CN104195106A - Comprehensive model for protecting mice and curing mice macrophage system UC (Ulcerative Colitis) by C-BF (Cathelicidin-BF) - Google Patents

Abstract

The invention discloses a comprehensive model for protecting mice and curing mice macrophage system UC (Ulcerative Colitis) by C-BF (Cathelicidin-BF). C-BF has broad-spectrum antibacterial activity, especially for gram-negative bacteria. IBDS (Inflammatory Bowel Diseases) include UC and CD (Crohn's Disease), and the pathological mechanisms of IBDS are not clear. The comprehensive model provided by the invention combines in-vivo mice experiments and in-vitro cell experiments to evaluate the effects of C-BF on relieving inflammations, reducing the cell apoptosis level and improving the intestinal barrier function.

Description

Antibacterial peptide protection mouse and mouse macrophage ulcerative colitis generalized model

Technical field

The invention belongs to biomedicine field, be specifically related to the provide protection of C-BF antibacterial peptide to UC mouse.

Background technology

IBD is the abbreviation of inflammatory bowel disease, comprises ulcerative colitis and segmental colitis, and its pathomechanism it be unclear that.IBD is defined as chronic recurrent gastrointestinal tract inflammation.There are some researches show recently, various immunity, h and E factor can affect germinating, the progress of colitis.Sickness rate at western countries IBD is higher, but other regional sickness rate are also more and more higher in the world due to the IBD that affects of Western lifestyle.Ulcerative colitis, is the relevant chronic gastrointestinal diseases of a kind of and mucous membrane of colon surface inflammation, and people infects ulcerative colitis and may be transformed into the mankind's the third-largest malignant tumour---large bowel cancer.

Antibacterial peptide has two extended familys: Cathelicidin and beta-alexin, and the former has possessed innate immune response and prevent the antibacterial of host infection microorganism.The antibacterial peptide that originates from Cathelicidin has antibacterial, antiviral and antifungic action.In Cathelicidin, the antibacterial peptide LL-37 of wide coverage derives from people, and mCRAMP is mouse derived antimicrobial peptide, represents mouse kind.Cathlicidin-BF, derives from Gold-banded Krait, is a kind of new antibacterial peptide, it is reported that C-BF has broad spectrum antibiotic activity, particularly to gram-negative bacteria.Cathlicidin-BF can become effective medicine (plos one) of acne vulgaris.Up to now, other potential source biomolecule medical science functional studies that the non-anti-microbial activity mechanism of C-BF causes are still in the blank stage.

Summary of the invention

In order to overcome the deficiencies in the prior art, the object of the invention is by the effect of the colitis of intravital mouse model and external Caco-2 cell and mouse macrophage model detection Cathelicidin-BF adjusting DSS induction.Result of study shows that colon administration exogenous Cathelicidin-BF significantly reduces inflammatory reaction that DSS causes and the infringement of intestinal barrier function.In body, with in vitro results combination, we also find that Cathelicidin-BF can reduce the inflammation that DSS cause by the activation that suppresses NF-κ B path.At present, the treatment of IBD is mainly to only limit to temporary amelioration of inflammation reaction, and, colonic inflammation finally very likely causes colorectal carcinoma, therefore, the medicine of we safer and more effective treatment and prevention IBD in the urgent need to one, antibacterial peptide may be desirable selection as a potential Substitutes For Antibiotic.

A kind of antibacterial peptide C-BF protection mouse and mouse macrophage ulcerative colitis generalized model,

1), for mouse, set up 3% dextran sulfate sodium (DSS) inducing mouse Ulcerative Colitis Model; The PBS solution of mouse rectal injection C-BF, once a day, the dosage of C-BF is 5mg/kg, and injection continues 1 week, and experiment periods is weighed and record weight data every day, and observes mouse proctorrhagia, ight soil moulding situation;

2) for mouse macrophage, in substratum, the concentration of antibacterial peptide C-BF is 25ug/ml, after antibacterial peptide processing RAW264.7 cell 4h, processes cell 3h with the LPS of 1 μ g/ml concentration.

