WO2018233618A1 - Method for establishing serum glycoprotein glycome profile model of liver cirrhosis - Google Patents

Abstract

Disclosed is a method for establishing a serum glycoprotein glycome profile model of liver cirrhosis. The method uses a G-test detection method to detect a serum glycoprotein glycome profile, establishes a glycome profile model, with liver cirrhosis patients and normal controls having a significant difference therebetween, and screens for NA3, the expression thereof being significantly different between the liver cirrhosis group and the normal control group.

Description

Method for establishing serum glycoprotein glycoprotein map model of liver cirrhosis Technical field

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.

Background technique

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. At present, the three most important causes of cirrhosis are alcohol, hepatitis B virus (HBV) and hepatitis C virus (HCV). Worldwide, 57% of cirrhosis is caused by hepatitis B virus (30%) and hepatitis C virus infection (27%). In developing countries, cirrhosis is caused mainly by hepatitis B virus.

The clinical diagnosis of cirrhosis requires comprehensive consideration including clinical manifestations, laboratory tests, histology, imaging and histopathology. 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. can be found that 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. Although many studies have found that liver ultrasound semi-quantitative scoring has a good correlation with liver fibrosis grading, it is currently not sensitive enough to early cirrhosis, and the diagnosis of fibrosis is difficult to quantify. 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. At present, 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. (2) Mass spectrometry Method (MS): 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; (3) 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 (Glycan-Test) is a capillary micro-electrophoresis technique (DSA-FACE) based on DNA sequencer. 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 (2μl 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.

Summary of the invention

In view of the existing cirrhosis detection, 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. By establishing the model, NA3 (three antennas) was selected as a specific marker by the model, which can be used for the diagnosis of cirrhosis.

The technical solution of the present invention is as follows:

The method for establishing a serum glycoprotein glycoprotein map model of liver cirrhosis, the specific steps are as follows:

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/μL of PNGaseF and 3.33% of NP-40. Formulated in a 1:20 volume ratio, 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/ μL 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;

In 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.

In step 1, the serum is inactivated.

In 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.

In 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.

In step 7, the cut-off value of the relative content of NA3 is 6.18.

Compared with the prior art, 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. In the subsequent application, 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.

DRAWINGS

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.

Detailed ways

The invention will be further described in detail below with reference to the embodiments and the accompanying drawings. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually tested according to conventional conditions or according to the conditions recommended by the manufacturer, and the reagents are exclusively for cell culture.

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.

(1) Test samples:

Sixty patients with cirrhosis caused by hepatitis B virus, and 70 patients without normal hepatitis B virus.

(2) Experimental equipment:

ABI3500dx DNA Sequencer (Applied Biosystems), PCR, centrifuge.

(3) Reagent preparation:

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/μL 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/μL) and hydrogen peroxide were mixed at a volume ratio of 5:1:14.

(4) G-Test detection

Step 1. Preparation of oligosaccharide chain: 2 μL of reagent A was added to 4 μL of diluted serum, and denatured at 95 ° C for 5 minutes, then an equivalent volume (4 μL) 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 μL of reagent C to the liquid of step 1, reacting at 65 ° C for 3 hours for fluorescent labeling, and then adding 200 μL of water to terminate the labeling reaction;

Step 3, post-labeling treatment: take 2 μL of fluorescently labeled step 2 liquid, add 2 μL of reagent D, react at 45 ° C for 3 hours, then add 200 μL of water to terminate the reaction;

Step 4: Oligosaccharide chain separation analysis: 10 μL 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.

In 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.

As shown in Figure 1, 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. In Fig. 1, the relative content of NA3 in the normal control group is 7%, and 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.

The peaks of the glycan map were quantified, and then statistical analysis was performed on the cirrhosis group (60 cases) and the normal control group (70 cases). It was found that the single-peak NA3 was statistically significant in the distinction between the two groups (p< 0.05). ROC curve analysis showed that unimodal NA3 has a significant clinical significance in the detection of patients with cirrhosis, ie AUC up to 0.912 (Figure 2). When the model was used to detect NA3, when the cut-off value was 6.18, the sensitivity and specificity for detection of cirrhosis were 84.3% and 85.0%, respectively, while the sensitivity and specificity of the existing Zeng Score and Hui Score serum markers were detected. The sexes are 59.1%, 60%, and 56.8, 68.9%, respectively. (European Association for the Study of the Liver. EASL-ALEH Clinical Practice Guidelines: Non-invasive tests for evaluation of liver disease severity and prognosis [J]. Hepatology, 2015, 63:237-264.), obviously, the sensitivity and specificity of the model are higher. The results indicate that there is a significant correlation between changes in serum NA3 levels and the occurrence of disease in patients with cirrhosis.

Claims (5)

1. A method for establishing a serum glycoprotein glycoprotein map model of liver cirrhosis, characterized in that the specific steps are as follows:

   Step 1. Collect serum from patients with cirrhosis and normal controls;

   Step 2, preparing a NH ₄ HCO ₃ solution containing 5% SDS at a concentration of 10 mM and having a pH of 8.3 as reagent A, and the reagent B is mixed by a ratio of 2.2 U/μL of PNGaseF and 3.33% of NP-40 by a ratio of 1:20. Formulation, reagent C is prepared by mixing equal volumes of 20 mM APTS and 1 M NaCNBH ₃ , and reagent D is mixed by 100 mM NH ₄ AC, 2 mU/μL of sialidase and hydrogen peroxide at 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;

   In 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.
2. The method of claim 1 wherein in step 1, said serum is inactivated.
3. The method of claim 1 wherein in step 4, the fluorescent labeling time of the serum sample is 2.5 h.
4. The method according to claim 1, wherein in step 5, the reaction time for removing the terminal sialic acid is 4 hours.
5. The method of claim 1 wherein in step 7, the cut-off value of the relative amount of NA3 is 6.18.

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