WO2018233619A1 - Method for establishing serum glycoprotein glycome profile model of liver failure - Google Patents

Abstract

Disclosed is a method for establishing a serum glycoprotein glycome profile model of liver failure. The method uses a G-Test detection method to detect a serum glycoprotein glycome profile, establishes a glycome profile model, with liver failure patients and normal controls having a significant difference therebetween, and screens for NA2, the expression thereof being significantly different between the liver failure group and the normal control group. Based on the constructed N-glycome profile model, the detection sensitivity and specificity for liver failure reach 100.0% and 97.6%, respectively. In subsequent applications, the degree of liver failure of persons to be tested can be detected by comparing the peak value of the singlet NA2 in the N-glycome profile of the serum of the persons to be tested with that thereof in the established profile model. The method can enable many patients suffering from liver failure to undergo routine, non-invasive detection, helps doctors and patients to monitor the occurrence and disease progression of liver failure in a timely manner, and is expected to be generalized and used clinically.

Description

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

The invention belongs to the technical field of biomedicine and relates to a method for establishing a serum glycoprotein glycoprotein pattern model of liver failure.

Background technique

Liver failure is a serious liver damage caused by a variety of factors, leading to serious disorders or decompensation of the liver's own synthesis, detoxification, excretion and biotransformation. Clinically, there are coagulation disorders, jaundice, A group of syndromes characterized by hepatic encephalopathy and dehydration have extremely high mortality. Liver failure in Europe and the United States is mainly caused by drugs. The cause of liver failure in China is mainly caused by hepatitis virus infection (mainly hepatitis B virus HBV). Hepatic failure may occur in HBV serological markers positive infections, a large number of viral replication leads to hepatocyte nutrient depletion, immune paralysis is a prerequisite for liver injury, followed by liver failure caused by drugs and hepatotoxic substances (such as ethanol, chemicals, etc.) . At present, the medical treatment of liver failure still lacks special effects drugs and means. In principle, it emphasizes early diagnosis and early treatment, and adopts corresponding etiological treatment measures and comprehensive treatment measures for different causes, and actively prevents various complications. After the diagnosis of liver failure is clear, the condition assessment and intensive care should be performed.

Histopathological examination has important value in the diagnosis, classification and prognosis of liver failure. However, due to the severely low blood coagulation function in patients with liver failure, hepatic puncture has certain risks, which may cause complications such as bleeding and infection. There is sampling error, because the liver biopsy center only accounts for 1/200,000-150,000 of the liver, which can not reflect the whole liver lesion; the patient's dependence is poor, and the single pathological examination results can not reflect the whole degree of liver failure. At present, the clinical diagnosis of liver failure needs to be determined based on comprehensive analysis such as medical history, clinical manifestations and auxiliary examination. Patients with liver failure usually have clinical symptoms, such as jaundice, fatigue, bloating, etc., and further examination is needed to confirm the diagnosis. It usually includes: (1) Hepatitis virus standard test to understand the specific virus type causing liver failure, such as HBV or HCV-positive, further quantitative detection of virus to assess the degree of viral replication; (2) complete blood cell analysis, including white blood cells, hemoglobin, platelets, white blood cell classification, etc., to understand whether there is hypersplenism and infection; (3) urine routine, including Specific gravity, pH value, urobilinogen, urinary bilirubin, etc., indirectly judge the type of jaundice, preliminary judgment of the body's metabolic status; (4) liver function, including ALT, AST, TBIL, ALB, CHE, CHO, PALB, etc. The degree of liver damage and liver combined reserve capacity; (5) ultrasound or CT, nuclear magnetic, evaluation of liver size, degree of injury and blood vessels, bile duct diameter, and exclusion of malignant obstructive lesions. In addition, for long-term alcoholics who have a history of chronic liver disease, electronic gastroscopy or gastrointestinal angiography is needed to understand varicose veins and gastric mucosa.

The above diagnostic methods have their own limitations, and it is necessary to use a variety of diagnostic methods to confirm the diagnosis based on the doctor's clinical experience. It takes a long time, the examination cost is high, and it is not convenient to carry out the screening of liver failure, so it is urgent to explore for assistance. A new noninvasive indicator for the diagnosis of liver failure with high sensitivity and specificity.

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 the existing liver failure test, each diagnostic method has its own limitations. It is necessary to use a variety of diagnostic methods to confirm the diagnosis based on the doctor's clinical experience. It takes a long time and the examination cost is high, and it is not convenient to carry out the census of liver failure. The present invention provides a method for establishing a serum glycoprotein glycoprotein map model for liver failure. By establishing the model, NA2 (two antennas, bigalacto, and biantennary) is selected as a specific marker and can be used for liver failure. diagnosis.

The technical solution of the present invention is as follows:

The method for establishing a serum glycoprotein glycoform map model of liver failure is as follows:

Step 1. Collect serum from patients with liver failure 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 liver failure group and The peak value of NA2 in the sugar group 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 NA2 is 35.6.

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 failure patients and normal control personnel. The model was screened for a significant difference in NA2 between the liver failure group and the normal control group. In the subsequent application, the degree of liver failure of the test subject can be detected by the peak of the single peak NA2 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 liver failure reached 100.0% and 97.6%, respectively. The glycoform map model constructed based on the method of the present invention can enable many patients with chronic hepatitis and liver failure to undergo routine and non-invasive testing, and help doctors and patients to timely monitor the occurrence of failure and progression of the disease, 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 failure group (B).

Figure 2 is a graph showing the ROC curve for the differential diagnosis of liver failure by NA2.

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.

Serum samples from 92 patients with liver failure and control group were treated by G-Test. Among them, 42 patients with liver failure and 50 patients without normal hepatitis B virus. The glycan profiles obtained from the G-Test detection technique were statistically analyzed.

(1) Test samples:

There were 40 patients with serum in liver failure and 52 patients in normal control group without 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 liver failure group. In Fig. 1, the relative content of NA2 in the normal control group was 47%, and the relative content of NA2 in the liver failure group was 35%. It can be seen that the oligosaccharide content of the unimodal NA2 in the liver failure group and the normal control group was significant. gap.

The peaks of the glycan map were quantified, and then statistical analysis was performed on the liver failure group (42 cases) and the normal control group (50 cases). It was found that the single-peak NA2 was statistically significant in the distinction between the two groups (p< 0.05). ROC curve analysis showed that unimodal NA2 has significant clinical significance in detecting patients with liver failure, ie AUC up to 0.987 (Figure 2). When using the model to detect NA2, when the cut-off value is 35.6, the sensitivity and specificity for detection of liver failure are 100.0% and 97.6%, respectively, while the detection sensitivity and specificity of the existing M30 antigen detection method are 76.5. % and 72.04 (Nie Qinghe. Clinical value and evaluation of new indicators in laboratory tests of liver failure [J]. Clin Hepatol, 2013, 29(9): 666-669.), obviously, the sensitivity and specificity of this model More sexual. The results indicate that there is a significant correlation between changes in serum NA2 levels and the development of disease in patients with liver failure.

Claims (5)

1. The method for establishing a serum glycoprotein glycoform map model of liver failure is as follows:

   Step 1. Collect serum from patients with liver failure 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 liver failure group and The peak value of NA2 in the sugar group 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 NA2 is 35.6.

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