CN105534962A - Model establishment method of effect of curcumin on mouse UC (ulcerative colitis) PPAR-gamma and COX-2 induced by DSS (dextran sulphate sodium) - Google Patents
Model establishment method of effect of curcumin on mouse UC (ulcerative colitis) PPAR-gamma and COX-2 induced by DSS (dextran sulphate sodium) Download PDFInfo
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Abstract
The invention provides a model establishment method of effect of curcumin on mouse UC (ulcerative colitis) PPAR-gamma and COX-2 induced by DSS (dextran sulphate sodium). The method comprises the steps of observing the curative effect of curcumin on mouse UC induced by DSS, observing the weight, fecal characters, occult blood condition, clinical symptoms, colon histology damage, macro-behavior of a mouse; measuring expression condition of colonic mucosa layer PPAR-gamma and COX-2, and STAT3mRNA and phosphorylation STAT3, and discussing the possible mechanism, affecting UC, of the curcumin.
Description
Technical field
The present invention relates to ulcerative colitis intervention model method for building up, particularly curcumin is on the method for establishing model of colitis in mice PPAR-γ and the COX-2 impact that DSS induces.
Background technology
Ulcerative colitis (ulcerativecolitis, UC) be a kind of cause of disease rectum still not fully aware of and colon chronic nonspecific inflammation disease; pathological changes is mainly limited to colorectal mucosa and tela submucosa; pathological changes becomes seriality to fill the air distribution; scope is many from anus end rectum, drives in the wrong direction to nearly section and develops, even involve total colectomy and terminal ileum; belong to one of inflammatory bowel (inflammatoryboweldisease, IBD).Primary disease is chronic process, and most of patient's recurrent exerbation, in recent years because treatment level improves, case fatality rate obviously declines, but chronic sustained sexual activity or recurrent exerbation are frequently, and prognosis is poor, and the course of disease is overflow, and elder's canceration is dangerous to be increased.
Peroxisome proliferation-activated receptors (peroxisomeproliferator-activatedreceptor, PPAR) be one group of nuclear receptor superfamily member with sophisticated functions, there is PPAR α, PPAR β/δ, PPAR γ 3 kinds hypotype, respectively by different gene codes, PPAR is the transcription factor that part regulates, PPAR γ is by ligand activation, main participation inflammation, immunoreactive adjustment, have the inflammatory reaction effect alleviating patients of ulcerative colitis and colitis animal model.
COX-2 (cyclooxygenase-2, COX-2) be that the one that promoter contains NF-κ B binding site induces type enzyme, do not express under normal circumstances, and highly express when there being inflammation, play an important role in the morbidity of UC, COX-2 stimulates reaction early to express as proinflammatory mediators and short cell division, the activity of COX-2 is depended at the Prostaglandin product of chronic colitis increase, COX-2 is that conversion of arachidonic acid generates PGE2 (prostaglandinE2, PGE2) key enzyme, PGE2 has expansion blood vessel, the effect increasing intestinal permeability and stimulate enterocyte to secrete, may suffer from abdominal pain with UC, the generation of diarrhoea is relevant.
STAT is an important intracellular signal transduction molecule, stat protein matter is a protein families containing STAT1/2/3/4/5a/5b/6, when not having specific receptor to stimulate, STAT keeps inactive form in Cytoplasm, relevant to the expression of multiple inflammatory factor, it plays an important role in the pathogenesis of UC.In different stimulations as cytokine, somatomedin, hormones etc., stat protein matter is activated by tyrosine phosphorylation rapidly.In the mechanism of action of UC, the important function of one of family member of STAT STAT3 in UC pathogenesis is confirmed in experimentation and clinical research.
Curcumin is a kind of pigment extracted from zingiberaceous plant Rhizoma Curcumae Longae, for orange-yellow crystalline powder, taste is slightly bitter, acid polyphenols class material, main chain is unsaturated aliphatic and aromatic group, and large quantity research proves that it has antioxidation, antiinflammatory, anticoagulant, blood fat reducing, atherosclerosis, effect such as defying age elimination free radical and Tumor suppression growth etc.