Further, 3) for mouse, fluorescent mark C-BF, concentration is 2mg/ml, rectal injection, by living imaging, HE dyeing, immunofluorescence, immunohistochemistry, flow cytometer, Tunnel, ELISA, utilize cell resistance instrument to transepithelial electrical resistance value measure, western-blot, Real-Time Fluorescent Quantitative PCR Technique method;

4) for RAW264.7 cell, utilize ELISA test kit to PGE2 in culture supernatant, the concentration of NO is measured, and extracts total RNA simultaneously the gene expression dose of TNF-α is measured, and western-blot measures NF-kB signal path;

5) mouse experiment and In vitro cell experiment in synthesis, evaluate C-BF at amelioration of inflammation, reduce Level of Apoptosis and improve the effect of gut barrier function.

Beneficial effect of the present invention is:

After 3%DSS administration 7 days, compared with other groups, DSS model group colitis is more serious, and the mCRAMP in colonic mucosa expresses and raises simultaneously.Compared with DSS group, C-BF+DSS group colonic epithelium form is eased to a great extent, in blood, the ratio of B, T, scavenger cell returns to normal level, (the TNF-α of pro-inflammatory cytokine in serum, IL-6, IL-8) and the protein level of anti-inflammatory factors IL-10 and the mRNA level of these cytokines significantly reduce.By immunohistochemical methods, we find, compared with DSS group, C-BF+DSS group apoptosis index and inflammatory cell obviously weaken at colon epithelial cell as scavenger cell and neutrophil infiltration index, and the COX-2 that inflammation is relevant and the enzymic activity of INOS obviously decline.Simultaneously, we test the classical downstream signal factor (c-jun and NF-kB) phosphorylation level, find that DSS can activate the phosphorylation level of these two typical transcription factors and C-BF can effectively suppress the phosphorylation (P65) of NF-κ B but can not suppress the phosphorylation of c-jun.Compared with DSS group, Ig A in the secretory volume of ileum stage casing sIgA and serum, G, the secretion level of M obviously raises.On the other hand, we have analyzed the impact of C-BF on gut barrier function, the detection of the dynamic change trend by the fluorescence intensity of the dextran to marked by fluorescein isothiocyanate in serum and the cross-film epithelium resistance value of colonic epithelium, we find that C-BF can effectively reduce the barrier function related gene expression level that intestinal permeability increases and significantly rise is caused by DSS, in vivo test result is further verified in the foundation of RAW264.7 cell model, comprehensive evaluation, C-BF can pass through regulating intestinal canal immunologic function, alleviate intestinal epithelial cells apoptosis, strengthen the ulcerative colitis that gut barrier function alleviates DSS induction.

Brief description of the drawings

Fig. 1 is for utilizing living imaging technology to confirm that antibacterial peptide C-BF can be by peritonaeum absorbing state;

Fig. 2 is for setting up 3%DSS inducing mouse Ulcerative Colitis Model, divide two kinds of processing, one is freely to drink sterilized water (control group, C-BF group) or freely drink water (the DSS group that is dissolved with DSS, C-BF+DSS group) 1 week, another kind of processing is that C-BF group and C-BF+DSS group rectal injection C-BF(are dissolved in PBS) or control group and DSS group inject separately PBS.

Fig. 3-1 is the hemorrhage situation of the large intestinal segment of mouse; Fig. 3-2 are day weight gain; Fig. 3-3 are colon length; Fig. 3-4 are colon weight; Fig. 3-5 are colon length/weight; Fig. 3-6 are disease activity index;

Fig. 4-1 is the HE of colon dyeing (100 ×); Fig. 4-2 are the HE of colon dyeing (400 ×);

For ileum is organized, HE dyes (200 ×) in Fig. 5-1; For ileum is organized, HE dyes (40 ×) in Fig. 5-2; For ileum is organized, HE dyes (200 ×) in Fig. 5-3;

Fig. 6-1 is the ratio of scavenger cell; Fig. 6-2 are the lymphocytic ratio of T; Fig. 6-3 are the ratio of bone-marrow-derived lymphocyte; Fig. 6-4 are the ratio of NK cell;

Fig. 7-1 is the immunofluorescence dyeing (200 ×) of colon; Fig. 7-2 are the expression amount of Western-blot analysis mCRAMP, and swimming lane 1 is DSS group, and swimming lane 2 is control group; The immunofluorescence dyeing (200 ×) that Fig. 7-3 are ileum tissue;

Fig. 8-1 is the expression amount of TNF-α in mice serum; Fig. 8-2 are the expression amount of IL-6 in mice serum; Fig. 8-3 are the expression amount of IL-8 in mice serum; Fig. 8-4 are the expression amount of IL-10 in mice serum;

