CN103087173B - Synthetic peptide vaccine of B cell epitope based on TGF(transforming growth factor)-beta1 and application thereof - Google Patents

Abstract

The invention discloses a synthetic peptide vaccine of B cell epitope based on TGF(transforming growth factor)-beta1 and application thereof, inhibits auxo-action of the cell factor TGF-B1-BB during the development process of liver fibrosis so as to inhibit the development of liver fibrosis to a some extent by constructing two epitope peptides of AY25 and LS30, coupling carrier proteins to synthesize an TGF-beta1 peptide vaccine, immunizing an ICR mice by intraperitoneal injection to induce the same to generate an anti-TGF-beta1 antibody, and neutralizing the cell factor TGF-B1-BB over expressed during CCL4 induced hepatic fibrosis process.

Description

Synthetic peptide vaccine and the application thereof of the B cell antigen epi-position based on TGF-β 1

Technical field

The invention belongs to the Prevention Technique field of hepatic fibrosis, relate to synthetic peptide vaccine and the application thereof of the B cell antigen epi-position based on TGF-β 1.

Background technology

Hepatic fibrosis be multiple chronic hepatopathy to liver cirrhosis development must through pathologic process, be the main factor that determines chronic hepatopathy prognosis.At present, most of chronic hepatopathy all lacks effective etiological treatment, and studies confirm that part hepatopathy removes heptic fibrosis in the cause of disease and still can continue progress, therefore thereby retardance or the generation development that delays hepatic fibrosis prevent progress becomes one of main path that chronic hepatopathy treats for liver cirrhosis.But there is no at present truly effectively anti-hepatic fibrosis medicines or measure, the anti-hepatic fibrosis measure safely and effectively of exploring is the important topic that current hepatopathy research field faces.

Cytokine vaccine is a focus in vaccinology research in recent years, it is mainly the method active immunity body that the cytokine playing a major role in a certain disease is adopted to vaccine, induction body produces neutrality antibody, neutralize this cytokine to stop the performance of its corresponding biological function, thereby play the object of control relative disease.Synthetic peptide vaccine is comparatively extensively and day by day one of ripe new generation vaccine of Recent study, and in stability, easily the property obtained, security, purifying be simple etc., and aspect has obvious advantage, existing lot of documents is reported its feasibility and validity.This vaccine, containing nucleic acid, is not a class vaccine of the epitope development and Design based on protein molecular, is ideal safe new generation vaccine.

Transforming growth factor-beta 1 (transforming growth factor beta, TGF-β 1) be the polypeptide that a class can regulate Growth of Cells and differentiation, possess various biological effect, in hepatic fibrosis generation, evolution, possess activated hepatic stellate cells, promote collagen gene expression.Promoting extracellular matrix to synthesize with deposition and wait effect, is the current known the strongest cytokine of short fibrosis effect.The effect of TGF-β 1 in hepatic fibrosis is in particular in:

Promote the various kinds of cell epimatrix compositions such as hepatic stellate cell expression and secretion I, III, type Ⅳ collagen fiber, ln;

Suppress the activity of proteolytic enzyme in extracellular matrix, as the expression of matrix metalloproteinase and tissue plasminogen activator is had to restraining effect, suppress the degraded of extracellular matrix;

Strengthen some cytokine as the expression of PDGF mRNA;

Inoblast, monocyte are had to chemotaxis;

Promote the expression of TGF-β 1 self by Autocrine.

The keying action of TGF β 1 in liver tissue during hepatic fibrogenesis obtained generally acknowledging of investigators, and the focus and the utmost point that also become anti-hepatic fibrosis research for the anti-hepatic fibrosis research of TGF β 1 have one of research of application prospect.The subject matter facing is at present to explore to block safely and effectively TGF β 1 measure.When having been reported TGF beta II type acceptor with TGF beta 1 antibodies, soluble T GF β1receptor and adenovirus mediated brachymemma etc. and making hepatic fibrosis by neutralizing effect, the TGF β 1 of excessive generation reduces, in experimentation on animals, show significant anti hepatic fibrosis and reduced hepatocellular apoptosis effect, and having found no obvious liver part or systemic side effects.There is the research of the organ fibrosis such as the lung, kidney, the heart of similar genesis mechanism also to draw similar conclusion to hepatic fibrosis.But from the angle of clinical application, above method all has preparation method's complexity, needs repeated multiple times administration, uses the outstanding shortcomings such as inconvenience, and application prospect is not good enough.Finding the more convenient means of effectively blocking TGF β 1 is one of current problem demanding prompt solutions.

Summary of the invention

The problem that the present invention solves is to provide synthetic peptide vaccine and the application thereof of the B cell antigen epi-position based on TGF-β 1, produce the antibody of anti-TGF-beta 1 by induction body, with in and the promoter action of TGF-β 1 in liver fibrosis process, reach the object that suppresses hepatic fibrosis process.

