CN110850086B - Application of serum diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation - Google Patents
Application of serum diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation Download PDFInfo
- Publication number
- CN110850086B CN110850086B CN201911129331.4A CN201911129331A CN110850086B CN 110850086 B CN110850086 B CN 110850086B CN 201911129331 A CN201911129331 A CN 201911129331A CN 110850086 B CN110850086 B CN 110850086B
- Authority
- CN
- China
- Prior art keywords
- serum
- ischemic
- acly
- liver transplantation
- biliary tract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an application of a serum polypeptide molecular diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation, wherein the amino acid sequence of the serum polypeptide molecular diagnostic marker ACLY is shown in SEQ.ID.NO. 1. This molecule is called ACLY, which is ATP citrate lyase with an exact molecular weight of 1949.9 daltons. ACLY shows specific low expression in serum detection of patients with ischemic biliary tract diseases after liver transplantation, and the method for detecting ACLY expression level by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or ELISA can be used as a method for detecting serum of patients with ischemic biliary tract diseases after liver transplantation.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of an ACLY (ACLY) diagnostic marker for ischemic biliary tract lesion serum polypeptide molecules after liver transplantation.
Background
Liver transplantation (liver transplantation, LT) has been the only effective means to treat end-stage liver disease since the first clinical application of Starzl in 1963, with half a century of development. With the development of novel immunosuppressants and the maturation of surgical techniques, rejection reactions and surgical technique related complications have been significantly reduced, and the death rate after surgery has been reduced to below 5% in 1 year. The ischemic biliary tract lesions (ischemic type biliary lesion, ITBL) are localized or diffuse biliary tract damage caused by biliary tract blood supply disorder, so that the transplanted hepatic biliary tract has non-anastomotic segmental stenosis, dilatation and intrahepatic biliary tract disappearance, and finally causes biliary tract mechanical obstruction and secondary biliary tract infection, and the incidence rate is 2% -19%; because the etiology and pathogenesis are not clear, early diagnosis is difficult, clinical treatment is troublesome, and the disease has become one of the main reasons for influencing the long-term survival of liver transplant patients and causing the loss of the transplant. Therefore, the research on the exact molecular mechanism of occurrence and development of the ITBL is carried out, and the search of a new specific diagnostic marker has important clinical significance for the early diagnosis and treatment of ITBL, the improvement of the curative effect of liver transplantation and the improvement of the survival quality of patients.
The intracellular proteins change in composition and quantity before any disease develops pathology and are reflected by the pattern of proteins in serum. Thus, by comparing the expression of different proteins in serum of different disease populations, it is possible to screen for disease-related marker molecules. Sero-proteomics refers to research on all proteins expressed in serum of a selected target population, searches for differential protein spots on the basis of establishing normal protein expression patterns (protein expression map, PEM), identifies disease-related proteins, further researches on the structure and the function of the proteins, and opens up a new way for researching pathophysiological mechanisms of major diseases, specific markers for early diagnosis, drug action targets and the like. A large number of proteins and polypeptides exist in human serum, wherein the existence and the absence of part of proteins and polypeptides and the expression level are closely related to the human health degree, and the proteins and the polypeptides become biomarkers for disease diagnosis.
Serum diagnosis is considered to be the latest and most effective method for early diagnosis of cancer. The early diagnosis of the tumor is realized by searching tumor markers in blood, particularly protein markers in the blood to judge the occurrence and development of the tumor. A large number of proteins and polypeptides exist in human serum, wherein the existence and the absence of part of proteins and polypeptides and the expression level are closely related to the human health degree, and the proteins and the polypeptides become biomarkers for disease diagnosis.
Currently, screening for ischemic biliary lesions following liver transplantation lacks a sensitive and specific diagnostic method.
Disclosure of Invention
The invention aims to provide a serum polypeptide molecular diagnostic marker for ischemic biliary tract lesions after liver transplantation and application thereof.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention discloses an application of a serum polypeptide molecular diagnosis marker ACLY in preparing a serum diagnosis medicament for ischemic biliary tract lesions after liver transplantation, wherein the amino acid sequence of the serum polypeptide molecular diagnosis marker is shown as SEQ ID No. 1.
