the labeling reaction;
  • Step 3 post-labeling treatment: take 2 ⁇ L of fluorescently labeled step 2 liquid, add 2 ⁇ L of reagent D, react at 45 ° C for 3 hours, then add 200 ⁇ L of water to terminate the reaction;
  • Step 4 Oligosaccharide chain separation analysis: 10 ⁇ L of the liquid after the step 3 reaction was taken, and the N-oligosaccharide chain was separated by an ABI 3500dx sequencer to obtain a sugar group map.
  • step 5 the obtained sugar group map is subjected to peak quantification, and the relative content of each peak is quantitatively calculated by dividing the peak height value of each peak by the sum of the heights of all the peaks, and statistical analysis is performed.
  • the glycan profile of human serum probably shows nearly 9 N-oligosaccharide chain peaks.
  • the oligosaccharide chains exhibit different mobility due to different molecular sizes, that is, they are expressed on the glycan map.
  • the different peaks represent different oligosaccharide chains, and the measured peak height represents the relative concentration of oligosaccharide chains, A is the normal control group and B is the cirrhosis group.
  • the relative content of NA3 in the normal control group is 7%
  • the relative content of NA3 in the hepatitis B cirrhosis group is 3%. It can be seen that the monomodal NA3 oligosaccharide in the hepatitis B cirrhosis group and the normal control group. There is a significant gap in content.

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  • Chemical & Material Sciences (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne une méthode d'établissement d'un modèle de profil de glycome de glycoprotéine sérique de la cirrhose du foie. La méthode utilise une méthode de détection de test G pour détecter un profil de glycome de glycoprotéine sérique, établit un modèle de profil de glycome, avec des patients atteints de cirrhose du foie et des contrôles normaux ayant une différence significative entre eux, et procède à un examen à la recherche de NA3, dont l'expression est significativement différente entre le groupe de cirrhose du foie et le groupe de contrôles normaux.
PCT/CN2018/091921 2017-06-20 2018-06-20 Méthode d'établissement d'un modèle de profil de glycome de glycoprotéine sérique de cirrhose du foie WO2018233618A1 (fr)

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CN109490548A (zh) * 2018-12-29 2019-03-19 江苏先思达生物科技有限公司 一种肝硬化检测试剂及其在肝硬化检测中的应用
CN112782297A (zh) * 2020-12-24 2021-05-11 郭继生 一种肝硬化相关生物标志物及其筛选方法和应用
CN114032284A (zh) * 2021-09-15 2022-02-11 陈翠英 一种食管癌检测试剂及其在食管癌检测中的应用
CN114032282A (zh) * 2021-09-15 2022-02-11 陈翠英 一种前列腺癌检测试剂及其在前列腺癌检测中的应用
CN114032283A (zh) * 2021-09-15 2022-02-11 陈翠英 一种肠癌检测试剂及其在肠癌检测中的应用
CN114058673A (zh) * 2021-09-15 2022-02-18 江苏先思达生物科技有限公司 一种脂肪肝检测试剂及其在脂肪肝检测中的应用

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