The treatment of ulcerative colitis key agents has aminosalicyclic acid supplement, but this type of adverse effect is many, and clinical efficacy is very not satisfactory, and the untoward reaction of 5-ASA new formulation obviously reduces, shortcoming is expensive, therefore need to research and develop new medicine, this medicine both can better play good efficacy to ulcerative colitis, simultaneously few side effects and economical and practical.Curcumin is to the research of ulcerative colitis progress to some extent in recent years.Ulcerative colitis (UC) mice induced due to dextran sulfate sodium (DSS) and the histology of people UC and clinical manifestation similar, for the animal model of current preferably research people UC, DSS guidance model is why welcome in IBD research is rapid, simple, repeatability, controllability due to it.This problem is by respectively organizing Biology seed coating, pathology, immunohistochemistry, molecular biology research before and after treating to normal mouse, DSS model mice, measure the expression of PPAR γ, COX-2, STAT3, observe curcumin to the intervention effect of ulcerative colitis, inquire into the mechanism of curcumin treatment ulcerative colitis.
Summary of the invention
For solving above-mentioned prior art Problems existing, the object of the present invention is to provide the method for establishing model that curcumin affects colitis in mice PPAR-γ and COX-2 that DSS induces.By observing curcumin to the curative effect of DSS inducing mouse ulcerative colitis; Observe the body weight of mice, feces character and situation of occulting blood, clinical symptoms, colon histology damage, macro manifestations; Measure colonic mucosa layer PPAR γ, COX-2 expression, and STAT3mRNA and pSTAT3, inquire into the mechanism that curcumin affects ulcerative colitis.
For achieving the above object, technical scheme of the present invention is:
The method for establishing model that colitis in mice PPAR-γ and COX-2 that curcumin is induced DSS affects, comprises the steps:
Step one, experiment grouping and model manufacturing;
Select female BAl BIc/c mice tens of, body weight 16-20g, mice adaptability is fed one week, and table of random number is divided into A, B, C, D, E, F six groups, often organizes number identical;
A group: Normal group, every day freely enters drinking water and feedstuff, and every day, lumbar injection 1ml normal saline, was total to 7d;
B group: model group, every day freely drinks 5%DSS solution, substitutes drinking water, ad lib feedstuff, every day lumbar injection 1ml 2.5% alcoholic solution, altogether 7d;
C group: dexamethasone in treatment group, drinks 5%DSS solution every day and substitutes drinking water, ad lib feedstuff, lumbar injection every day 1 dexamethasone injection, and dosage is 1.5mg/kg.d, is dissolved in 1ml normal saline, altogether 7d.
L group: curcumin low dose group, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time, curcumin is dissolved in 1ml2.5% alcoholic solution by the dosage of 25mg/kg.d every day, altogether 7d.
M group: dosage group in curcumin, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time, curcumin is dissolved in 1ml2.5% alcoholic solution by the dosage of 50mg/kg.d every day, altogether 7d.
H group: curcumin high dose group, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time, curcumin is dissolved in 1ml2.5% alcoholic solution by the dosage of 100mg/kg.d every day, altogether 7d.
Step 2, colon's sampling;
8th day, cervical dislocation put to death mice, cuts off abdominal cavity, free colon and distal ileum, took out anus to the whole intestinal segment of cap end, cut open, repeatedly rinse well with ice-cold PBS along the mesentery longitudinal axis, then with clean filter paper suck dry moisture.Macro-graph after marking, clip ulcer the most seriously locates colon 1cm, is placed in 4% formalin and fixes, and routine paraffin wax embeds, and section, gives over to immunohistochemical staining.Ling Quliangduan colon puts into cryogenic refrigerator-80 DEG C and deposits, and is RT-PCR and ELISA.