Fig. 8-5 are the relative expression quantity of TNF-α/GAPDH; Fig. 8-6 are the relative expression quantity of IL-6/GAPDH; Fig. 8-7 are the relative expression quantity of IL-8/GAPDH; Fig. 8-8 are the relative expression quantity of IL-10/GAPDH;

TUNNEL immunohistochemical staining (400 ×) is carried out for mouse colon in Fig. 9-1; Fig. 9-2 are mouse cell apoptotic index;

Figure 10-1 is the concentration of INOS in mouse colon; Figure 10-2 are the concentration of COX-2 in mouse colon; Figure 10-3 are the expression amount of Western-blot analysis genes involved;

Figure 11-1 is the immunofluorescence dyeing (CD177 400 ×) of colon; Figure 11-2 are neutrophil leucocyte invasion index; Figure 11-3 are the immunofluorescence dyeing (F4/80 400 ×) of colon; Figure 11-4 are scavenger cell invasion index; Figure 11-5 are the mensuration of MPO enzymic activity; Figure 11-6 are the mensuration of NAG enzymic activity; Figure 11-7 are the mensuration of EPO enzymic activity;

Figure 12-1 is the concentration of sIgA in serum; Figure 12-2 are the concentration of IgA in serum; Figure 12-3 are the dense of IgG in serum; Figure 12-4 are the concentration of IgM in serum;

Figure 13-1 is the concentration of FITC-dextran in serum; Figure 13-2 are TEER; Figure 13-3 are the immunofluorescence dyeing (ZO1 200 ×) of colon; Figure 13-4 are the relative expression quantity of ZO1/GAPDH in mice serum; Figure 13-5 are the relative expression quantity of ZO2/GAPDH in mice serum; Figure 13-6 are the relative expression quantity of Claudin-1/GAPDH in mice serum; Figure 13-7 are the relative expression quantity of Mucin1/GAPDH in mice serum; Figure 13-8 are the relative expression quantity of Mucin2/GAPDH in mice serum;

Figure 14-1 is the concentration of PGE2; Figure 14-2 are the concentration of NO; Figure 14-3 are the concentration of PGE2; Figure 14-3 are the relative expression quantity of TNF-α/GAPDH in mice serum; Figure 14-4 are the expression amount of Western-blot analysis genes involved;

Embodiment

Below in conjunction with accompanying drawing, the present invention is described in detail, embodiment describes and non-limiting the present invention.

1, first we utilize living imaging technology to confirm that antibacterial peptide C-BF can be absorbed by peritonaeum, but after retaining in vivo 24h, metabolism is complete.Fig. 1 is for utilizing living imaging technology to confirm that antibacterial peptide C-BF is by enteric cavity absorbing state; From Fig. 1, we can confirm that C-BF spreads all over whole body by enteric cavity in about 10 points, but in the interior C-BF metabolism of 24h or discharge body, therefore per injection C-BF should not exceed 24 hours;

2, we set up 3%DSS inducing mouse Ulcerative Colitis Model on this basis, and experiment is divided into groups and designed as Fig. 2:

C-BF dosage: 5mg/kg Mouse Weight, C-BF final concentration is 200 μ g/ml, DSS dosage: 3% (m/v)

Ulcerative Colitis Model is set up as mentioned above, four groups are respectively control group, DSS group, C-BF group and C-BF+DSS group, point two kinds of processing, and a kind of is the water of freely drinking distilled water or being dissolved with DSS, another kind of process be rectal injection C-BF or PBS in contrast, mouse is put to death on the 8th day.

3, experiment periods is weighed and record weight data every day, and observes mouse proctorrhagia, ight soil moulding situation, the mental status.

The respectively hemorrhage situation of intestinal segment (Fig. 3-1) greatly:

In experimentation, the 6th day control group, the DSS group verification model of taking pictures is successfully established, on photo anus place we can find that DSS group has obvious bloodstain, and control group is normal.Subsequently, put to death this two groups of mouse, find large intestine (caecum, colon, rectum) severe haemorrhage, and successful from the data validation modeling of day weight gain and colon length/weight ratio.

From Fig. 3-2, Fig. 3-3, Fig. 3-4, Fig. 3-5, Fig. 3-6, we find that DSS group colon length/weight ratio significantly reduces, and C-BF+DSS group symptom is improved, almost consistent with control group level, DSS group disease activity index (DAI, ight soil moulding situation, the comprehensive grading of colonorrhagia, body weight) worsen day by day, and the decay to a certain extent of C-BF+DSS group.