The present invention is achieved through the following technical solutions:

A B cell antigen epi-position of TGF-β 1, comprising:

The AY25 epi-position of amino acid residue sequence as shown in SEQ.ID.NO.1;

The LS30 epi-position of amino acid residue sequence as shown in SEQ.ID.NO.2.

A kind of synthetic peptide vaccine, comprises the B cell antigen epi-position of TGF-β 1, and is coupled on carrier proteins;

Shown epitope is the AY25 epi-position as shown in SEQ.ID.NO.1, or LS30 epi-position as shown in SEQ.ID.NO.2.

Described carrier proteins is BSA or KLH.

The application of described synthetic peptide vaccine in the antibody of preparing anti-TGF-beta 1.

The application of described synthetic peptide vaccine in the medicine of preparing anti-hepatic fibrosis.

Described medicine is the medicine of inhibition or anti-TGF-beta 1B activity short of money.

Described medicine is the medicine that suppresses hepatocellular apoptosis.

Compared with prior art, the present invention has following useful technique effect:

The B cell antigen epi-position of TGF-β 1 provided by the invention, adopts synthetic its peptide sequence of chemical process, preparation coupling peptide vaccine, and by the method immune mouse of abdominal injection, inducing mouse body produces corresponding antibody, and passes through CCl ₄build Liver Fibrosis Model, anti-TGF-beta 1 antibody being produced to observe can in and TGF-β 1 to reach the effect of anti-hepatic fibrosis.

Result shows, the epi-position AY-25 screening and LS-30 all have antigenicity, with ELISA and the detection of Western blot method, for control group and normal group, after immune four times, experimental mice internal antibody titre can reach 1:5120, sustainable 4-6 week of this antibody titers, then decay gradually, after 6 months, antibody titers decays to 1:640-1:1280.Can be with commercialization, have the restructuring TGF-β 1B specific reaction of biologic activity; Can be combined with the TGF-of prokaryotic expression β 1 purifying protein; And the antibody producing shows good reactivity and biologic activity.

TGF-β 1 synthetic peptide vaccine provided by the invention, immune mouse, inducing mouse produces anti-TGF-beta 1 antibody, in and at CCL ₄the cytokine TGF-β 1 of overexpression in the liver fibrosis process of induction, suppresses its promoter action in hepatic fibrosis evolution, thereby suppresses to a certain extent the development of hepatic fibrosis:

After immunity, at CCL ₄in the liver fibrosis process of induction, hepatic tissue structure is more complete, and in liver, deposition of fibrous tissue is few, and degree of hepatic fibrosis classification significantly reduces; The expression of α-SMA and interstitial district PCNA obviously reduces; PCNA index in interstitial district obviously reduces; Hepatic tissue Hyp content all significantly reduces.Confirm can significantly suppress CCl after TGF-β 1 synthetic peptide vaccine immune mouse ₄the hepatic fibrosis in mice of induction.Show that TGF-β 1 vaccine is expected to become a kind of simple, safe and effective hepatic fibrosis and prevents and treats novel method

Brief description of the drawings

Each group for ELISA detects of Fig. 1-1 immune serum antibody titers.

Fig. 1-2 is the reactivity that Western blot detects each group of mice serum antibody.

Fig. 1-3 are the biologic activity of each group of mice serum antibody of MV1LU Growth of Cells experiment detection.

Fig. 2 is each group of murine liver tissue Masson coloration result.

Fig. 3 is each group of murine liver tissue hydroxyproline content result.

Fig. 4-1 is each group of murine liver tissue α-SMA coloration result.

Fig. 4-2 are each group of murine liver tissue α-SMA positive region statistic analysis result.

Express for Western blot detects each group of murine liver tissue pSmad2/3 and Smad2/3 Fig. 4-3.

Express for Western blot detects each group of HSC-T6 cell pSmad2/3, Smad2/3 and α-SMA Fig. 5-1.

Express for real-time fluorescence quantitative PCR method detects COL1A2mRNA Fig. 5-2.

Express for real-time fluorescence quantitative PCR method detects PAI-1 Fig. 5-3.

Express for real-time fluorescence quantitative PCR method detects TIMP-1 Fig. 5-4.

Fig. 6-1 is each group of murine liver tissue TUNEL coloration result.

Fig. 6-2 are each group of mouse liver cell apoptotic index statistic analysis result.

Fig. 7-1 is each group of murine liver tissue PCNA coloration result.

Fig. 7-2 are each group of mouse liver cell PCNA index statistic analysis result.

Fig. 8-1 is each group of murine liver tissue DESMIN coloration result.

Fig. 8-2 are each group of murine liver tissue DESMIN stained positive range statistics analytical results.

Embodiment

The invention provides synthetic peptide vaccine and the application thereof of the B cell antigen epi-position based on TGF-β 1, screening has antigenic epitope peptide synthetic TGF-β 1 peptide vaccine of coupling carrier albumen, adopt the method immune mouse of abdominal injection, repeat immunity and detect mice serum antibody titers, CCl ₄inducing mouse Liver Fibrosis Model.HE, Masson dyeing is observed level of fibrosis and is learned change.Immunohistochemical staining detects in hepatic tissue.Alkali hydrolysis method detects each group of hepatic tissue hydroxyproline content, observes each group of murine liver tissue fibroplasia situation, relatively the difference of degree of hepatic fibrosis between vaccine immunity group and modeling group, KLH group.Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.