Step 3, observation index;
Observe the body weight of primary part observation mice every day during modeling, feces character and situation of occulting blood, and carry out DAI integration, feces/occult blood symptom scores; Macro-graph is marked; Score of tissue damage;
Step 4, Biochemical Indexes;
According to above-mentioned anomalous integral scoring, qualitative analysis is carried out to testing result, meanwhile,
Further, in described step one, selected mice is 60, often organizes 10.
Relative to prior art, beneficial effect of the present invention is:
The invention provides the method for establishing model that curcumin affects colitis in mice PPAR-γ and COX-2 that DSS induces.By observing curcumin to the curative effect of DSS inducing mouse ulcerative colitis; Observe the body weight of mice, feces character and situation of occulting blood, clinical symptoms, colon histology damage, macro manifestations; Measure colonic mucosa layer PPAR γ, COX-2 expression, and STAT3mRNA and pSTAT3, inquire into the mechanism that curcumin affects ulcerative colitis.
Accompanying drawing explanation
Fig. 1 is the expression schematic diagram of the colon p-STAT3 albumen that the embodiment of the present invention provides;
Fig. 2 is the schematic diagram of the colon STAT3 expression (RT-PCR) that the embodiment of the present invention provides;
Fig. 3 is the schematic diagram of colon's light Microscopic observation (HE × 200) that the embodiment of the present invention provides;
Fig. 4 is the schematic diagram of the expression (SP × 400) of the colon PPAR γ that the embodiment of the present invention provides;
Fig. 5 is the schematic diagram of the expression (SP × 400) of the colon STAT3 that the embodiment of the present invention provides.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical solution of the present invention is described in further detail:
Test example:
One, experiment material
1. laboratory animal: female BAl BIc/c mice 60, cleaning grade, Mus 6-7wk in age, body weight 16-20g.
2. major experimental reagent
Dextran sulfate sodium, curcumin, dexamethasone, PPAR γ rabbit against murine polyclonal antibody, STAT3 rabbit against murine polyclonal antibody, COX-2ELISA detection kit, SP immunohistochemical staining test kit, DAB colour reagent box, 4% paraformaldehyde fixative, Trizol test kit, cDNA synthetic agent box, STAT3 upstream and downstream gene primer, Traizol total RNA extraction reagent, Reverse Transcription box, pSTAT3 polyclonal antibody.
Two. experimental technique
1 experiment grouping and model manufacturing
Select female BAl BIc/c mice 60, body weight 16-20g.Mice adaptability is fed one week, and table of random number is divided into A, B, C, D, E, F six groups, often organizes 10.
A group: Normal group, every day freely enters drinking water and feedstuff, and every day, lumbar injection 1ml normal saline, was total to 7d;
B group: model group, every day freely drinks 5%DSS solution, substitutes drinking water, ad lib feedstuff, every day lumbar injection 1ml 2.5% alcoholic solution, altogether 7d;
C group: dexamethasone in treatment group, drinks 5%DSS solution every day and substitutes drinking water, ad lib feedstuff, lumbar injection every day 1 dexamethasone injection (dosage is 1.5mg/kg.d, is dissolved in 1ml normal saline), altogether 7d.
L group: curcumin low dose group, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time (curcumin is dissolved in 1ml2.5% alcoholic solution [2] by the dosage of 25mg/kg.d every day, altogether 7d.
M group: dosage group in curcumin, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, lumbar injection curcumin suspension 1 time every day (curcumin is dissolved in 1ml2.5% alcoholic solution [2,3] by the dosage of 50mg/kg.d every day), altogether 7d.
H group: curcumin high dose group, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, lumbar injection curcumin suspension 1 time every day (curcumin is dissolved in 1ml2.5% alcoholic solution [2] by the dosage of 100mg/kg.d every day), altogether 7d.
2. colon's sampling
8th day, cervical dislocation put to death mice, cuts off abdominal cavity, free colon and distal ileum, took out anus to the whole intestinal segment of cap end, cut open, repeatedly rinse well with ice-cold PBS along the mesentery longitudinal axis, then with clean filter paper suck dry moisture.Macro-graph after marking, clip ulcer the most seriously locates colon 1cm, is placed in 4% formalin and fixes, and routine paraffin wax embeds, and section, gives over to immunohistochemical staining.Ling Quliangduan colon puts into cryogenic refrigerator (one 80 DEG C) and deposits, and is RT-PCR and ELISA.