4, on this basis, first we carry out HE dyeing observation after taking colon stage casing paraformaldehyde fixing:

Find that by (Fig. 4-1) observation colonic epithelium structural form under 100 times of light microscopics DSS processes postcolon mucous layer enteric epithelium morphological structure and destroys serious, and there is obvious oedema phenomenon in submucosa, and its enteric epithelium form obtains recovery to a certain extent after adding antibacterial peptide C-BF, and submucosa showed edema situation is obviously alleviated.

Further we are amplified under 400 times of light microscopics (Fig. 4-2), find that equally DSS group mucous layer intestinal epithelial tissue form destruction is serious, and after adding antibacterial peptide C-BF, its form are improved to a certain extent.

5, same, we have taked ileum stage casing to be fixed HE dyeing observation:

First we find the obvious enhancing compared with control group of DSS treatment group ileal epithelium tissue secretion intensity under 200 times of light microscopics (Fig. 5-1), and edge has a lot of secretory cells to assemble.Illustrate and have inflammation to occur herein.

Meanwhile, we are respectively in 40(Fig. 5-2) and 200 times (Fig. 5-3) ileum tissue is observed to discovery, DSS group ileal epithelium length seriously shortens, and after adding antibacterial peptide, its length has had obvious increase.

6, further, we carry out eye socket to mouse and get blood, whole blood carries out flow cytometer showed B after removing red corpuscle, T, scavenger cell, the ratio of NK cell, we find significantly to have suppressed B after DSS processes, T, the ratio of scavenger cell, and add the ratio of three kinds of immunocytes after antibacterial peptide obviously to promote (Fig. 6-1, Fig. 6-2, Fig. 6-3, Fig. 6-4).

7, simultaneously, the gene expression abundance of we the Endogenous antimicrobial polypeptide mCRAMP to mouse colon stage casing carries out immunofluorescence detection, found that in 200 times of situations (Fig. 7-1, Fig. 7-2, Fig. 7-3), the gene expression abundance of DSS treatment group mCRAMP obviously raises, and its gene expression abundance significantly reduces after adding antibacterial peptide to process, and studies have reported that the high expression level of derived antimicrobial peptide and the positive correlation that becomes of inflammation in mouse.

8, by ELISA to proinflammatory factor TNF-α in mice serum, IL-6, IL-8, press down scorching factor IL-10 and detect (Fig. 8-1, Fig. 8-2, Fig. 8-3, Fig. 8-4), colon is extracted to RNA simultaneously and carry out qRT-PCR detection (Fig. 8-5, Fig. 8-6, Fig. 8-7, Fig. 8-8), result shows, DSS treatment group inflammatory cytokine significantly improves, and adding the secretion level of inflammatory factor after antibacterial peptide C-BF significantly to decline, gene expression dose is consistent with serum protein level.

9, mouse colon is carried out to TUNNEL immunohistochemical staining (Fig. 9-1), detect colon epithelial cell apoptosis situation, result shows that DSS processes has significantly increased Level of Apoptosis (brown), and adds after antibacterial peptide C-BF its apoptotic index significantly decline (Fig. 9-2).

10, the inflammatory relevant enzyme work of mouse colon is detected and finds that the enzyme work of DSS group INOS and COX-2 significantly improves (Figure 10-1, Figure 10-2), and add its enzyme running water aobvious decline dawn after antibacterial peptide, further extract tissue protein and carry out Western-blot analysis discovery (Figure 10-3), DSS by the activation of MAPK and NF-kB signal path approach is raised, live and the expression of cytokine by inflammatory relevant enzyme, and C-BF lowers inflammatory relevant enzyme by the phosphorylation of inhibition NF-kB (p65) to live, simultaneously may be relevant with the down-regulated expression of above-mentioned several cytokines.