1, the screening of the B cell antigen epi-position of TGF-β 1 and synthetic

First adopt bioinformatics method analysis, specifically by the online B cell antigen epi-position of webserver TGF-β 1 albumen and the physicochemical property of TGF-β 1 albumen, screening has immunogenic epi-position, has specifically screened two epi-position AY25 and LS30:

Shown in TGF-β 125:41ANFCLGPCPYIWSLDTQYSKVLALY65(SEQ.ID.NO.1);

Shown in TGF-β 1-30:83LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS112(SEQ.ID.NO.2);

And the epi-position of screening be also T and mouse TGF-β 1 molecule reply fragment height homology, and be the position playing a crucial role in TGF-β 1 its receptor corresponding with it.

Then adopt automatic Peptide synthesizer to synthesize corresponding peptide sequence, synthetic polypeptide is analyzed through high performance liquid chromatography (high performance liquid chromatography, HPLP), and purity all can reach 99.00%.

2, the preparation of coupling peptide

Epitope peptide is coupled on carrier proteins, because carrier proteins has immunogenicity, is conducive to body and produces the antibody for epitope peptide.

The operation steps of the coupling of KLH/BSA-AY25 and KLH/BSA-LS30:

(1) take: AY255mg, LS305mg, EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) 20mg, KLH(Yao Kong Qi hemocyanin) 4mg, BSA(ovalbumin) 4mg(please provides respectively Chinese), be loaded on respectively in corresponding bottle and mark.

(2) dissolve: in KLH and BSA, add respectively 400ul0.1M MES damping fluid (laboratory preparation), visible dissolving rapidly.

(3) dissolve: in AY25 and LS30, add respectively about 1.2ml0.1M MES damping fluid, fully vibration impels it to dissolve, and it not exclusively dissolves as seen; Add wherein 100ul dinethylformamide (organic solvent) dissolution, it dissolves rapidly as seen; Add wherein more a small amount of hydrochloric acid to regulate its pH value, it is fully dissolved.

(4) mix: AY25 and LS30 are divided into respectively to two equal portions (about 600ul/ part), add respectively in KLH solution and BSA solution, be labeled as KLH-AY25, BSA-AY25, KLH-LS30 and BSA-LS30.

(5) coupling: add 2ml deionized water dissolving in 20mg EDC, respectively get immediately 100ul and add respectively in KLH-AY25, BSA-AY25, KLH-LS30 and BSA-LS30, mix rear room temperature and leave standstill 2 hours.

(6) dialysis: liquid in each bottle is put in and in dialysis tubing, uses the PBS damping fluid of 0.01M in 4 DEG C of dialysis 24 hours, within every 8 hours, change liquid 1 time, totally 3 times, visible dialysis tubing bulging gradually.

(7) packing: dialyzate is pressed to the packing of 0.5ml/ pipe, each 8 pipes.Frozen for subsequent use in-20 DEG C of refrigerators.

3, immune mouse

Male ICR mouse is divided into normal group, CCl at random ₄group, KLH group, KLH-AY25 group and KLH-LS30 group, wherein 6 of normal group, CCl ₄group and KLH organize each 8, and KLH-AY25 group and KLH-LS30 organize each 10.

Immunity flow process:

1) first week: OVA-VF16 and KLH-QK16 are respectively got to 2 (1ml), mix respectively with isopyknic Freund's complete adjuvant, give mouse peritoneal injection after fully emulsified, 0.2ml/ only.

2) second week: OVA-VF16 and KLH-QK16 are respectively got to 1 (0.5ml), with mixing with isopyknic Freund's incomplete adjuvant respectively after equivalent PBS dilution, give mouse peritoneal injection, 0.2ml/ after fully emulsified.

3) the 3rd week: same to second week.

4) 4th week: OVA-VF16 and KLH-QK16 are respectively got to 1 (0.5ml), give mouse peritoneal injection after mixing with equivalent PBS dilution, 0.1ml/ only.

5) the 5th week: same to 4th week.

6) the 8th week: same to second week.

7) the tenth week: same to second week.

4, indirect ELISA detects

Blank while collecting mouse mouse tail serum for ELISA detection before immune mouse, frozen for subsequent use in-20 DEG C of refrigerators.Adopted mouse tail blood respectively at after immunity the 4th week, the 6th week, the 8th week and carry out ELISA detection serum antibody titer, after experiment finishes, put to death mouse, collect serum and carry out indirect ELISA detection.