3. observation index
3.1 is accurate with reference to the mark such as OsmanN [3], during modeling every day primary part observation mice body weight, fecal
Shape and situation of occulting blood, and carry out DAI integration, standards of grading are shown in (table 1)
Table 1DAI integration
※ normally defecates: be shaped stool; Loose stool: do not attach to the pasty state of anus, half form stool; Loose stool: the stool that can attach to anus
3.2 symptom scores (feces/occult blood) (see table 2)
Table 2 symptom scores
3.3 macro-graph standards of grading (see table 3)
Table 3 macro-graph standards of grading
Also need the colon lengths measuring animal simultaneously.
3.4 histological examination standards of grading [4] (see table 4) HE carries out histological score after dyeing
Table 4 score of tissue damage standard
4, Biochemical Indexes
The HE dyeing of 4.1 colon specimen
1) Paraffin tissue block 4 μm of serial section, tissue slice is put baking box 60 DEG C of 30min and is put into dimethylbenzene dewaxing twice, each 10min immediately.
2) dehydrated alcohol (I) 10 minutes, dehydrated alcohol (II) 10min, each 1min of 95%, 85%, 75% graded ethanol, washes 2-3 time successively.
3) haematoxylin dyeing 3-5min, tap water 2-3 time.(blotting water)
4) 0.25% ammonia returns blue 1s, washes 3 times.
5) 1% eosin stains 2min, washes 3 times.
6) conventional dehydration, transparent, mounting: 75%, 80%, 85%, 95% ethanol, dehydrated alcohol II, dehydrated alcohol I dewater, 2-3min at different levels, dimethylbenzene transparent twice, each 10min, neutral gum mounting, microscopy.
4.2ELISA method measures mucous membrane of colon COX-2 (COX-2) content
4.2.1 the process of sample
Get 1cm colon, clean and take weight, add a certain amount of PBS (10%, 100mg adds 1ml), in buffer, add 1 μ g/L protease inhibitor.Under condition of ice bath, by homogenizer, specimen homogenate is abundant, centrifugal about 20 minutes (2000-3000 rev/min), carefully collects supernatant
4.2.2 concrete steps are as follows:
1) take out test kit from refrigerator, room temperature rewarming balances 30 minutes.
2) dosing: the cleaning mixture with distilled water, 20 times of concentrated cleaning solutions being diluted to former times.
3) add standard substance and testing sample: the enzyme mark bag getting sufficient amount, by plate, is fixed on framework, establishes gauge orifice, sample to be tested hole and blank control wells respectively, records each hole site, in standard sample wells, add standard substance 50 μ L; First add sample to be tested 10 μ L in sample to be tested hole, add Sample dilution 40 μ L (i.e. Sample Dilution 5 times); Blank control wells does not add.
4) incubation: 37 DEG C of calorstat incubation 30min.
5) wash plate: discard liquid, absorbent paper pats dry, cleaning mixture is filled it up with in every hole, leaves standstill 1min, gets rid of cleaning mixture, absorbent paper pats dry, so repeat to wash plate 4 times.
6) enzyme-added mark working solution: every hole adds enzyme mark working solution 50 μ L, and blank control wells does not add.
7), incubation: 37 DEG C of calorstat incubation 30min.
8), wash plate: discard liquid, absorbent paper pats dry, cleaning mixture is filled it up with in every hole, leaves standstill 1min, gets rid of cleaning mixture, absorbent paper pats dry, so repeat to wash plate 4 times.
9) develop the color: every hole first adds developer A liquid 50 μ L, then adds developer B liquid 50 μ L, gently shake mixing 30s with have gentle hands, 37 DEG C of lucifuge colour developing 15min.
10) stop: take out ELISA Plate, every hole adds stop buffer 50 μ L, cessation reaction.