11, further by CD177 neutrophilic granulocyte (Figure 11-1), the specific antibody of F4/80 scavenger cell carries out immunohistochemical methods (Figure 11-3) to colon, analyze neutrophilic granulocyte invasion intestinal tissue situation (Figure 11-2, Figure 11-4), simultaneously, extract detection (Figure 11-4 that colon's total protein is lived for significant enzyme, (Figure 11-5, Figure 11-6, Figure 11-7), the data that binding immunoassay group and enzyme are lived show, DSS processes neutrophilic granulocyte invasion obviously to be increased, and alleviated to a certain extent this phenomenon after adding antibacterial peptide, simultaneously, measuring the significant enzyme slip-knot fruit of this neutrophilic granulocyte of MPO is consistent with groupization result.Detect the significant enzyme of eosinophilic granulocyte EPO alive and find that its expression rule is consistent with MPO, detect the significant enzyme of scavenger cell simultaneously and live, but find that its expression is inconsistent with the result of immunohistochemical methods, but consistent with bibliographical information.

12, extract nearly cecum ileum total protein simultaneously, separation of serum, measurement result shows, the secretion level of DSS treatment group ileum sIgA significantly reduces (Figure 12-1), IgA in serum, G, the level of M tri-antibody is obviously suppressed, and adds rear its sIgA secretion level of antibacterial peptide processing and three kinds of immunoglobulin levels all to have to a certain degree raise (Figure 12-2, Figure 12-3, Figure 12-4).

13, simultaneously, we detect in the effect aspect barrier antibacterial peptide, mainly be divided into 4 points of experiments, experiment one is the dextran of the front 2h rectal injection FITC mark of sampling, by detecting FITC concentration Indirect evaluation intestinal permeability (Figure 13-1) in serum, when experiment two is experiment, get the fresh and alive 1cm of organizing of colon ²left and right, utilize USING Champer to carry out enteron aisle cross-film epithelium resistance change trend and measure (Figure 13-2), experiment three is for getting colon, after 4% paraformaldehyde is fixing, carry out the immunofluorescence experiment of barrier tight junction protein ZO-1, detect its gene expression abundance (Figure 13-3).Experiment four is for extracting the RNA of colon, and the gene expression dose of the tight junction protein of being correlated with detects (Figure 13-4, Figure 13-5, Figure 13-6, Figure 13-7, Figure 13-8).The result of experiment one shows, after DSS processes, its fluorescence intensity significantly improves, and prove that gut barrier is damaged, and after adding antibacterial peptide, its fluorescence intensity significantly reduces, illustrate that its barrier function has obtained certain improvement, show in conjunction with the result of testing two, DSS processes intestines cross-film epithelium resistance value and significantly declines, and corresponding intestinal permeability obviously rises, and antibacterial peptide treatment group has obviously improved cross-film epithelium resistance value, indirectly reflect that its gut barrier function improves.Can find out from testing three, after DSS processes, the gene expression abundance of tight junction protein ZO-1 obviously reduces, antibacterial peptide treatment group gene expression abundance increases, and tests three and shows in conjunction with the result of experiment four, and DSS processes and significantly lowered tight protein related gene Claudin-1, ZO-1, the genetic expression of ZO-2, with the closely-related Saliva Orthana mucin-1 of barrier of intestinal epithelial cells monolayer, 2 expression level is also suppressed, and after adding antibacterial peptide to process, the expression level of five kinds of genes has all obtained remarkable rise.

14, last, six orifice plates are cultivated RAW264.7 cell, add 25ug/mL C-BF to cultivate after 4h, add 1 μ g/ml LPS to stimulate 3h, ELISA kit measurement PGE2 and NO concentration (Figure 14-1, Figure 14-2) for collecting cell supernatant, scrape cell extraction RNA and analyze the expression level (Figure 14-3) of TNF-α gene.From result, we may safely draw the conclusion: after LPS individual curing, PGE2, NO and TNF-α significantly raise, and are eased after C-BF advanced processing, and mouse COX-2 and PGE2 enzymic activity that this and DSS process strengthen, and C-BF+DSS group is effect improved consistent.C-BF mechanism of action may be the reduction (Figure 14-4) of the phosphorylation level of NF-κ B p65 after processing based on C-BF.

Finally it should be noted that above content is only in order to technical scheme of the present invention to be described, but not limiting the scope of the invention, some improvement and retouching that those of ordinary skill in the art carries out technology of the present invention, be all considered as protection scope of the present invention.

The present invention probes into the effect of antibacterial peptide C-BF to enteritis UC model.

In order to realize object of the present invention, the invention provides following technical scheme:

Set up 3% dextran sulfate sodium (DSS) inducing mouse Ulcerative Colitis Model, by living imaging, HE dyeing, immunofluorescence, immunohistochemistry, flow cytometer, Tunnel, ELISA, western-blot, Real-Time Fluorescent Quantitative PCR Technique method, probe into the effect of the colitis of C-BF to DSS induction.In vitro, we study the effect of C-BF to Caco-2 cell system.