Preferably, the serum polypeptide molecular diagnostic marker ACLY is ATP citrate lyase with a molecular weight of 1949.9 daltons.
Preferably, the detection parameter of the serum polypeptide molecular diagnosis marker ACLY in serum is 456.07-802.50 pg/mL.
Preferably, the serum diagnostic medicine for ischemic biliary tract lesions after liver transplantation is a serum polypeptide molecular medicine for ELISA detection of ischemic biliary tract lesions after liver transplantation.
The invention also discloses application of the molecule combined with the serum polypeptide molecular diagnosis marker ACLY in preparing a serum diagnosis medicament for ischemic biliary tract lesions after liver transplantation.
The invention also discloses application of the molecule combined with ACLY protein in preparing a blood serum diagnostic medicine for ischemic biliary tract lesions after liver transplantation.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses an ischemic biliary tract lesion serum polypeptide molecule after liver transplantation, the amino acid sequence of which is shown as SEQ.ID.NO.1, and the molecule is called ACLY. ACLY is an ATP citrate lyase with a precise molecular weight of 1949.9 daltons. ACLY exhibits specific low expression in serum detection of patients with ischemic biliary lesions following liver transplantation: the expression range in serum of normal healthy population is: 863.21-1291.79 pg/mL; the expression range in serum of patients with good postoperative recovery is as follows: 631.07-1006.79 pg/mL; the expression range in serum of patients with ischemic biliary tract lesions after liver transplantation is as follows: 456.07-802.50pg/mL, and the expression between the different groups has very significant differences (p < 0.01).
Given that ACLY is specifically expressed low in serum of ischemic biliary tract lesions after liver transplantation, ACLY can be used as a serum diagnostic marker of ischemic biliary tract lesions after liver transplantation; the method for detecting the expression level of ACLY by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or an ELISA method can be used as a method for detecting patients with ischemic biliary tract lesions after liver transplantation. And aiming at ELISA detection of serum diagnosis of ischemic biliary tract lesions after liver transplantation, ACLY can be used as a new target of ELISA detection drugs.
Drawings
FIG. 1 is a graph showing the results of Flex analysis of specific low-expression of 7 protein polypeptides in serum of patients with ischemic biliary tract disorders: through the comparison of the expression of M/Z:1949.9 in ischemic biliary lesions patients (red), postoperative recovery good patients (green) and normal healthy people (blue), the protein polypeptide peak diagram of M/Z:1949.9 is found to be remarkably low-expressed in serum of the ischemic biliary lesions patients, so that the sequence identification is carried out and is used as the first choice for further identification of markers.
FIG. 2 shows the results of gel chromatography of serum protein polypeptides from patients with ischemic biliary tract disorders: the abscissa in the chromatogram represents the sample outflow time, the ordinate represents the relative abundance of the polypeptide, the chromatographic setting time is 78min, the collection fraction is collected from 5min, the polypeptide components are mainly separated after 6min and the gradient elution mode is adopted, so that the elution efficiency is improved, and the collection fraction is collected at the set trapping time: 25 peptide fractions were collected and the peak for the target peptide of 1.9K Da was eluted at about 62 min.
FIG. 3 is an MS/MS mass spectrum identification profile of ACLY.
FIG. 4 shows the expression levels of ACLY protein in the different groups: the results of the ELISA analysis show that the expression level of ACLY in different detection groups is normal healthy people > good postoperative recovery patients > ischemic biliary tract lesion patients after liver transplantation, and the three groups have obvious differences.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, as used in the description and claims of the present invention, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements that are expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the attached drawing figures:
the serum polypeptide molecule of ischemic biliary tract lesions after liver transplantation provided by the invention is a newly screened serum diagnostic marker of ischemic biliary tract lesions after liver transplantation, has specificity in expression, and can be applied to diagnosis of ischemic biliary tract lesions after liver transplantation.