Operation steps:

1) antigen coated: the carbonate buffer solution dilution coupling peptide OVA-AY25 of 0.05M PH9.6, not coupling of OVA-LS30(OVA) (50ng/ hole) and commercial TGF-β 1 recombinant cytokine (20ng/ hole) the different hole of coated polystyrene board respectively that has biologic activity.In the reacting hole of each polystyrene board, add 0.1ml, 4 DEG C are spent the night.Next day, discards solution in hole, adds wherein 300ul/ hole 10% bovine serum/PBS sealing, and 4 DEG C are spent the night.The 3rd day, discard solution in hole, pat dry, frozen for subsequent use in-20 DEG C of refrigerators.(with before need wash 1 time by washing lotion).

2) add primary antibodie: add and in above-mentioned coated reacting hole, (do blank hole and negative control hole) by each experimental mice serum 0.1ml of gradient dilution proportion with 10% bovine serum/PBS liquid simultaneously, put 37 DEG C and hatch 1 hour.After washing 4 times, pat dry.

3) add ELIAS secondary antibody: in each reacting hole, add the goat anti-mouse IgG 0.1ml (with 10% bovine serum/PBS liquid by 1:5000 dilution) of the HRP mark of fresh dilution, hatch 1 hour for 37 DEG C, pat dry after washing 4 times.

4) add substrate solution and nitrite ion: in each reacting hole, add each 1 of substrate solution and nitrite ion, 37 DEG C of 10min.

5) termination reaction: add 1 of stop buffer in each reacting hole.

6) result is judged: a, direct observations in white background with the naked eye: reacting hole in, color is darker, and positive degree is stronger, and negative reaction is colourless or extremely shallow, and the depth of color that foundation is, represents with "+", "-" number.B, microplate reader are measured: measure its OD value in 450nm wavelength, and positive to treat gaging hole OD value >=2.1 times negative control hole OD value.

Mice serum after OVA-AY25, OVA-LS30 epitope peptide vaccine immunity is test positive in the coated hole of corresponding antigens all, and negative in the coated hole of KLH, PBS, after being described, epi-position peptide vaccine immune mouse produces the specific antibody for corresponding epitope peptide.

The detected result that ELISA detects each group of immune serum antibody titers as Figure 1-1, after immune four times, experimental mice internal antibody titre can reach 1:5120, sustainable 4-6 week of this antibody titers, then decay gradually, after 6 months, antibody titers decays to 1:640-1:1280.And can be with commercialization, have the restructuring TGF-β 1 of biologic activity that specific reaction occurs.

5, Western blot detects

1) preparation 12%Tris-glycine SDS-polyacrylamide gel:

(1) preparation separation gel 5m1:H ₂o1.6ml, 30% acrylamide 2.0ml, 1.5M Tris (PH8.8) 1.3ml, 10%SDS50ul, 10% Ammonium Persulfate 98.5 50ul, TEMED2ul, adds in order successively, mixes rear ordinary method encapsulating, and after 30min, gelling is solid.

(2) preparation spacer gel 2ml:H2O1.4ml, 30% acrylamide 330ul, 1.0M Tris (PH6.8) 20ul, 10%SDS20ul, 10% Ammonium Persulfate 98.5 20ul, TEMED2ul, adds in order successively, mixes rear ordinary method encapsulating, insert comb, 30min after coagulation, can loading.

2) sample preparation: 6his-TGF-β 1 chain gene Prokaryotic expression, purification protein sample is mixed with isopyknic sample-loading buffer (6 × loading buffer:DTT is 9:1), in metal bath, boil 5min, ice bath 5min, littlely can be used for loading after centrifugal.

3) loading: electrophoresis apparatus is installed, is poured wherein into 1 × SDS-glycine electrophoretic buffer, by 3ul/ hole sample size loading, 2 × loading buffer loading of equivalent for blank well.

4) protein electrophoresis: connect electrophoresis apparatus, voltage 80V, stops electrophoresis after about 120min, takes off gel, carries out transferring film.

5) transferring film

(1) take out gel, first gel is put into transferring film damping fluid balance 30min, carry out according to a conventional method transferring film.

(2) be followed successively by 3MM Whatman filter paper (3 layers), gel (connecing negative pole), nylon membrane (NC film) (connecing positive pole), 3MM Whatman filter paper (3 layers) from bottom to upper strata.

(3) constant current transferring film, 1mA/cm2,30min.

(4) take out NC film, use the TBST liquid that contains 5% skim-milk in room temperature sealing 1 hour.

6) dye film

(1) after NC film is washed once with TBST liquid, add the mice serum (primary antibodie) diluting by 1:1000 with the TBST liquid that contains 5% skim-milk, after incubated at room 1h, spend the night in 4 DEG C.

(2) wash film 4 times with TBST liquid, each 5-10min, adds with the TBST liquid that contains 5% skim-milk by the goat anti-mouse IgG of the HRP mark of 1:2000 dilution, incubated at room 1h.

(3) wash film 4 times with TBST liquid, each 5-10min, presses exposure, observations scan image after adding ECL colour developing lmin in darkroom.