-BF withdSS preparation

Cathelicidin-BF(C-BF) and purifying synthetic by Chinese Peptide Co., Ltd.'s (Hangzhou China), its molecular weight passes through substance assistant laser desorpted/ionization time of flight mass spectrometry (MALDI-TOF MS using alpha-cyano-4-hydroxycinnamic acid as matrix, Model Autoflex, Brooker dalton company, the U.S.) measure.Purity by anti-phase-high-efficient liquid phase chromatogram technique analysis peptide is more than 95%.Peptide is dissolved in PBS damping fluid, and concentration is 200 μ μ g/ml, uses 80 ° of C of Qian – to store.Dextran sulfate sodium (being called for short DSS) is purchased from the MP(U.S.), molecular weight is 36000-50000 Da, is dissolved in sterilized water, (3g DSS/100ml sterilized water) with 3%.

after ulcerative colitis induction, measure the effect of C-BF by DAI points-scoring system

Test is grouped into control group, DSS group C-BF group, C-BF+DSS group, raise in advance after 1 week, induce 1 week, point two kinds of processing, one is freely to drink sterilized water (control group, C-BF group) or freely drink the water (DSS group, C-BF+DSS group) 1 week that is dissolved with DSS, another kind of processing is that C-BF group and C-BF+DSS group rectal injection C-BF(are dissolved in PBS) or control group and DSS group inject separately PBS, once a day, injection continues 1 week, and the dosage of C-BF is 5mg/kg, and total injection volume is 500ul.Experiment periods is weighed and record weight data every day, and observes mouse proctorrhagia, ight soil moulding situation.DAI represents the mean value of these three indexes.

utilize noclilucence imaging that C-BF is absorbed and evaluated

Six C57/BL6 male mices are divided into three groups, rectal injection 100ul PBS respectively, FITC, FITC-C-BF, concentration is 2mg/ml, after rectal injection FITC-C-BF 10 minutes, 30 minutes, 2 hours, 24 hours difference abdominal injection tranquillizer vetanarcol, then open the power supply of noclilucence imager and fluorescence light source, click living imaging icon, IVIS system initialization, machine initialize and state of temperature lamp are from redness becomes green, anesthetized mice is placed on to observation place, visit in pass, excite with emission wavelength and be respectively 488nm and 525nm, select fluorescent sample and time shutter and rank value are set, F/stop value being set to 2, then obtain corresponding picture, comprise fluoroscopic image and laser data.

histopathological evaluation

Intestinal tissue is taken from five mouse, one every group, carries out histology measurement.Ileum stage casing and colon holostrome section cut and longitudinally cut open, are fixed on immediately 4% paraformaldehyde solution, paraffin embedding, HE dyeing.Epithelium morphological specificity is amplified different multiples by come card NEWDM 4500BR microscope and is observed.

flow cytometry analysis immunocyte (B, T lymphocyte, scavenger cell, NK cell)

Eye socket is got blood and is placed in the 1.5ml pipe that contains heparin sodium (Sangong Chinese Shanghai), getting blood 50ul mixes with specific surface antibody, this mixture is hatched 15 minutes in room temperature lucifuge, antibody CD3(PE-Cy5), CD45R(FITC), CD49B(PE), F4/80(PE) purchased from (connection section, Chinese Shanghai), consumption is respectively 0.25ug/5ul, 0.5ug/5ul, 0.5ug/5ul, 0.25ug/5ul, then add 1 × FCM lysate, room temperature lucifuge is hatched 10 minutes, the centrifugal 5min of 300-400g, abandon supernatant, 2ml PBS is resuspended, the centrifugal 5min of 300-400g, abandon supernatant, 500ul PBS is resuspended, flow cytometer fluorometric analysis.

colon's immunofluorescence and immunohistochemical staining

The mCRAMP(Abcam U.S.) and the ZO-1(Abcam U.S.) immunofluorescence analysis, first, with the PBS sealing non-specific binding site of containing 1% w/v BSA 30 minutes, then with 1:1000 ratio, rabbit polyclonal antibody is joined in Cathelicidin antibody, with 1:500 ratio, rabbit polyclonal antibody is joined in ZO-1 antibody, solution is the PBS containing 1% w/v BSA, the 4 DEG C of overnight incubation of cutting into slices, PBS washes 5 times, each 3 minutes, then dry sample PBS around with thieving paper, add the goat anti-rabbit igg (the JIR U.S.) of TRITC mark with 1:100 ratio, room temperature lucifuge is hatched 1h, again washing two resists, then with the dyeing of DAPI nucleus, finally, wash away unconjugated DAPI, glycerine mounting, use immediately fluorescence microscope stained.

CD177 and F4/80 immunohistochemical analysis, first, with the PBS sealing non-specific binding site of containing 1% w/v BSA 30 minutes, then with 1:100 ratio, sheep polyclonal antibody is joined to the CD177(Santa U.S.) in antibody, with 1:100 ratio, sheep polyclonal antibody is joined to the F4/80(Santa U.S.) in antibody, solution is the PBS containing 1% w/v BSA, the 4 DEG C of overnight incubation of cutting into slices, PBS washes 5 times, each 3 minutes, then dry sample PBS around with thieving paper, add the Tu Kangyang IgG(JIR U.S. of HRP mark with 1:100 ratio), hatch 1h for 4 DEG C, PBS rinses three times, then add the 50-100ul DAB(DAKO U.S.), after color forms, Hematorylin suppresses section, with the dehydration of alcohol of the different gradients of 70-100%, each gradient 10 minutes, make to cut into slices transparent with dimethylbenzene afterwards, neutral gum mounting.Level of Apoptosis in evaluation of tissue, paraffin section de-waxing, aquation and antigen retrieval, TDT and TUNEL(test kit, the Roche U.S.) mix with the ratio of 1:9, hatch after 60min for 37 DEG C, that box keeps humidity.Endogenous peroxydase sealing, chip drying, transforming agent-POD(test kit, the Roche U.S.) cover tissue, to hatch 30 minutes for 37 DEG C, PBS washes three times, adds DAB, distilled water color development stopping, nucleus is redyed, section dehydration mounting.

cytokine of Serum FITC-dextran, IgA, IgG, IgM, MPO in terminal ileum SIgA secretion level and colon, EPO, the measurement of NAG enzymic activity

Cytokine of Serum TNF-α, IL-6, IL-8, the detection of IL-10 ELISA test kit (doctor's moral, Wuhan, China).Mensuration process is as follows: with 100ul standard substance production standard curve, testing sample joins corresponding antibody coated elisa plate, and 37 DEG C are reacted 90 minutes, abandon liquid in plate, add 100ul TNF α, IL-6, IL-8, IL-10 antibody, 37 DEG C are reacted 60 minutes, PBS washes plate three times, and each hole adds ABC working fluid, and 37 DEG C are reacted 30 minutes, again wash plate with PBS, add the tmb substrate solution of premix, 37 DEG C are reacted 25 minutes, add TMB stop buffer, measure OD value at microplate reader 450nm place.

Quantitative assay intestinal permeability indirectly, take a blood sample first 4 hours injection FITC-Dextran 200 ul to colon, eye socket venous blood collection, lucifuge is added to serum in 96 orifice plates, then use microplate reader (spectramax M5 Molecular devices, the U.S.) fluorescence intensity, excite, emission wavelength is respectively 488nm and 525nm.

Cytokine of Serum IgA, IgG, IgM, MPO in terminal ileum SIgA secretion level and colon, EPO, the measurement of NAG enzymic activity is used ELISA test kit (the R & D U.S.), and simple operation step is as follows: 50ul standard substance and testing sample add antibody coated elisa plate, each hole adds 50ul enzyme marking reagent, hatch 1h for 37 DEG C, abandon residue, washings washing 5 times, each hole successively adds 50uL developer A and B, and 37 DEG C of lucifuges are hatched 15 minutes.Take out plate, add 50ul stop buffer, measure the right 450nm OD of place value.

iNOS and COX-2 enzyme assay in colon

INOS and COX-2 enzyme assay in colon, used the ELISA test kit (Biovalue Britain) of corresponding INOS and COX-2.Operate as follows: the preparation of tissue, with RIPA lysate (doctor's moral Wuhan, China) extraction colon's total protein and BCA test kit (triumphant base Nanjing of China) detection protein concn, quantitative, 50uL standard substance and testing sample join antibody coated elisa plate, each hole adds 25ul enzyme marking reagent, silver paper bag quilt and 37 DEG C are hatched 1h, scavenging solution is washed plate five times, add successively 50ul substrate one, substrate two, lucifuge is hatched 15 minutes, then add 50ul stop buffer termination reaction, in 30 minutes, measure the OD of 450nm place value.

electrophysiology is measured

Adopt previously described improved Ussing chamber (VCC MC6, PI) to measure membrane potential.Cut mouse colon, be immersed in immediately in oxygen containing Kreb damping fluid, put into Ussing chamber (world's precision instrument; Drugs science, Mississauga city, ontario, Canada).Under 37 ° of C, 0.6 square centimeter of stream chamber tissue surface is immersed in the Kreb buffer zone in the circulation oxygen of 8U ml.In serous coat damping fluid, contain 10ml glucose as energy derive, carry out equilibrium osmotic pressure and contain 10ml N.F,USP MANNITOL in mucous membrane damping fluid.The potential difference that perfusate chamber contains agar bridge monitoring different tissues two ends, indicates by automatic electric pressing tongs, injects required Isc and keeps isoelectric.Organize electricity to lead, be equivalent to passive infiltration ion, calculated by Ohm's law.As the backstepping measure of tissue resistance, the baseline value of ISC indicates clean ion secretion and electricity to lead, and intestinal segment balance is record after 20 minutes.

analyze Western blot

Grind colon stage casing, the complete arrestin enzyme mixture that extracts test kit (triumphant base Nanjing of China) with total protein dissolves.After quantitatively diluting, guarantees each tissue protein content that 7.5%/10% the every hole of SDS-PAGE gel contains 30 μ G albumen.Albumen separates by SDS-PAGE, transfers on nitrocellulose filter the skim-milk sealing of 5% (w/v) of TBS configuration, 4 ° of C overnight incubation of primary antibodie (NF-κ B P65, p-p65, c-jun, P-c-jun, the Santa Cruze U.S.).Horseradish peroxidase-labeled two is anti-hatches, colour developing.

isolation of RNA and quantitative fluorescent PCR

(the total RNA of (Invitrogen Corporation, Carlsbad, CA) separation and Extraction, M-MuLV reversed transcriptive enzyme test kit (Fermentas, EU, Glen Burnie, Maryland, USA) reverse transcription synthesizes cDNA to Trizol reagent.SYBR ^?premix Ex Taq ^tMtest kit (Applied Biosystems, Foster City, CA, USA) detects the mRNA level of individual gene by real-time fluorescence quantitative PCR.At ABI the first step PLUSTM real-time PCR system (Applied Biosystems company, Foster City, CA, USA).Data are according to cycle threshold (CT) and internal reference (GAPDH) comparative analysis.The primer of the present invention is listed at table 1.

 

Claims (2)

1. antibacterial peptide C-BF protection mouse and a mouse macrophage ulcerative colitis generalized model, is characterized in that,

1), for mouse, set up 3% dextran sulfate sodium (DSS) inducing mouse Ulcerative Colitis Model; The PBS solution of mouse rectal injection C-BF, once a day, the dosage of C-BF is 5mg/kg, and injection continues 1 week, and experiment periods is weighed and record weight data every day, and observes mouse proctorrhagia, ight soil moulding situation;

2) for mouse macrophage, in substratum, the concentration of antibacterial peptide C-BF is 25ug/ml, after antibacterial peptide processing RAW264.7 cell 4h, processes cell 3h with the LPS of 1 μ g/ml concentration.

2. antibacterial peptide C-BF protection mouse according to claim 1 and mouse macrophage ulcerative colitis generalized model, is characterized in that, further,

3) for mouse, fluorescent mark C-BF, concentration is 2mg/ml, rectal injection, by living imaging, HE dyeing, immunofluorescence, immunohistochemistry, flow cytometer, Tunnel, ELISA, utilize cell resistance instrument to transepithelial electrical resistance value measure, western-blot, Real-Time Fluorescent Quantitative PCR Technique method;

4) for RAW264.7 cell, utilize ELISA test kit to PGE2 in culture supernatant, the concentration of NO is measured, and extracts total RNA simultaneously the gene expression dose of TNF-α is measured, and western-blot measures NF-kB signal path;

5) mouse experiment and In vitro cell experiment in synthesis, evaluate C-BF at amelioration of inflammation, reduce Level of Apoptosis and improve the effect of gut barrier function.