The specific screening of the serum diagnostic markers of ischemic biliary tract lesions after liver transplantation is as follows:
firstly, separating and extracting serum protein polypeptide of a good patient and normal healthy people after liver transplantation by using a liquid protein chip technology, capturing serum protein polypeptide maps of the good patient and the normal healthy people after liver transplantation by using a matrix-assisted laser analysis ionization time-of-flight mass spectrometry technology, comparing and analyzing serum protein polypeptide spectrogram differences of the good patient and the normal healthy people after liver transplantation by using ClinProTools2.1 software, finding out protein polypeptide molecules which are obviously differentially expressed among groups, and screening out serum markers of the ischemic biliary lesions after liver transplantation from protein polypeptide peaks which are obviously and lowly expressed in serum of the ischemic biliary lesions after liver transplantation.
Verification of the screened post-liver-transplant ischemic biliary tract lesion polypeptide serum diagnostic markers is:
the protein polypeptide mixture separated from the serum of the ischemic biliary tract disease patient after the liver transplantation is divided into 20-30 components by using HPLC, the secondary mass spectrum identification is carried out on the protein polypeptide, the serum regression analysis is carried out on the identified protein polypeptide by adopting an enzyme-linked immunosorbent assay, and the result serum regression verification proves that the protein polypeptide is obviously low-expressed in the serum of the ischemic biliary tract disease patient after the liver transplantation, has specificity, and can be used as a biomarker for screening the serum of the ischemic biliary tract disease patient after the liver transplantation.
1. Sample collection and processing:
9 cases (7 men; 2 women; average age 53 years) of the liver-biliary surgery-confirmed ischemic biliary lesions patients from the hepatobiliary surgery of the first affiliated hospital of the university of western traffic (3 months of 2016 to 12 months of 2016), 10 cases of the postoperative recovery good patients (8 men; 2 women; average age 51 years) and 10 cases of the normal healthy population (7 men; 3 women; average age 50 years) were collected. The sample considers the factors such as age, sex, collection time, whether the storage conditions are consistent or not, whether basic diseases exist or not, and the like. The person to be collected takes blood in an empty stomach in the morning, and takes 5mL of whole blood by a vacuum blood taking tube (yellow cap and isolation glue), and stands for 30min at room temperature; centrifuging at room temperature for 5min (3000 g), packaging the upper serum into 100 μl/tube, and immediately storing at-80deg.C to avoid repeated freeze thawing.
Reagents and instrumentation:
serum proteins were extracted using the magnetic bead kit "weak cation" (MB-WCX) from Bruce, germany, as well as spectrally pure (HPLC grade) acetonitrile, trifluoroacetic acid (Merck, germany), α -cyano-4-hydroxycinnamic acid (HCCA) (Sigma, USA).
2. Preparation of serum protein samples
The serum protein polypeptide is captured by using weak cation (MB-WCX) magnetic beads, and the specific operation steps are as follows:
(1) completely mixing the magnetic bead suspension with a mixer for 1min;
(2) adding 10 mu L of MB-WCX binding solution and 10 mu L of MB-WCX magnetic beads into a PCR tube, uniformly mixing, adding 5 mu L of serum, uniformly mixing for at least 5 times, and standing for 5min;
(3) placing the PCR tube into a magnetic column separator, enabling the magnetic beads to be attached for 1min, and discarding supernatant after the liquid is clear;
(4) adding 100 mu LMB-WCX flushing liquid, moving the PCR tube for 10 times back and forth on the magnetic column separator, adhering the magnetic beads, discarding the supernatant, and repeating the steps (3) and (4) twice;
(5) washing the attached magnetic beads with 5 mu LMB-WCX eluent, repeatedly blowing for 10 times, attaching the magnetic beads for 2min, and transferring the supernatant into a clean centrifuge tube;
(6) adding 5 mu LMB-WCX stabilizing solution into a centrifuge tube, mixing, and directly detecting by MALDI-TOF-MS or freezing in a refrigerator at-20deg.C for 24 hr for mass spectrometry.
