CN103116028B - Use of annexin A3 for detecting alcoholic liver fibrosis - Google Patents

Use of annexin A3 for detecting alcoholic liver fibrosis Download PDF

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CN103116028B
CN103116028B CN201110366184.XA CN201110366184A CN103116028B CN 103116028 B CN103116028 B CN 103116028B CN 201110366184 A CN201110366184 A CN 201110366184A CN 103116028 B CN103116028 B CN 103116028B
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annexin
fibrosis
liver
liver fibrosis
antibody
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CN103116028A (en
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张丽军
袁正宏
马芳
贾小芳
姚亚敏
刘晓茜
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SHANGHAI PUBLIC HEALTH CLINICAL CENTER
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SHANGHAI PUBLIC HEALTH CLINICAL CENTER
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Abstract

The invention belongs to the technical field of biology and relates to use of annexin A3 for detecting alcoholic liver fibrosis. Low-expressed protein, namely annexin A3 (ANXA3), in liver nonparenchymal cells of alcoholic liver fibrosis is obtained by screening differentially expressed protein in liver nonparenchymal cells of alcoholic liver fibrosis and normal livers. Western blot further proves that ANXA3 has lower expression level in the liver nonparenchymal cells of alcoholic liver fibrosis. The experimental result proves that annexin A3 can be used as a molecular mark for detecting alcoholic liver fibrosis. A specific antibody of annexin A3 comprises a monoclonal antibody and a polyclonal antibody and can be used for preparing a preparation for detecting alcoholic liver fibrosis. An antibody of annexin A3 comprises a monoclonal antibody and a polyclonal antibody and can be used for preparing a kit for detecting alcoholic liver fibrosis.