Western Blot detected result as shown in Figure 1-2, from left to right be respectively No. KLH-AY25-1, No. KLH-AY25-2, No. KLH-AY25-3, No. KLH-LS30-1, No. KLH-LS30-2, No. KLH-LS30-3, KLH-1 mice serum antibody and PBS, antibody dilution is 1:800.Result shows: the KLH group of comparing, the TGF-beta 1 antibodies in experimental mice immune serum can be combined with the TGF-of prokaryotic expression β 1 purifying protein, shows good reactivity and specificity.

6, detect the impact of TGF-beta 1 antibodies on MV1LU cell proliferation

(this is antibody neutralization test, because TGF-β 1 can promote cell proliferation, add after TGF-beta 1 antibodies can anti-TGF-beta 1 short of money proliferation, for detection of antibodies specific, reactivity and antibody biologic activity)

Taking the logarithm goes down to posterity vegetative period cultivates MV1LU cell, and after tryptic digestion, counting cells is also adjusted into 1 × 10 with DMEM serum-free medium by cell concn ³/ ml, adds 96 aseptic porocyte culture plates with every hole 200 μ l volumes by cell suspension, is placed in 37 DEG C, 5%CO ₂cell culture incubator in cultivate 48 hours.

Discard nutrient solution, cultivate 30 minutes with containing the sero-fast DMEM nutrient solution of different Dilution ratios.

To the TGF-Β 1-BB and the BrdU that add 3ng/ml in each hole, continue to cultivate 48 hours.

Discard nutrient solution, wash with PBS, detect Growth of Cells situation to adding in each hole with anti-BrdU Ad and ELISA.More than experiment repeats 2 times.

Experiment grouping situation is: blank group, KLH group, KLH-AY25 group and KLH-LS30 group.Establish 3 multiple holes for every group.In the time starting 2w, 4w before immune animal and after immunity, 6w, 8w, 10w, 12w, 14w, 16w, 18w, 20w, 22w, 24w, take mice serum to repeat above-mentioned experimentation respectively.

Each group MV1LU Growth of Cells experimental result is shown in Fig. 1-3.With KLH group and CCL ₄group is compared, and KLH-AY25 group and KLH-LS30 group all can obviously suppress Mv1Lu cell proliferation, significant difference (P < 0.05); And the extent of dilution of 1:250 can suppress 50% Mv1Lu cell proliferation.

Above-mentioned detected result shows, two epitope peptide AY25 of synthesized and LS30, after vaccine is made in coupling, afterwards all can be in the middle of body all can inducing mouse produce the antibody of high titre in immunity, and the antibody producing shows good reactivity and specificity, and all can suppress the growth of MV1LU cell, show the activity of anti-TGF-beta 1.

7, CCl ₄the foundation of inducing mouse Liver Fibrosis Model

Detect KLH-AY25 group and KLH-LS30 in the 8th week of immunity and organize mice serum antibody titers to reaching 1:5120, start to use CCl ₄inducing mouse Liver Fibrosis Model.CCl ₄be mixed with by volume 20% mixed solution with peanut oil, every modeling group mouse is pressed 5ml/kg abdominal injection CCl ₄/ peanut oil mixed solution, 1 week 2 times, continuous 6 weeks.

8, laboratory animal is got blood and sample

1) after experiment finishes, eyeball is got blood system and is not put into the corresponding clean EP pipe that mark is good, places about 30min under room temperature, observes blood coagulation in EP pipe, while having as seen serum to separate out, and centrifugal 5min, 3000rpm/min.

2) supernatant after centrifugal is drawn in the EP pipe of respective markers, recentrifuge 5min,, 5000rpm/min.

3) supernatant after centrifugal is drawn in corresponding EP pipe ,-20 DEG C save backup.

4) get each group of mouse liver, right lobe of liver is placed in to 10% formalin fixing; Lobe of the liver corresponding to residue liver collected respectively, puts into-70 DEG C and saves backup.

Visual inspection: normal group mouse liver smooth surface, ruddy, matter is soft.OVA-VF16 group and KLH-QK16 group mouse liver color depth, surface is more smooth without adhesion, and volume has no obviously and dwindles.KLH group and CCl ₄group mouse liver gross specimen shows that liver loses normal ruddy gloss, is brown color, and there are many white particles on surface, and quality is more tough, not easily broken.

9, paraffin section and HE dyeing

Carry out paraffin section and HE dyeing according to conventional treatment method, can entrust laboratory or company to carry out, specifically make section and dyeing by Xi'an Communications University's Pathological Staff Room.

Each group mouse HE coloration result: in the section of normal group murine liver tissue, liver lobule form shows no obvious abnormalities, without the infiltration of hepatic necrosis and inflammatory cell.OVA-VF16 group and the most of form of KLH-QK16 group liver cell are normal, small part hepatocellular degeneration, necrosis, cell infiltration.KLH group and CCl4 group Microscopic observation liver lobule normal configuration disappear, and pseudolobuli forms, and visible significantly liver cell cavity sample sex change is even downright bad, cell infiltration.

10, Masson dyeing

1) the hepatic tissue paraffin section that is 6um by thickness is placed in 37 DEG C of incubators and toasts 1h.