Mass spectrometry:
1. Mu.L of the protein sample obtained by separation and collection was mixed with 10. Mu.L of matrix alpha-cyano-4-hydroxycinnamic acid, 1. Mu.L of the mixture was spotted on an Anchor target plate (Bruker, germany), and three targets were spotted on each sample for three replicates. And after the target plate is dried at room temperature, placing the target plate into a mass spectrometer for flight time mass spectrometry analysis, correcting a standard product by using FlexControl 2.0 software, and then starting sample detection, wherein each sample is subjected to total 300 times of laser targeting (5 times of targeting, 2 times of targeting each time) to generate a mass spectrum, and a protein polypeptide spectrum consisting of different mass nuclear ratios (m/z) is obtained. Protein polypeptide profiles of two sets of serum samples were analyzed using ClinProTools2.1 software in combination with biometric and bioinformatic methods such as genetic algorithm. Carrying out normalization smoothing treatment on the total ion flow graph to eliminate chemical and electro-physical noise; the protein difference between groups is analyzed and the size of the difference is calculated, and protein polypeptide peaks (P < 0.001) with significant difference in the expression between groups are found out according to the arrangement of the sizes of the differences from large to small.
After the serum samples of the postoperative ischemic biliary tract lesion patient, the postoperative recovery good patient and the normal healthy population are treated by a magnetic bead separation system, after MALDI-TOF-MS analysis, protein polypeptide map drawing is carried out on each sample of the postoperative ischemic biliary tract lesion patient, the postoperative recovery good patient and the normal healthy population, 76 protein polypeptide peak maps are totally detected in the molecular weight range of 1000 Da-10000 Da, and the three-time repeated stability of each sample is higher.
The serum protein polypeptide patterns of the post-operative recovery good patient and the normal healthy population of the post-operative recovery good patient are analyzed by adopting ClinProTools2.1 software, the serum polypeptide patterns of the post-operative recovery good patient are compared and analyzed with the serum polypeptide patterns of the normal healthy population, 9 protein polypeptide peak patterns (P < 0.05) with obvious differences are detected totally, 4 protein polypeptide peak patterns have extremely obvious differences (P < 0.001) among three groups, 7 protein polypeptides are obviously down-regulated in the post-operative recovery good patient and the expression of the other 2 protein polypeptides is obviously up-regulated in the post-operative ischemic biliary lesion patient, and the method is specifically shown in table 1:
flex analysis software analysis was performed on 7 protein polypeptides specifically expressed in serum of patients with ischemic biliary tract lesions in Table 1, and the results are shown in FIG. 1, and the protein polypeptide peak pattern of M/Z1949.9 was found to be significantly expressed in serum of patients with ischemic biliary tract lesions by comparing the expression of M/Z1949.9 in patients with ischemic biliary tract lesions (red), recovering good patients after surgery (green) and normal healthy people (blue), so that the sequence was identified and further identified as a first choice of markers.
3. Sequence identification of serum potential markers of ischemic biliary tract lesions after liver transplantation
Specifically, a liquid chromatography separation and mass spectrometry combined technology is adopted to identify a serum polypeptide marker M/Z:1949.9 of a patient with ischemic biliary tract lesions after liver transplantation, a Waters company Nano Acquity UPLC is adopted to carry out two-dimensional gel chromatography separation on the serum protein polypeptide which is remained after mass spectrometry and is collected by magnetic bead separation, and 25 peptide fraction fractions are collected: the 1.9kDa peak was eluted at about 62min and detected in the collection; and then carrying out sequence identification on protein polypeptide M/Z1949.9 with the expression down-regulated in serum of the ischemic biliary tract lesion patient after the liver transplantation by using a LTQ Orbitrap XL mass spectrometry system of Thermo Fisher company.
3.1 sample pretreatment
Combining the extracted protein samples, 13300 r for 5min, taking supernatant, drying by a freeze dryer to obtain a final volume of 50ul, obtaining liquid A, and concentrating and treating the liquid A by using an Agilent ziptip extraction column. The treatment method comprises the following steps: (1) the ziptip column was blown 5 times with 100% acetonitrile to activate the column; (2) repeatedly blowing and sucking the activated ziptip in the liquid 1 for 10 times, and avoiding bubbles as much as possible; (3) washing 3 times the ziptip column with 40% acn 0.2% tfa in water; (4) repeatedly blowing and sucking the ziptip column in 0.2% TFA to elute the sample to obtain eluent 2; (5) repeating the steps 1-4 for 30 times; (6) the 30 times eluate 2 was pooled, lyophilized to 10ul and used for mass spectrometry.