Description

Annexin A 3 is detecting the purposes in alcoholic fibrosis
Technical field
The invention belongs to biological technical field, relate to annexin A 3 and detecting the purposes in alcoholic fibrosis, be specifically related to annexin A 3 as the purposes detecting alcoholic fibrosis molecular marker.
Background technology
Modern medicine study finds, alcoholic fibrosis shows as chronic inflammation in liver and Progressive symmetric erythrokeratodermia liver tissue fibrosis, along with the progress of the course of disease can cause alcoholic fibrosis, is chronic alcoholism patients's main causes of death.Have statistics display, excessive drinking (alcohol abuse and alcohol dependence) has become the important public health problem in one, the world today; In the U.S., the adult of 67% has alcohol drinking patterns, adult's excessive drinking of 7.4%, and nearly 100,000 people die from excessive drinking every year; And in China, along with the raising of people's living standard and the day by day frequent of social activity, the incidence of disease of alcoholic fibrosis increases year by year, the health of serious threat compatriots; Meanwhile, the prognosis of alcoholic fibrosis patient allows of no optimist, and 4 ~ 5 annual survival rates are still very low.
The hepatic injury of alcohol induction is the major reason causing liver fibrosis, although the clinical manifestation of liver fibrosis has sorted out comparatively clear and definite at present, its early diagnostic rate, the particularly diagnosis of alcoholic fibrosis generation commitment still have much room for improvement.
At present, be the process of how to bring out liver fibrosis about alcohol factor, finally cause liver fibrosis to occur, Study on Molecular Mechanism wherein lacks still very much.Now there are some researches show, alcohol can cause liver plasma membrane, mitochondria and nuclear change.Alcohol can cause the change of liver plasma membrane permeability, the permeability of cell membrane is increased, cause the defect (Molleken on membrane structure simultaneously, Sitek et al.2009), and alcohol can also cause the phosphatide total amount of cell surface to increase, cell surface charge density is caused to raise and the increasing (Szachowicz-Petelska, Dobrzynska et al.2008) of lipid peroxidase body.Currently also studies have found that, alcohol also carrys out the apoptosis (Dan, Popov et al.2005) of active cell by activating caspase-9 and caspase-3 approach.Therefore, it is very complicated that alcohol affects hepatocellular mechanism, need to utilize high-throughout way to find the marker molecule in alcoholic fibrosis generation, evolution, thus be the pathogenesis of in-depth explanation alcoholic fibrosis, the gene that final screening and exploitation are expressed with treatment and the alcoholic fibrosis relevant difference that is diagnosed as object or/and protein, this area in the urgent need to the gene of new differential expression in alcoholic fibrosis or/and protein.
The accession number of annexin A 3 SwissProt is ANXA3_RAT, and cytoplasma membrane has distribution, its be one of annexin family member (see https:// myhits.vital-it.ch/cgi-bin/view_seq_entry? name=sw:ANXA3_RAT & view_sw=UniProt); This protein family plays regulating action in Growth of Cells and signal transduction.The function of described annexin A 3 is that inhibition of phospholipase A 3 is cracked into inositol 1,2-recycled phosphoric acid salt and 1-phosphoinositide, also plays a role in anticoagulation simultaneously.
Up to now, there is not yet about the directly or indirectly relevant report of annexin A 3 and alcoholic fibrosis.
Summary of the invention
The object of this invention is to provide the new purposes of annexin A 3, be specifically related to the purposes that annexin A 3 is used as the molecular marker detecting alcoholic fibrosis.
Another object of the present invention is to provide annexin A 3 specific antibody, comprises monoclonal antibody and polyclonal antibody, and this specific antibody detects the purposes in alcoholic fibrosis preparation in preparation.
Another object of the present invention with the antibody that a kind of anti-annexin A 3 is provided, comprise monoclonal antibody and polyclonal antibody, detect the purposes in alcoholic fibrosis kit in preparation.
The present invention is by the protein of screening differential expression in the liver non-parenchymal cell of alcoholic fibrosis and normal liver, obtaining a kind of protein (in alcoholic fibrosis cell low expression) that there are differences expression in alcoholic fibrosis and normal liver nonparenchymal cell, is annexin A 3 through Mass Spectrometric Identification; Further immunoblot experiment confirms, this protein exists the difference (downward of alcoholic fibrosis cells) of protein expression level in the liver non-parenchymal cell of alcoholic fibrosis and normal liver; Based on the correlativity of annexin A 3 and alcoholic fibrosis, carry out quantitatively detecting to its transcriptional level using the transcript of this albumen as a molecular marker and can be used as detecting alcoholic fibrosis.
Object of the present invention is carried out by following technical proposals:
The present invention collects the liver non-parenchymal cell of isolated experiment animal normal liver and alcoholic fibrosis with the method for the sucrose density gradient centrifugation of improvement, after cracking obtains cell protein, with the method isolated protein of protein two-dimensional gel electrophoresis, by the protein of ImageMaster software analysis differential expression, and Mass Spectrometric Identification is carried out to obtained differential protein, result shows, and annexin A 3 is low expression in the liver non-parenchymal cell of alcoholic fibrosis; Immunoblotting reaction experiment confirms further, the low expression of annexin A 3 in the liver non-parenchymal cell of liver fibrosis; Experimental result shows, annexin A 3 can be used as the molecular marker of alcoholic fibrosis, carries out detection can be used for detecting alcoholic fibrosis to its expression; Further, detection alcoholic fibrosis preparation can be prepared.
In the present invention, adopt the method for sucrose density gradient centrifugation to be first separated liver non-parenchymal cell, then obtain the plasmalemma protein matter of liver non-parenchymal cell in the mode of enzymolysis;
In the present invention, by the protein that two-dimensional gel electrophoresis method separating and cracking obtains, by ImageMaster 2D Platinum6.0 software analysis, screening obtains the protein of differential expression, carries out separations identify with Dai Anna upgrade liquid chromatogram Ultimate 3000 high power capacity ion trap mass spectrometry (LC-MS/MS) of connecting;
Experimental identification obtains 14 kinds of non-redundant proteins differential expression in the nonparenchymal cell of normal liver and alcoholic fibrosis liver, comprising the annexin A 3 expression of expressing reduction in the liver non-parenchymal cell of liver fibrosis; The appraisal result of the annexin A 3 that mass spectrophotometry obtains is as shown in table 1.
Table 1: the detailed qualification result of annexin A 3
Protein ID Protein describes Search storehouse score Isoelectric point Molecular weight (kDa) Sequential covering rate
ANXA3_RAT Annexin A 3 86 5.96 36.6 20%
The present invention also demonstrates annexin A 3 differential expression: the protein extracting liver fibrosis nonparenchymal cell, first SDS-PAGE is carried out, protein transduction in gel is moved on on pvdf membrane, carry out immunoblotting reaction, result and internal reference β-Actin contrast, and determine the expression of annexin A 3 in fibrosed tissue and normal liver tissue, result shows, annexin A 3 is low-level expression in alcoholic fibrosis tissue, and result is consistent with the result of protein two-dimensional gel electrophoresis;
Experimental result shows, described annexin A 3 there are differences expression in the liver non-parenchymal cell of normal liver and alcoholic fibrosis, with the generation of alcoholic fibrosis, develops and has close correlativity; Carry out detection using annexin A 3 as a molecular marker to its expression to can be used for detecting alcoholic fibrosis, wherein, annexin A 3 can be used as detecting alcoholic fibrosis as its expression in liver non-parenchymal cell of molecular marker, wherein, especially detect annexin A 3 and whether there is downward at liver non-parenchymal cell transcription; Further, the antibody of corresponding specific detection annexin A 3, comprising monoclonal antibody and polyclonal antibody, because it can detect the expression of annexin A 3, thus can be used for detecting alcoholic fibrosis or for the preparation of detecting the preparation of alcoholic fibrosis or kit etc.
A further object of the present invention is to provide the method that vitro detection annexin A 3 is expressed, and particularly relate to a kind of method that in vitro detection alcoholic fibrosis liver non-parenchymal cell, whether annexin A 3 expression is abnormal, it comprises step:
(1) by the quantity of annexin A 3 albumen in detection of specific antibody liver non-parenchymal cell to be checked;
(2) in amount detection step (1) obtained and normal liver liver non-parenchymal cell, the quantity of annexin A 3 compares, and the result recorded lower than normal value, then represents the abnormal expression of annexin A 3 in tested sample.
Experimental result of the present invention confirms the correlativity of annexin A 3 and alcoholic fibrosis, for the diagnosis of alcoholic fibrosis and/or treatment provide brand-new reference approach; Described annexin A 3 can be used as the application of the molecular marker detecting alcoholic fibrosis; The specific antibody of described annexin A 3, can be used for preparation and detect alcoholic fibrosis preparation, and preparation detects alcoholic fibrosis kit.
Accompanying drawing explanation
Fig. 1 shows the expression of annexin A 3 in normal liver and alcoholic fibrosis liver non-parenchymal cell, wherein, N represents normal liver, A represents alcoholic fibrosis liver, 6,96,9 weeks liver specimens of getting after liver fibrosis modeling are represented, 1,2,3 represent 3 repetitions, circle and arrow indication place represent the position at annexin A 3 place.
Fig. 2 shows the immunoblot results of annexin A 3 in normal liver and alcoholic fibrosis, wherein, N represents normal liver, and A represents alcoholic fibrosis liver, 6,9 represent 6,9 weeks liver specimens of getting after liver fibrosis modeling, β-Actin is internal reference contrast.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following example, usually the suggestion condition of the conveniently condition described in conditioned reference " Molecular Cloning: A Laboratory guide " (third edition, Cold Spring Harbor Publications) or manufacturer.
Embodiment 1 prepares alcoholic fibrosis liver non-parenchymal cell protein example
The urea used in the present embodiment, thiocarbamide, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT) (DTT), 3-[(3-courage amido propyl)-diethyl ammonium]-1-propane sulfonic acid (CHAPS) be available from Sigma all, and DNA enzymatic is purchased from Takara.
First the present embodiment is separated liver non-parenchymal cell with the method for sucrose density gradient centrifugation, then obtains the plasmalemma protein matter of liver non-parenchymal cell in the mode of enzymolysis, and concrete steps are as follows:
Get in vitro alcoholic fibrosis animal experimental model liver organization to shred in ice-cold physiological saline, cold buffer A (50mM HEPES is suspended in Potter-Elvehjem Tissue Grinders homogenate, 1mM CaCl2 and 1mM PMSF, pH7.4) fragment of tissue in, the suspension that homogenate is good is placed in 50ml centrifuge tube, under 4 DEG C of conditions, the centrifugal 15min of 15000g, get precipitation to mix with 60% sucrose, extremely to the sucrose concentration in potpourri be 44%.The sucrose solution of 42.3% is carefully added drop-wise to 44% sucrose mixture upper strata, under 4 DEG C of conditions, the centrifugal 2.5h of 100000g, the fragment of tissue getting the centrifuge tube the superiors is and is separated the plasmalemma protein matter obtained and slightly gets sample this, by this sediment at buffer B (50mM HEPES, 1mM PMSF, pH 7.4) in washing twice.Originally mixing slightly getting sample with the sucrose of 60%, until the sucrose concentration in potpourri is 44%, being added drop-wise to 44% sucrose mixture upper strata successively by 42.3%, 41.0%, 39.0%, 37.0%.The centrifugal 6h of 100000g, the fragment of tissue got in the superiors 37.0% sucrose washs twice in buffer B, is extracted plasma membrane.
The liver insubstantial plasma membrane of acquisition is added lysis buffer (8mol/L urea, 4%CHAPS, 65mmol/L DTT, 2mol/L sulphur urine, 1%NP-40,0.5mmol/L PMSF, 1%DNA enzyme), act on 30min on ice, more at room temperature hatch 30min.After the protein Bradford method that cracking obtains measures protein content ,-80 DEG C save backup.
Embodiment 2 screens differential expression protein
The acrylamide used in the present embodiment, N, N '-di-2-ethylhexylphosphine oxide (acrylamide), glycocoll, lauryl sodium sulfate (SDS), trishydroxymethylaminomethane (Tris), urea, glycerine purchased from American Amresco company, Ammonium Persulfate 98.5 (AP), TEMED are purchased from Bio-Rad.
Protein cracking obtained is separated by the method for two-dimensional gel electrophoresis, by ImageMaster 2D Platinum 6.0 software analysis, find out the protein of differential expression, and separation qualification is carried out to obtained differential protein Dai Anna upgrade liquid chromatogram Ultimate3000 series connection high power capacity ion trap mass spectrometry (LC-MS/MS), concrete steps are as follows:
First of two dimensional gel electrophore-sis adopts the non-linear adhesive tape of pH 3 ~ 10 to isoelectric focusing electrophoresis, and electrophoretic procedures is arranged: aquation 30V, 12h; Electrophoresis: 500V, 1h; 1000V, 1h; 8000V, 30min, gradient; 8000V, 5h, linearly; General power reaches 44 kilowatts.Second is separated to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and resolving gel concentration is 11.5%.After electrophoresis is complete, stripping glue, contaminates glue with Coomassie brilliant blue, the imaging of Image scanner scanner.Electrophoresis pattern is divided into 2 classifications by gained image ImageMaster 2D Platinum 6.0 software to carry out looking for the analyses such as point, coupling, finds out differential protein particle.
Get differential protein particle decolouring dewatered freeze-dried after, often add about 5 μ L pancreatin (0.02 μ g/ μ L) in pipe.37 DEG C of enzymolysis spend the night (16 ~ 18h).Sample after enzymolysis is first through C18 pre-column (300 μm of id x5mm, 5 μm) desalination, and flow velocity is 20 μ L/min, and mobile phase is 2% ACN, 0.1%FA.Then be separated through the reverse post of C-18 (75 μm of id x 15cmlength, 3 μm, PepMapTM) with the flow velocity of 300nL/min, the gradient of chromatogram is 4-48%, and gradient timetable is 40min.The peptide section be separated by C18 chromatographic column is entered HCT mass spectrum and carries out real-time ionization by receiving stream nozzle needle and analyze and detect.HCT mass spectrum carries out an one-level scanning and 5 secondary scanning analysis p.s..
Peptide fingerprinting spectrum and tandem mass spectrum second order spectrum is integrated, by Mascot software inquiry SwissProt database with DataAnalysis 3.2 software.MS error: ± 0.6Da, it is that halfcystine is modified to urea methyl cysteine (Carbamidomethyl-Cys) that MS/MS error: 1.2Da. fixes modification, variablely to be modified to methionine oxidized (Oxidation (M)), each peptide section has allowed 1 incomplete cracking site, ion selects (+1, + 2, + 3), pattern is: single isotope, fiducial interval is 95%, P < 0.05, protein score more than 36 points reliable results.
14 kinds of non-redundant proteins differential expression in the nonparenchymal cell of normal liver and alcoholic fibrosis liver is identified, comprising the annexin A 3 expression of expressing reduction in the liver non-parenchymal cell of liver fibrosis with said method.As shown in Figure 1, two dimensional gel electrophore-sis display annexin A 3 down-regulated expression (caption: 6,9 different time points representing alcoholic fibrosis in fibrotic processes of coomassie brilliant blue staining, N represents tissue-derived in normal liver, A represents tissue-derived in liver fibrosis, and circle represents the position at annexin A 3 place).
The detailed appraisal of the annexin A 3 that mass spectrophotometry obtains is as shown in table 2.
Table 2: the detailed qualification result of annexin A 3
Protein ID Protein describes Search storehouse score Isoelectric point Molecular weight (kDa) Sequential covering rate
ANXA3_RAT Annexin A 3 86 5.96 36.6 20%
Embodiment 3 verifies annexin A 3 differential expression
The antibody of the anti-annexin A 3 used in the present embodiment is purchased from ProteinTech Group Inc.; The antibody of β-Actin and two of horseradish peroxidase-labeled resists all purchased from Santa; Pvdf membrane is purchased from Millipore.
Extract the protein of liver fibrosis nonparenchymal cell, first SDS-PAGE is carried out, protein transduction in gel is moved on on pvdf membrane, carry out immunoblotting reaction, result and internal reference β-Actin contrast, to determine the expression of annexin A 3 in fibrosed tissue and normal liver tissue, comprise the following steps:
50mg protein is carried out electrophoresis, electrophoresis adopts 11.5% separation gel, electrophoresis terminates rear transferring film, pvdf membrane is spent the night closed, film after closing washes film 3 times in TBST, 37 DEG C of antibody 2h of hatching anti-annexin A 3, then pvdf membrane is washed 3 times in TBST, in two of horseradish peroxidase-labeled resists, hatch 2h, after washing film with above-mentioned steps, carry out exposure colour developing in darkroom.
The expression of annexin A 3 is by being the protein content of surveyed annexin A 3 divided by the ratio measured of internal reference β-Actin, result as shown in Figure 2, compared with the relative content of the annexin A 3 in normal liver, content in alcoholic fibrosis is obviously lower, result shows, annexin A 3 has the expression of reduced levels in alcoholic fibrosis tissue, and the above results is consistent with the result of protein two-dimensional gel electrophoresis.
Experimental result confirms, annexin A 3 there are differences expression in the liver non-parenchymal cell of normal liver and alcoholic fibrosis, obviously and the generation of alcoholic fibrosis, develop and have close correlativity, therefore, carry out detection using annexin A 3 as a molecular marker to its expression to may be used for detecting alcoholic fibrosis; The antibody of corresponding specific detection annexin A 3, because it can detect the expression of annexin A 3, thus can be used for detecting alcoholic fibrosis or for the preparation of detecting the preparation of alcoholic fibrosis or kit etc., this is apparent for a person skilled in the art.

Claims (5)

1. annexin A 3 is being prepared as the purposes in the molecular marker of alcoholic fibrosis.
2. the specific antibody of annexin A 3 detects the purposes in alcoholic fibrosis preparation in preparation.
3., by purposes according to claim 2, it is characterized in that, described specific antibody comprises monoclonal antibody and polyclonal antibody.
4. the antibody of anti-annexin A 3 detects the purposes in alcoholic fibrosis kit in preparation.
5., by purposes according to claim 4, it is characterized in that, the antibody of described anti-annexin A 3 comprises monoclonal antibody and polyclonal antibody.
CN201110366184.XA 2011-11-17 2011-11-17 Use of annexin A3 for detecting alcoholic liver fibrosis Expired - Fee Related CN103116028B (en)

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US9487831B2 (en) * 2013-09-12 2016-11-08 Versitech Limited Use of annexin A3 as a diagnostic and prognostic biomarker and therapeutic target for treating hepatocellular carcinoma
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US6843980B2 (en) * 2001-04-03 2005-01-18 Theseus Imaging, Corp. Methods for using annexin for detecting cell death in vivo and treating associated conditions
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CN100571785C (en) * 2006-09-06 2009-12-23 中国医学科学院北京协和医院 The dependency of the platinum-based chemotherapy drug resistance of Annexin A3 and cancer
WO2011094579A2 (en) * 2010-01-29 2011-08-04 New Chapter Inc. Mushroom compositions and methods for making and using
US20130274215A1 (en) * 2010-04-08 2013-10-17 Fate Therapeutics, Inc. Pharmaceutical compositions to treat fibrosis

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