2) dewaxing: order is: dimethylbenzene 2min → dimethylbenzene 2min → dimethylbenzene 2min → dehydrated alcohol 2min → dehydrated alcohol 2min → 95% alcohol 2min → 85% alcohol 2min → tap water is washed till few 3min → distillation washing 1min.

3) section is put in to about 5min in masson dye liquor, the about 5s of distillation washing.

4) section is put in to about 3min in 1% phospho-molybdic acid, the about 5s of distillation washing.

5) section is put in to about 6min in 2% BG, the about 5s of distillation washing.

6) section is put in to about 5s in 0.2% glacial acetic acid, the about 5s of distillation washing.

7) dehydration: order is: 85% alcohol 5s → 95% alcohol 5s → 95% alcohol 5s → dehydrated alcohol 5s → dehydrated alcohol 5s → propyl carbinol 5s → dimethylbenzene 5s → dimethylbenzene 5s.

8) neutral gum is fixed, conventional mounting.

9) micro-Microscopic observation coloration result: under opticmicroscope, visible endochylema, myofiber are red, collegen filament, mucus are green.Liver cirrhosis pathology grade scale is as follows:

(1) 0 grade is that hepatic tissue is normal, collagen-free fibroplasia;

(2) I level is that collegen filament slightly stretch out from portal area or around central vein;

(3) II level is that collegen filament extend obviously, but not yet connects, and holds whole liver lobule;

(4) III level is that collegen filament extend obviously, interconnection, holds whole liver lobule;

(5) IV level is that collegen filament hold and cut apart liver lobule, and normal hepatocytes leaflet structure is destroyed, and pseudolobuli forms, but taking large square pseudolobuli as main;

(6) V level is that liver lobule structure is destroyed completely, and pseudolobuli forms large square pseudolobuli and small circular pseudolobuli respectively accounts for 50%;

(7) VI level is in liver, to be covered with small circular pseudolobuli, has the collegen filament of thick hyperplasia in pseudolobuli.

Respectively organize mouse Masson coloration result as shown in Figure 2, no abnormality seen fibroplasia after the dyeing of normal group murine liver tissue, portal area is without obvious proliferation of fibrous tissue.A small amount of proliferation of fibrous tissue is seen in KLH-AY25 group and KLH-LS30 group portal area, and liver lobule is cut apart not exclusively by fibrous septum.KLH group and CCl ₄the visible a large amount of proliferations of fibrous tissue of group, liver lobule is cut apart and is held into the liver cell group of differing in size by the fibrous tissue of hyperplasia, forms pseudolobuli, pseudolobuli endite central vein lack as, off normal or more than two.

Each group murine liver tissue is carried out pathological grading, by ANOVA one-way analysis of variance method, classification results is carried out to statistical procedures, result shows: 1. significantly (P<0.05) of normal group mouse liver and other 4 experimental mice liver case classification othernesses; 2. KLH-AY25 group, KLH-LS30 group and KLH group, CCl ₄group is compared, and significant difference (P<0.05) 3. KLH-AY25 group is compared with KLH-LS30 group, KLH group and CCl ₄group is compared, and there are no significant for difference (P>0.05).

11, hydroxyproline determination

1) get 40 clean glass test tubees mark.

2) accurately weigh hepatic tissue weight in wet base 60 ㎎ and put into the glass test tube that mark is good, add the hydrolyzed solution of about 1ml, fully mix.

3) test tube is put into 95 DEG C of constant water bath box water-bath 20min, mixed once every 10min, make the more thorough of tissue hydrolysis.

4) after test tube is cooling, in each pipe, add 1 of indicator, fully mix.

5) in each pipe, add and regulate pH value first liquid 1ml, fully mix, now in visible test tube, solution is red.

6) in each test tube, dropwise add the second liquid of adjusting pH value, need fully to mix at once liquid after being often added dropwise to, until liquid becomes yellow-green colour in each test tube, every Guan Zhongyue adds 500 μ l to adjust the second liquid of pH values, now in each pipe the pH value of liquid about 6.0-6.8 left and right.

7) in each pipe, adding distilled water to final volume is 10ml, fully mixes.

8) get 40 5ml centrifuge tubes that mark is good, get the diluent of 3ml and put into respectively centrifuge tube, weigh 25 ㎎ gacs and pour in each centrifuge tube, fully mix, centrifugal, 3500rpm/min, 10min.

9) get supernatant liquor 1ml and do testing sample.

10) according to the form below operation:

Table 3-1 oxyproline operation table

11) by liquid blending in above-mentioned each pipe, water-bath 15min in 60 DEG C of constant water bath box, cooling rear centrifugal, 3500rpm/min, 10min.

12) open spectrophotometer, get in each centrifuge tube supernatant liquor at 550nm place, 1cm optical path, after distilled water zeroing, adds liquid in each pipe in cuvette successively, measures each pipe light absorption value, records each pipe light absorption value.