3.2 chromatographic separation
The original sample was added with 10ul of mobile phase A and transferred to a sample bottle for a total of 20ul.
One-dimensional ultra-high performance liquid phase system: waters company Nano air UPLC (Waters Corporation, milford, USA). Chromatographic column:
trapping column:
C18,3μm,0.10×20mm,nanoAcquity TM Column
analytical column:
C18,1.9μm，0.15×120mm,nanoAcquity TM Column
mobile phase a:5% acetonitrile, 0.1% formic acid in water
Mobile phase B, 95% acetonitrile, 0.1% formic acid aqueous solution; all solutions were HPLC grade.
The trapping flow rate is 600nl/min, the trapping time is 3min, and the analysis flow rate is 400nl/min; analysis time is 60min, chromatographic column temperature is 35 ℃; partial Loop mode sample injection was performed with a sample injection volume of 18 μl.
The gradient elution procedure is shown in table 2 below:
TABLE 2
| Time (min) | A | B | Flow rate (nL/min) |
| 0 | 95％ | 5％ | 600 |
| 16 | 90％ | 10％ | 600 |
| 51 | 78％ | 22％ | 600 |
| 71 | 70％ | 30％ | 600 |
| 72 | 5％ | 95％ | 600 |
| 78 | 5％ | 95％ | 600 |
The gel chromatography results are shown in FIG. 2. The abscissa in the chromatogram represents the sample outflow time, the ordinate represents the relative abundance of the polypeptide, the chromatographic setting time is 78min, the collection fraction is collected from 5min, the polypeptide components are mainly separated after 6min and the gradient elution mode is adopted, so that the elution efficiency is improved, and the collection fraction is collected at the set trapping time: 25 peptide fractions were collected and the peak for the target peptide of 1.9K Da was eluted at about 62 min.
3.3 LTQ-Orbitrap XL mass spectrometry
A Thermo Fisher company Q-exact HF mass spectrometry system was used. Nano-liter flow HPLC liquid phase system Easy nLC 1000, spray voltage 1.8kV; mass spectrum scanning time is 78min; the experimental mode is Data dependence (Data dependence) and dynamic exclusion (Dynamic Exclusion), and parent ions are added into the exclusion list for 80 seconds after 2 cascade steps are performed within 60 seconds; scanning range is 300-1400m/z; the primary scan (MS) uses Obitrap with resolution set to 70000; CID and secondary scan use LTQ; the monoisotope of the 20 ions with the strongest intensity is selected as the parent ion in the MS spectrogram to carry out MS/MS (single charge exclusion, not as the parent ion).
Data analysis: database search authentication was performed using data analysis software, mascot, to incorporate PD 2.0. The parent ion error was set to.+ -.15 ppm, the fragment ion error was set to 20mmu, the cleavage mode was non-cleavage, and the modification was changed to M (Methionine) Methionine oxidation. The search result parameter is set to be less than or equal to 0.01 of Peptide FDR.
The search result is: m/z 1950.106; uniprot, P53396; gene symbol=acly; the sequence is as follows: ILIIGGSIANFTNVAATFK.G.
The isolated M/Z1949.9 is called ACLY, which is ATP citrate lyase with an exact molecular weight of 1949.9 daltons and an amino acid sequence of: ILIIGGSIANFTNVAATFK.G (shown as SEQ. ID. NO. 1).
Therefore, the method for detecting the expression level of ACLY by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or an ELISA method can be used as a method for detecting ischemic biliary tract lesions patients after liver transplantation.
ACLY was suggested to be a protein specifically associated with ischemic biliary lesions following liver transplantation, and was further validated by ELISA detection.