11) measure: with blank well zeroing, after termination in 15min, with the light absorption value (OD value) in each hole of 450nm wavelength measurement.
12) calculate: according to the concentration of standard substance and the OD value of correspondence, calculate the linear regression equation of standard curve, then according to the OD value of sample, regression equation calculates the COX-2 sample concentration of correspondence.Ultimate density is that practical measurement concentration is multiplied by extension rate
4.3 immunohistochemical stainings observe the expression of PPAR γ, STAT3 in colon.
4.3.1 carry out Immunohistochemical detection according to SP immunohistochemical staining test kit and DAB colour reagent box description, step is as follows:
1), before dewaxing and aquation dewax, organization chip should be toasted 20 minutes in 60 DEG C of calorstats.Organization chip is placed in dimethylbenzene and soaks 10 minutes, soaks 10 minutes again after changing dimethylbenzene; 5 minutes are once soaked in dehydrated alcohol, 95% ethanol, 70% ethanol.
2) PBS liquid rinse 3 times each 5 minutes.
3) drip 3%H2O2 incubated at room 5-10min, eliminate endogenous peroxidase activity.
4) PBS rinses 2-3 each 5min.
5) antigen retrieval: add 0.01M citrate buffer (pH6.0) in boiling water.Cover the lid of rustless steel pressure cooker, do not lock, be placed in by slide on metallochromy frame, slowly pressurize, make slide soak 5 minutes in buffer, then by cover lock, paddle can rise.After 10 minutes, remove thermal source, insert in cold water, uncap after valve sinks.
6) PBS liquid rinse 3 times each 5 minutes.
7) Normal Goat Serum at room temperature closes 15min, removes unnecessary serum;
8) drip sheep anti mouse PPAR γ/STAT3 monoclonal antibody (primary antibodie), put 4OC refrigerator overnight.
9) will cut into slices 37OC refrigerator rewarming 20min, PBS liquid rinse 3 times each 5 minutes.
10) drip the anti-goat-anti body of biotin labeled rabbit (two resist), incubated at room 20min.PBS liquid rinses 3 times 5 minutes
11) drip the streptavidin working solution of horseradish peroxidase-labeled in section, 37OC hatches 20 minutes; Section is washed 5 minutes, 3 times with PBS buffer;
12) DAB developer color development at room temperature, about 5 minutes time; Clean with tap water after completion of the reaction;
13) section haematoxylin dyeing 2 minutes, tap water 10 minutes, 1% acidic alcohol breaks up tens of second, then washes from the beginning;
14) give successively rise alcohol concentration dehydration: 75% ethanol 2min, 80% ethanol 2min, 85% ethanol 2min, 95% ethanol 2min, dehydrated alcohol (II) 2min, dehydrated alcohol (I) 2min; Soak 5 minutes in dimethylbenzene (I) again, soak in dimethylbenzene (II) 5 minutes transparent;
15) rear neutral gum mounting is dried, then Microscopic observation.
4.3.2 evaluation of result: often open section random selecting 5 high power light microscopic downward views, final coloration result positive expression location: occur that cell that sepia dyes is as positive cell at cell nuclear administration l:l/ or cell cytosol, score by staining power and positive cell range percentage, specifically in table 5, final result is the product of said two devices score.The average that the appraisal result of getting 5 visuals field at random is finally marked in each section represents.And judge: the product >3 of staining power and positive cell percentage is divided into the positive.
Table 5 SABC standards of grading
| Staining power | Score | Positive cell percentage (%) |
| Colourless | 0 | 0 |
| Faint yellow | 1 | ≤10 |
| Brown color | 2 | 11-50 |
| Sepia | 3 | 51-75 |
| ------- | 4 | 76-100 |
The expression of STAT3mRNA in 4.4RT-PCR technology for detection colon:
4.4.1 extract colon's total serum IgE
Total serum IgE in mouse Colon tissue samples is extracted with RNAsimpleTotalRNAKit total RNA extraction reagent box.Experimental implementation is undertaken by product description, and step is as follows:
1. sample treatment
Colon: will be organized in liquid nitrogen and grind.Every 50 – 100mg tissues add 1ml lysate RZ, carry out homogenized with Syrup-homogenizing instrument.Sample volume should not exceed 1/10th of lysate RZ volume.