13) calculate as follows every mouse liver hydroxyproline content:

In each group murine liver tissue hydroxyproline content as shown in Figure 3, KLH group and CCl ₄organize Hyp content apparently higher than normal group, significant difference (P<0.05); But between these two groups, relatively, difference is not remarkable; KLH-AY25 group and KLH-LS30 group hydroxyproline content and KLH group and CCl ₄group is compared, significant difference (P<0.05); Between KLH-AY25 group and KLH-LS30 group, relatively, difference is not remarkable.KLH-AY25 group is with KLH-LS30 group hydroxyproline content compared with normal group, and difference is not remarkable.

12, immunohistochemical staining detects the expression of α-SMA in liver

1) hepatic tissue paraffin section is placed in to 37 DEG C of incubators and toasts 1h.

2) section is put into dimethylbenzene and soak 60min, change dimethylbenzene and soak 10min, again change dimethylbenzene and soak 5min.

3) section is put into dehydrated alcohol and soak 5min, change soaked in absolute ethyl alcohol 5min.

4) section is put into 95% alcohol-pickled 5min.

5) section is put into 75% alcohol and soak 5min.

6) section is put into the about 30min of 3% hydrogen peroxide incubated at room, distilled water flushing.

7) sealing: with 10% bovine serum/PBS sealing 1 hour, discard this confining liquid; Again seal 1 hour with the lowlenthal serum of the sealing in SP detection kit again, discard this serum.

8) add primary antibodie: dilute primary antibodie with 10% bovine serum/PBS, wherein anti-α-SMA Dilution ratio is 1:800, and anti-PCNA Dilution ratio is 1:400, and anti-Desmin Dilution ratio is 1:400, section is put into wet box, room temperature is placed after 1 hour and is spent the night in 4 DEG C.

9) take out section and at room temperature hatch 45min, with PBST flushing, 5min/ time, totally 3 times.

10) adding two resists: in section, drip biotin labeled two anti-working fluids, in 37 DEG C of constant water bath box, hatch 1 hour.

11) rinse section with PBST, each 5min, washes 3 times altogether.

12) in section, drip the chain enzyme avidin working fluid of horseradish peroxidase-labeled, in 37 DEG C of constant water bath box, hatch 1 hour.

13) DAB dyeing: get 1ml deionized water in 1.5ml EP pipe, add successively wherein A liquid, B liquid, each 1 of C liquid (often adding 1 fully mixes afterwards), after mixing for section statining.Dyeing 5-10min, controls dye levels under microscope.

14) tap water fully rinses section.

15) phenodin dye liquor is redyed 2-10min, hydrochloride alcohol differentiation.

16) tap water fully rinses section.

17) section is put into successively to 75% alcohol, 95% alcohol, dehydrated alcohol and dewater, each 5min.

18) taking out section, to put into dimethylbenzene transparent.

19) neutral gum is fixed, conventional mounting.

20) optical microphotograph Microscopic observation section.

Each group murine liver tissue α-SMA coloration result, as shown in Fig. 4-1, has no obvious α-SMA positive cell in normal group mouse liver; KLH group and CCl ₄the group visible portal area of murine liver tissue has obvious brown yellow granule spaced apart along fibrosis around, and painted darker; In KLH-AY25 group and KLH-LS30 group murine liver tissue, visible positive cell distributes around portal area, but painted more shallow, and positive cell number is starkly lower than CCl ₄group.

Each group murine liver tissue α-SMA stained positive region adopts the system statistical analysis of area of computer aided semi-quantitative analysis to the results are shown in Figure 4-2.With KLH group and CCl ₄group is compared, and KLH-AY25 group and KLH-LS30 group murine liver tissue α-SMA stained positive region area are less, and its difference has significance (P<0.001).

Western-blot detects each group of murine liver tissue pSmad2/3 and Smad2/3 expression.

The phosphorylation of Smad2/3 is one of mark of TGF-signal β Signal Transduction Pathways activation, respectively organizes murine liver tissue pSmad2/3 and Smad2/3 expression as shown in Fig. 4-3.Repeat to give CCL ₄abdominal injection can be induced the phosphorylation of Smad2/3, activates TGF-signal β Signal Transduction Pathways, and gives the phosphorylation that KLH-AY25 and KLH-LS30 can suppress Smad2/3 in Fibrotic murine liver tissue.

13, cell in vitro is learned the biologic activity of the specific antibody of experiment detection TGF-β 1 synthetic peptide vaccine inducing mouse generation.

As shown in Fig. 5-1,5-2,5-3,5-4, the HSC-T6 cell Smad2/3 phosphorylation of TGF-β 1 being induced by Western-blot detection specificity antibody and the impact of α-SMA, the impact that HSC-T6 cell COL1A2, PAI-1 TGF-β 1 being induced by real-time fluorescence quantitative PCR method detection specificity antibody and TIMP-1mRNA express.

Result is visible, 6ng/ml TGF-β 1 can significantly induce HSC-T6 cell Smad2/3 phosphorylation, promote the expression of α-SMA, COL1A2, PAI-1 and TIMP-1, give TGF-β 1 synthetic peptide specific antibody (1:200 dilution) and can obviously suppress the Smad2/3 phosphorylation of HSC-T6 cell and the expression of α-SMA, COL1A2, PAI-1 and TIMP-1.

The above results shows, by and TGF-β 1 active and then the activation that suppresses hepatic stellate cell to CCL ₄the hepatic fibrosis of induction has obvious restraining effect.

14, TGF-β 1 synthetic peptide vaccine is to CCL ₄the impact of the hepatic fibrosis mouse liver cell apoptosis of induction.

Each group mouse TUNEL coloration result is as shown in Fig. 6-1 and 6-2.The per-cent that apoptotic index accounts for the total cell number of counting with TUNEL positive cell represents.Repeat to give CCL as seen ₄abdominal injection can obviously increase the hepatocellular quantity of mouse apoptosis, gives TGF-β 1 synthetic peptide vaccine and can significantly reduce CCL ₄the hepatocellular ratio of apoptosis in the mouse liver fibrosis of induction.The above results shows that crossing of TGF-β 1 expressed can increase hepatocellular apoptosis in Fibrotic mouse liver, and in and the activity of TGF-β 1 can suppress its apoptosis.

15, TGF-β 1 synthetic peptide vaccine is to CCL ₄the impact of the hepatic fibrosis mouse liver cell propagation of induction.

PCNA and cell DNA compositive relation are close, in the startup of cell proliferation, play an important role, and are the good index of reflection proliferative activity; Positive painted being mainly distributed in karyon of PCNA, it is painted that endochylema has no, and occurs the positive cell signal of brown with karyon.

Each group mouse PCNA coloration result is shown in Fig. 7-1: the section of normal group murine liver tissue has no obvious PCNA positive cell; Repeat to give CCL ₄abdominal injection can obviously increase PCNA positive cell number, and KLH-AY25 group and KLH-LS30 group are compared with KLH group and CCl ₄group is compared, and the increase of PCNA positive cell number is more remarkable, and prompting is by the propagation giving in TGF-β 1 synthetic peptide vaccine and too much TGF-β 1 can accelerating fibers mouse liver cell.Every PCNA stained random 5 visuals field of solid area and visual field, 5, interstitial district selected under 40 times of object lens, count respectively PCNA positive cell and total cell under each visual field, through one-way analysis of variance, PCNA index and KLH group and the CCl of KLH-AY25 group and KLH-LS30 group mouse liver cell ₄group PCNA index differential has significance (P<0.001), as shown in Fig. 7-2.

16, respectively organize murine liver tissue Desmin dyeing.

Each group murine liver tissue Desmin coloration result is shown in Fig. 8-1.In normal group mouse liver, have no obvious Desmin positive cell; KLH group and CCl ₄organize the interior visible positive cell of murine liver tissue more and painted darker, KLH-AY25 organizes and the interior positive cell of KLH-LS30 group mouse liver is less and dyed particles is painted more shallow.Each group murine liver tissue Desmin stained positive region adopts the system statistical analysis of area of computer aided semi-quantitative analysis to the results are shown in Figure 8-2.With KLH group and CCl ₄group is compared, and KLH-AY25 group and KLH-LS30 group murine liver tissue Desmin stained positive region area are less, and difference has significance (P<0.001).

As can be seen here, in murine liver tissue, hydroxyproline content is consistent with HE dyeing, Masson dyeing and immunohistochemical staining result.Produce on the basis of antibody at AY25 and LS30 epitope peptide vaccine, experimentation on animals confirms can significantly suppress the hepatic fibrosis in mice that CCl4 induces after TGF-β 1 synthetic peptide vaccine immune mouse.

To sum up result shows, screen and in TGF-β 1 molecule, there is antigenic peptide sequence AY25 and LS30, after adopting chemical process synthetic with the coupling of carrier proteins phase after immune mouse, show that by ELISA and Western blot detection method 2 screened 16 peptides all can produce the antibody of high titre by inducing mouse, and the antibody producing shows good reactivity and specificity.Further, after QK16 and VF16 synthetic peptide vaccine immune mouse, can significantly suppress CCl ₄the hepatic fibrosis in mice of induction.

Confirm through the TGF-of abdominal injection AY25 and LS30 β 1 synthetic peptide vaccine, can produce specific antibody by inducing mouse, can significantly suppress CCl ₄the mouse experiment hepatic fibrosis of induction.Show that TGF-β 1 vaccine is expected to become a kind of simple, safe and effective hepatic fibrosis and prevents and treats novel method.

Claims (3)

1. the application of synthetic peptide vaccine in the medicine of preparing anti-hepatic fibrosis, described synthetic peptide vaccine comprises the B cell antigen epi-position of TGF-β 1, and is coupled on carrier proteins;

Described epitope is the AY25 epi-position as shown in SEQ.ID.NO.1, or LS30 epi-position as shown in SEQ.ID.NO.2.

2. application as claimed in claim 1, is characterized in that, described medicine is the medicine that suppresses TGF-β 1 activity.

3. application as claimed in claim 1, is characterized in that, described medicine is the medicine that suppresses hepatocellular apoptosis.