4. ELISA serum validation analysis of ischemic biliary tract lesion serum ACLY expression after liver transplantation
1) Serum samples: serum validation analysis of ELISA was performed by collecting 9 cases (8 cases in men, 1 case in women; average age 52) of patients with ischemic biliary lesions after liver transplantation, 10 cases (8 cases in men, 2 cases in women; average age 48) of patients with good postoperative recovery, and 10 cases (7 cases in men; 3 cases in women; average age 55.3) of serum of normal healthy people. All serum samples come from attached hospital of the university of western security traffic, and the collection time is 1 month in 2017 to 9 months in 2017;
2) The detection method comprises the following steps: the expression level of serum ACLY of patients with ischemic biliary tract lesions after liver transplantation, patients with good postoperative recovery and normal healthy people is detected by enzyme-linked immunosorbent assay (ELISA), and the kit is purchased from R & D company in the United states. The kit adopts a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA): to the coated microwells pre-coated with anti-human ACLY protein (ACLY) antibodies, samples, standards, HRP-labeled detection antibodies were added sequentially, incubated and thoroughly washed. The color is developed with the substrate TMB, which is converted to blue under the catalysis of the peroxidase and to the final yellow under the action of the acid. The intensity of the color and the ACLY protein (ACLY) in the sample were positively correlated. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the sample concentration was calculated. Specific experimental steps refer to the kit specification, and positive judgment standards are defined according to the kit specification;
3) The statistical method comprises the following steps: single-factor analysis of variance (ANOVA) and T-test of independent samples were performed using graphpad. Prism.v5.01 software;
4) Analysis of results: the results of the ELISA analysis show that the expression level of ACLY in different detection groups is normal healthy people > good postoperative recovery patients > ischemic biliary tract lesion patients after liver transplantation, and the three groups have obvious differences, and the specific results are shown in Table 3 and FIG. 4.
TABLE 3 Table 3
ELISA detection is carried out on ACLY in serum of ischemic biliary tract lesion patients after liver transplantation, patients with good postoperative recovery and normal healthy people, and the results show that the expression of ACLY has specificity: the expression range in serum of patients with ischemic biliary tract lesions after liver transplantation is as follows: 456.07-802.50pg/mL; the expression range in serum of patients with good postoperative recovery is as follows: 631.07-1006.79 pg/mL; the expression range in serum of normal healthy patients is: 863.21-1291.79 pg/mL, and there was a significant difference in expression between the different groups (p < 0.01). This indicates that: ACLY is a protein closely related to the occurrence of ischemic biliary tract lesions after liver transplantation, and can be used as an index for detecting ischemic biliary tract lesions after preliminary liver transplantation. Therefore, the ACLY expression of the serum sample to be detected can be preliminarily judged to be ischemic biliary lesions patients (456.07-802.50 pg/mL) after liver transplantation, patients with good postoperative recovery (631.07-1006.79 pg/mL) or normal healthy people (863.21-1291.79 pg/mL) through ELISA experiments.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<110> university of traffic of western Security
<120> application of serum polypeptide molecular diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Ser Pro Pro Met Gly Leu Val Val Ala Pro Thr Gly Leu
1 5 10
Claims (6)
1. The application of a serum polypeptide molecular diagnosis marker ACLY in preparing a serum diagnosis medicament for ischemic biliary tract lesions after liver transplantation is characterized in that the amino acid sequence of the serum polypeptide molecular diagnosis marker is shown as SEQ ID No. 1.
2. The use of claim 1, wherein the serum polypeptide molecular diagnostic marker ACLY is ATP citrate lyase with a molecular weight of 1949.9 daltons.
3. The use according to claim 1, wherein the serum polypeptide molecular diagnostic marker ACLY has a detection parameter in serum of 456.07-802.50 pg/mL.
4. The use according to claim 1, wherein the serum diagnostic drug for ischemic biliary lesions after liver transplantation is a serum polypeptide molecular drug for ELISA detection of ischemic biliary lesions after liver transplantation.
5. Use of a molecule that binds to the serum polypeptide molecular diagnostic marker ACLY of claim 1 for the preparation of a serum diagnostic medicament for ischemic biliary lesions following liver transplantation.