2. homogenised sample is placed 5 minutes 15 – 30 DEG C, nucleic acid-protein complex is separated completely.
3.4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 5 minutes, get supernatant, proceed to one new in the centrifuge tube of RNase.
4. add 200 μ l chloroforms, build pipe lid, thermal agitation 15 seconds, room temperature places 3 minutes.
5.4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 10 minutes, sample can be divided into three layers: yellow organic facies, intermediate layer and colourless aqueous phase, RNA is mainly in aqueous phase, and the volume of aqueous phase is about 50% of lysate RZ reagent used.Aqueous phase is transferred in new pipe, carries out next step operation.
6. slowly add 0.5 times of volume dehydrated alcohol, mixing (now may occur precipitation).To obtain solution proceeds in adsorption column CR3 together with precipitation, 4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 30 seconds.
8. in adsorption column CR3, add 700 μ l rinsing liquid RW, room temperature leaves standstill 2 minutes, and 4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 30 seconds, abandon waste liquid.
9. in adsorption column CR3, add 500 μ l rinsing liquid RW, room temperature leaves standstill 2 minutes, 4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 30 seconds, removes residual liquid.
10. adsorption column is put into 2ml collecting pipe, 4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 2 minutes, remove residual liquid.
Adsorption column CR3 to proceed in a new centrifuge tube by 11., and add 30 – 100 μ lRNase-freeddH2O, room temperature places 2 minutes, 4 DEG C 12,000rpm (~ 13,400 × g) centrifugal 2 minutes.Elution buffer volume should not be less than 30 μ l, and volume is too small affects organic efficiency.And RNA should be kept at-70 DEG C, in case degraded.
4.4.2. total RNA from animal tissues is identified:
5 μ lRNA+1 μ l sample-loading buffers, 1% agargel electrophoresis 70v10min identifies RNA.OD260/OD28O, calculates RNA concentration, and has judged whether DNA, protein contamination; Be qualified between 1.8 1 2.0, each experimental group of labelling also records concentration
4.4.3.cDNA synthesis and the PCR stage:
MusStat3F：AACCTCCAGGACGACTTTG
MusStat3R:CGCCCTTGTAGGACACTTT
A. reverse transcription reaction
1. the primer synthesizing cDNA can be chosen any one kind of them from OligodT-AdaptorPrimer, Random9mers or specific Down Stream primer in conjunction with practical situation.
By following composition preparation inverse transcription reaction liquid.
2. reverse transcription reaction is carried out by following condition.
B.PCR reacts
1. by following composition preparation PCR reactant liquor.
2. B-reactant liquor 1. join A-reverse transcription reaction 2. terminate after PCR reaction tube in, mix gently.
3. PCR reaction is carried out by following condition
4. after reaction terminates, get PCR reactant liquor (5 ~ 10l) and carry out agarose gel electrophoresis, confirm PCR product.If this PCR
Product is tested after need being used for, must by PCR primer freezen protective.
4.5Westernplot surveys the STAT3 of phosphorylation
4.5.1 protein example obtains: after induced expression in bacterium, by electrophoresis sample-loading buffer By Direct Pyrolysis cell, eukaryotic cell adds homogenate buffer, machinery or ultrasound wave room temperature homogenate 0.5-1min.Then 4 DEG C, the centrifugal 15min of 13,000g.Get supernatant as sample.
4.5.2 electrophoresis: PAGE production gel, carries out SDS-PAGE.
4.5.3 shift: (half dry type transfer)
1), adhesive tape cuts to suitable size after terminating by electrophoresis, by transferring film buffer balance, and 5min × 3 time.
2), film process: cut out the filter paper onesize with adhesive tape and NC film in advance, immerse 10min in transferring film buffer.
3), transferring film: membrane-transferring device is put well by the order of carbon anode plate, 24 metafiltration paper, NC film, gel, 24 metafiltration paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, NC film Accurate align, each step removes bubble, and upper pressure 500g weight, blots liquid unnecessary on carbon plate.Switch on power, constant current 1mA/cm2, transfer 1.5hr.After transfer terminates, film takes out by deenergization, extracts film bar to be measured and does immunoblotting.By there being the band of protein standard to dye, after putting into film dyeing liquor 50s, repeatedly decolour in 50% methanol, to clear background, then wash with distilled water, air-dry being sandwiched in two layers of filter paper is preserved, and stays and compares with the result that develops the color.
4.5.4 immunoreation:
1), with 0.01MPBS film is washed, 5min × 3 time.
2), add coating buffer, steadily shake, room temperature 2h.
3, abandon coating buffer, wash film with 0.01MPBS, 5min × 3 time.
4, add primary antibodie (dilute in appropriate dilutions ratio 0.01MPBS, liquid must coverlay whole), place more than 12hr for 4 DEG C.Negative control, replace primary antibodie with 1%BSA, all the other steps are identical with experimental group.
5, abandon primary antibodie and 1%BSA, wash film respectively with 0.01MPBS, 5min × 4 time.
6, add two anti-(diluting in appropriate dilutions ratio 0.01MPBS) of horseradish peroxidase, steadily shake, room temperature 2hr.
7, abandon two to resist, wash film with 0.01MPBS, 5min × 4 time.
8, add nitrite ion, lucifuge colour developing puts into distilled water cessation reaction to when there is band.Acquired results is as shown in the table:
Table1-1TheDAIscores
Note:
*p<0.01VSA group;
#p<0.01VSB group;
△p<0.05VSC group;
◇p<0.05VSL group.
Table 1-2 respectively organizes mouse tissue Injury score
Note:
※p<0.01VSA group;
△p<0.05VSB group;
zerop<0.01VSC group;
*p<0.05VSL group.
Table 1-3 respectively organizes the PPAR-γ expression contents of colon
Note:
※p<0.01VSA group;
△p>0.05VSA group;
□p<0.01VSB group;
#p>0.05VSC group;
﹡p>0.05VSL group.
The STAT3 that table 1-4 respectively organizes colon expresses
Note:
△p<0.01VSB group;
※p>0.05VSC group;
◇p<0.01VSL group.
Table 1-5 respectively organizes COX-2 content
Note:
※p<0.01VSA group;
△p<0.01VSB group;
﹟p>0.05VSC group.
The expression of accompanying drawing 1 colon p-STAT3 albumen
Figure1Theexpressionofp-STAT3proteinincolonictissues(SP×400)
(Westernblotting)
RT-PCR method detects the expression of colon STAT3mRNA
Accompanying drawing 2 colon STAT3 expression (RT-PCR)
Figure2TheexpressionofSTAT3mRNAincolonictissues
Accompanying drawing 3 colon light Microscopic observation (HE × 200)
Figure3Theobservationofcolonictissuesundermicroscope(HE×200)
The expression (SP × 400) of accompanying drawing 4 colon PPAR γ
Figure4TheexpressionofPPARgammaincolonictissues(SP×400)
The expression (SP × 400) of accompanying drawing 5 colon STAT3
Figure5TheexpressionofSTAT3incolonictissues(SP×400)
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any change of expecting without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (3)
1. curcumin is on the method for establishing model of colitis in mice PPAR-γ and the COX-2 impact that DSS induces, and comprises the steps:
Step one, experiment grouping and model manufacturing;
Select female BAl BIc/c mice 60, body weight 16-20g, mice adaptability is fed one week, and table of random number is divided into A, B, C, D, E, F six groups, often organizes 10;
A group: Normal group, every day freely enters drinking water and feedstuff, and every day, lumbar injection 1ml normal saline, was total to 7d;
B group: model group, every day freely drinks 5%DSS solution, substitutes drinking water, ad lib feedstuff, every day lumbar injection 1ml 2.5% alcoholic solution, altogether 7d;
C group: dexamethasone in treatment group, drinks 5%DSS solution every day and substitutes drinking water, ad lib feedstuff, lumbar injection every day 1 dexamethasone injection, and dosage is 1.5mg/kg.d, is dissolved in 1ml normal saline, altogether 7d;
L group: curcumin low dose group, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time, curcumin is dissolved in 1ml2.5% alcoholic solution by the dosage of 25mg/kg.d every day, altogether 7d;
M group: dosage group in curcumin, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time, curcumin is dissolved in 1ml2.5% alcoholic solution by the dosage of 50mg/kg.d every day, altogether 7d;
H group: curcumin high dose group, drinks 5%DSS solution and substitutes drinking water, ad lib feedstuff, every day lumbar injection curcumin suspension 1 time, curcumin is dissolved in 1ml2.5% alcoholic solution by the dosage of 100mg/kg.d every day, altogether 7d;
Step 2, colon's sampling;
8th day, cervical dislocation put to death mice, cuts off abdominal cavity, free colon and distal ileum, took out anus to the whole intestinal segment of cap end, cut open, repeatedly rinse well with ice-cold PBS along the mesentery longitudinal axis, then with clean filter paper suck dry moisture; Macro-graph after marking, clip ulcer the most seriously locates colon 1cm, is placed in 4% formalin and fixes, and routine paraffin wax embeds, and section, gives over to immunohistochemical staining; Ling Quliangduan colon puts into cryogenic refrigerator-80 DEG C and deposits, and is RT-PCR and ELISA;
Step 3, observation index;
Observe the body weight of primary part observation mice every day during modeling, feces character and situation of occulting blood, and carry out DAI integration, feces/occult blood symptom scores; Macro-graph is marked; Score of tissue damage;
Step 4, Biochemical Indexes;
According to above-mentioned anomalous integral scoring, qualitative analysis is carried out to testing result.
2. method according to claim 1, is characterized in that, in described step one, selected mice is 60, often organizes 10.
3. method according to claim 1, is characterized in that, in described step 4, concrete biochemical indicator and assay method are:
ELISA method measures mucous membrane of colon COX-2 (COX-2) content;
Immunohistochemical staining observes the expression of PPAR γ, STAT3 in colon;
RT-PCR technology detects the expression of STAT3mRNA in colon.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107367407A (en) * | 2017-05-25 | 2017-11-21 | 长沙金域医学检验所有限公司 | A kind of Pathologic specimen is fixed and dehydration treatment method |
| CN109432448A (en) * | 2018-11-29 | 2019-03-08 | 天津凯茵科技有限公司 | A kind of gastrointestinal contrast agent for X-ray examination |
| CN113416752A (en) * | 2021-06-23 | 2021-09-21 | 周娟 | Mog1 gene knockout zebra fish model and application |
-
2015
- 2015-12-24 CN CN201510990250.9A patent/CN105534962A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 何双艳等: "何双艳等", 《实用医学杂志》 * |
| 郭琳 等: "姜黄素对溃疡性结肠炎作用机制的研究进展", 《世界华人消化杂志》 * |
| 陈欧 等: "姜黄素对DSS诱导的小鼠溃疡性结肠炎的疗效", 《世界华人消化杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107367407A (en) * | 2017-05-25 | 2017-11-21 | 长沙金域医学检验所有限公司 | A kind of Pathologic specimen is fixed and dehydration treatment method |
| CN107367407B (en) * | 2017-05-25 | 2020-04-24 | 长沙金域医学检验所有限公司 | Pathological specimen fixing and dehydrating treatment method |
| CN109432448A (en) * | 2018-11-29 | 2019-03-08 | 天津凯茵科技有限公司 | A kind of gastrointestinal contrast agent for X-ray examination |
| CN113416752A (en) * | 2021-06-23 | 2021-09-21 | 周娟 | Mog1 gene knockout zebra fish model and application |
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