6. The application of the molecule combined with ACLY protein in preparing the blood serum diagnostic medicine for ischemic biliary tract disease after liver transplantation is provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911129331.4A CN110850086B (en) | 2019-11-18 | 2019-11-18 | Application of serum diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911129331.4A CN110850086B (en) | 2019-11-18 | 2019-11-18 | Application of serum diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110850086A CN110850086A (en) | 2020-02-28 |
| CN110850086B true CN110850086B (en) | 2023-05-02 |
Family
ID=69602074
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911129331.4A Active CN110850086B (en) | 2019-11-18 | 2019-11-18 | Application of serum diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110850086B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101495498A (en) * | 2005-02-07 | 2009-07-29 | 基因信息公司 | Mild osteoarthritis biomarkers and uses thereof |
| CN102643334A (en) * | 2012-03-29 | 2012-08-22 | 西安交通大学 | Polypeptide molecule for serum in gastric cancer and application |
| CN103224922A (en) * | 2013-04-11 | 2013-07-31 | 西安交通大学 | New stomach cancer marker, detection method and applications thereof |
| CN103492590A (en) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | Circulating biomarkers |
| CN104781670A (en) * | 2012-09-12 | 2015-07-15 | 博格有限责任公司 | Use of markers in the identification of cardiotoxic agents |
| CN105622743A (en) * | 2016-02-22 | 2016-06-01 | 西安交通大学 | Autistic serum polypeptide marker PF4-A and application thereof |
| CN105622742A (en) * | 2016-02-22 | 2016-06-01 | 西安交通大学 | Autistic serum polypeptide marker FABP1-A and application thereof |
| CN105652019A (en) * | 2016-02-22 | 2016-06-08 | 西安交通大学 | ASD (autism spectrum disorders) serum polypeptide marker APOC1-A and application thereof |
| CN105738631A (en) * | 2016-02-22 | 2016-07-06 | 西安交通大学 | Autism serum polypeptide marker SERPINA5-A and application thereof |
| CN107001414A (en) * | 2014-12-23 | 2017-08-01 | 伊玛提克斯生物技术有限公司 | For hepatocellular carcinoma (HCC) and the new type of peptides and peptide combinations of other cancer immunotherapies |
| CN107449914A (en) * | 2017-07-10 | 2017-12-08 | 北京大学人民医院 | The application of UCHL1 autoantibody |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050202485A1 (en) * | 2004-03-03 | 2005-09-15 | Ye Jack Z. | Method and compositions for detection of liver cancer |
| TW201100801A (en) * | 2009-06-18 | 2011-01-01 | Atomic Energy Council | Serum biomarkers and the testing methods for diagnosing liver fibrosis |
-
2019
- 2019-11-18 CN CN201911129331.4A patent/CN110850086B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101495498A (en) * | 2005-02-07 | 2009-07-29 | 基因信息公司 | Mild osteoarthritis biomarkers and uses thereof |
| CN103492590A (en) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | Circulating biomarkers |
| CN102643334A (en) * | 2012-03-29 | 2012-08-22 | 西安交通大学 | Polypeptide molecule for serum in gastric cancer and application |
| CN104781670A (en) * | 2012-09-12 | 2015-07-15 | 博格有限责任公司 | Use of markers in the identification of cardiotoxic agents |
| CN103224922A (en) * | 2013-04-11 | 2013-07-31 | 西安交通大学 | New stomach cancer marker, detection method and applications thereof |
| CN107001414A (en) * | 2014-12-23 | 2017-08-01 | 伊玛提克斯生物技术有限公司 | For hepatocellular carcinoma (HCC) and the new type of peptides and peptide combinations of other cancer immunotherapies |
| CN105622743A (en) * | 2016-02-22 | 2016-06-01 | 西安交通大学 | Autistic serum polypeptide marker PF4-A and application thereof |
| CN105622742A (en) * | 2016-02-22 | 2016-06-01 | 西安交通大学 | Autistic serum polypeptide marker FABP1-A and application thereof |
| CN105652019A (en) * | 2016-02-22 | 2016-06-08 | 西安交通大学 | ASD (autism spectrum disorders) serum polypeptide marker APOC1-A and application thereof |
| CN105738631A (en) * | 2016-02-22 | 2016-07-06 | 西安交通大学 | Autism serum polypeptide marker SERPINA5-A and application thereof |
| CN107449914A (en) * | 2017-07-10 | 2017-12-08 | 北京大学人民医院 | The application of UCHL1 autoantibody |
Non-Patent Citations (7)
| Title |
|---|
| Cornelia J Verhoeven, et al..MicroRNA profiles in graft preservation solution are predictive of ischemic-type biliary lesions after liver transplantation.J Hepatol.2013,第59卷(第6期),全文. * |
| Giancarlo Maccagno, et al..Ischemic-type biliary lesions after liver transplantation: a retrospective analysis of risk factors and outcome.Clin Lab.2013,第59卷(第7-8期),全文. * |
| Lucio Urbani, et al..The role of immunomodulation in ABO-incompatible adult liver transplant recipients.J Clin Apher.2008,第23卷(第2期),全文. * |
| Speranta Iacob, et al..Genetic, immunological and clinical risk factors for biliary strictures following liver transplantation.Liver Int.2012,全文. * |
| Tim O Lankisch, et al..MicroRNAs in the bile of patients with biliary strictures after liver transplantation.Liver Transpl.2014,第20卷(第6期),全文. * |
| Zhao-Wei Qu, et al..Preventive effects of autologous bone marrow mononuclear cell implantation on intrahepatic ischemic-type biliary lesion in rabbits.Hepatobiliary Pancreat Dis Int.2010,第9卷(第6期),全文. * |
| 史瑞.临床肝移植术后缺血型胆道病变的相关研究.中国博士学位论文全文数据库 医药卫生科技辑 (月刊).2016,第2016年卷(第04期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110850086A (en) | 2020-02-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cutillas et al. | Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome | |
| JP5194212B2 (en) | Methods associated with serum proteomics systems | |
| WO2003019193A1 (en) | Method of diagnosing nephrotic syndrome | |
| Tucholska et al. | Endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-of-flight | |
| CN111521828A (en) | Application of RSPH9 as diagnosis marker or therapeutic target of oligoasthenospermia | |
| KR100916651B1 (en) | Method for diagnosis of disease using quantitative monitoring of protein tyrosine phosphatase | |
| CN112063599A (en) | Acetylation modified SIRT2 protein marker molecule related to central nervous senescence and application thereof | |
| CN105622743B (en) | A kind of autism serum polypeptide marker PF4-A and its application | |
| CN103224922B (en) | New stomach cancer marker, detection method and applications thereof | |
| CN105652019B (en) | A kind of autism serum polypeptide mark APOC1 A and its application | |
| CN105738631B (en) | A kind of autism serum polypeptide mark SERPINA5 A and its application | |
| CN110850086B (en) | Application of serum diagnostic marker ACLY for ischemic biliary tract lesions after liver transplantation | |
| CN112940096A (en) | Bladder cancer serum polypeptide marker and application thereof | |
| EP1485461A2 (en) | Protein biomarkers that distinguish prostate cancer from non-malignant cells | |
| CN112063603B (en) | Acetylation modified CNP protein marker molecule related to central nervous senescence and application thereof | |
| CN102643334B (en) | Polypeptide molecule for serum in gastric cancer and application | |
| CN112881694A (en) | Renal cancer serum polypeptide marker and application thereof | |
| CN105622742A (en) | Autistic serum polypeptide marker FABP1-A and application thereof | |
| CN114487413A (en) | Application of serum polypeptide diagnostic marker Ezrin of brain glioma | |
| CN114354930A (en) | Application of serum polypeptide diagnostic marker AFP of brain glioma | |
| CN110850079A (en) | Application of diagnosis marker APOA1 for effect evaluation after liver transplantation | |
| CN117310183A (en) | Serum polypeptide marker SERPINA5 of immune-related pneumonia and application and detection kit thereof | |
| CN116298280A (en) | Application of liquid biopsy index in preparation of endometriosis screening and diagnosis products | |
| CN117304305A (en) | Serum polypeptide marker COL1A2 of immune-related pneumonia, application thereof and detection product | |
| Dillen et al. | Mass spectrometer characterization of bovine chromaffin granule peptides related to chromogranin B |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |