CN104587469A - Application of G-CSF antagonist in treatment of chronic inflammation and prevention of inflammation-associated tumor - Google Patents

Abstract

The invention discloses application of G-CSF antagonist in treatment of chronic inflammation and prevention of inflammation-associated tumor, and discloses a kit for preventing and/or treating inflammation or tumor. The kit comprises antagonist of granulocyte colony-stimulating factors. The G-CSF antagonist is used as an effective intervention means of the tumor, and can be applied to prevention and/or treatment of inflammation or tumor.

Description

The application of G-CSF antagonist in treatment chronic inflammatory disease, prevention of inflammation related neoplasms

Technical field

The present invention relates to the application of a kind of G-CSF antagonist in treatment chronic inflammatory disease, prevention of inflammation related neoplasms, belong to biological technical field.

Background technology

Increasing research in recent years shows, the morbidity of digestive tract chronic inflammatory disease and tumor has close relationship, as chronic reflux esophagitis and the esophageal carcinoma, Helicobacter pylori infection and gastric cancer and inflammatory bowel and colon cancer etc.Inflammatory reaction with multiple links of tumor, from the generation of tumor, development until the transfer of tumor.Inflammatory reaction and tumor are connected each other by two approach, are divided into intrinsic pathway and extrinsic pathway.Intrinsic pathway is mainly caused by the activation of epithelial cell proto-oncogene, comprises the inactivation etc. of the sudden change of oncogene, chromosomal rearrangement or amplification and tumor suppressor gene.The cell activated through above process produces proinflammatory inflammation factor, thus causes inflammatory microenvironment at tumor happening part.In tumor microenvironment, the lasting stimulation of inflammatory cell can provide a large amount of reactive oxygen free radical, and these free radicals can cause the epithelial cell generation tissue oxidizing damage closed on, thus cause gene mutation.Extrinsic pathway refers to inflammation or infects the incidence rate that can improve some tumor.These two kinds of approach can cause the Expression of Activated of tumor cell transcription factor, as NF-kB, Stat3 and HIF-alpha.The activation of these transcription factor thus inducing cell expresses inflammatory factor, comprises cytokine and chemotactic factor, produces Cox2 simultaneously.Panimmunity cell be raised and be activated to these inflammatory factors can, especially the monocytic series in marrow sample source.Inflammatory factor activates the monocytic series raised, the expression of the transcription factor such as activation NF-kB, Stat3 wherein, thus causes stronger inflammatory reaction, and produces the relevant inflammatory microenvironment of tumor, thus promotes generation and the development of tumor.

In normal body, the myeloid precursor in bone marrow promptly can be divided into ripe granulocyte, dendritic cell and macrophage, and enters corresponding organ, tissue, immunologic function of bringing into normal play.In pathological conditions, especially, when tumor occurs, the precursor anacmesis in these medullary systems source, thus rests on each differential period, become the T suppression cell (Myeloid-derived suppressor cell, MDSC) in the marrow sample source with immune suppression function.MDSCs by a group heterogeneity, comprise and ripe to form with the cell mass of immature cell, wherein have dendritic cell, granulocyte, immaturity macrophage, natural killer cell, T cell and myeloid precursor etc.In recent years, increasing evidence shows, MDSCs all plays critical function in inflammation and tumor, and becomes the bridge of both connections.MDSCs has the ability of very strong depression effect t cell response, can the anti-tumor immune response of negative regulation body, therefore MDSCs the quantity of periphery and pathological changes local increase and the generation of functional activation and tumor to develop be close positive correlation.MDSCs suppresses the approach of body's immunity and nonimmunologic function to have multiformity and more complicated, comprise and produce too much nitric oxide synthase type (Inducible NO synthase, iNOS), reactive oxygen free radical (Reactive oxygen species, ROS), arginase (Arginase, ARG) etc.MDSCs can high expressed iNOS, high-caliber iNOS cause NO generation in tumor microenvironment to increase, and acts on T cell, makes IL-2 receptor-mediated T cell mitogen activity suppressed.ARG can act on iNOS, and make it transfer to from the normal NO of generation and produce harmful substance ROS, ROS and NO combines and forms active nitrogen (Reactive nitrogen species, RNS), comprising Peroxynitrite salt etc.RNS can catalysis CD8 ⁺the receptor (T cell receptor, TCR) of T cell is nitrated and affect T cell-protein polypeptide-MHCI quasi-molecule and contact with each other, and therefore promotes t cell proliferation.But, when inflammation occurs, ROS belongs to the hazardous compound produced in body oxidation reaction, there is strong oxidizing property, tissue and the cell of body can be damaged, thus cause DNA damage, be i.e. DNA double chain break and base mutation, to go forward side by side one-step activation oncogene or cause the inactivation of antioncogene, finally cause epithelial cell generation vicious transformation.In addition, the number of different types cytokine in inflammation or tumor microenvironment can promote the raising of MDSCs, growth and differ entiation.At present, clinical and basic research shows, in digestive tract tumor's tissues such as the esophageal carcinoma, gastric cancer and colorectal cancer, MDSCs soakage water is on average increased significantly, and take part in the Fashion and Evolution of tumor with mechanism of action such as suppressing the function of antineoplastic immune cell or increase the weight of that local inflammation accelerates that normal cell cancerates.

Granulocyte colony-stimulating factor (granulocyte colony-stimulating factor, G-CSF) clinical existing more than 20 years are applied to, be so-called in the middle of oncotherapy " rising BAIYAO ", it is widely used in caused by radiotherapy and chemotherapy leukopenia and hemopoietic function of bone marrow obstacle.But recent research shows, G-CSF is high expressed in multiple solid tumor, and combined chemotherapy can promote revascularization and secondary tumors growth, weakens chemosensitivity.In Healthy Human Serum, G-CSF foundation level is very low, general <21pg/ml.The lung carcinoma cell of planting from reported first such as Asano.S in 1977 in nude mice can be secreted and produce G-CSF, at present both at home and abroad existing example up to a hundred about the report of high expressed in solid tumor.Multinomial research shows that the poor prognosis of malignant pleural mesothelioma, ovarian cancer, cervical cancer, colorectal cancer etc. may be relevant to the unconventionality expression of G-CSF, and after the operation in patients of G-CSF overexpression, G-CSF level has decline in various degree.Detect and find that the tumor tissues differentiation of generation G-CSF is poor, the neutral grain like cell of many companions infiltrates, and easily shifts, and treat insensitive to chemotherapy, anti-vegf, survival rate is low, poor prognosis.Multinomial research shows, G-CSF is by mobilizing positive bone marrow-derived cells (bonemarrow-derived cells, BMDCs), stimulation cycle endothelial progenitor cells (circulating endothehelialprogenitor cells, CEPs), Gr1 ⁺cD11b ⁺bone marrow derived T suppression cell (myeloid-derivedsuppressor cells, MDSCs) and VEGF-1 ⁺the activation of marrow sample hematopoietic cell, propagation, induction of vascular is formed and tumor proliferation.Wherein MDSCs forms poor prognosis the key link.Although G-CSF has been widely used in tumor and has been correlated with the clinical treatment of leukopenia, but the relation of G-CSF and tumorigenesis, also needs large quantity research clear and definite further with opportunity of chemicotherapy, targeted therapy use in conjunction, dosage, mechanism of action and effect etc.How to select suitable G-CSF antibody or inhibitor to block it and promote revascularization, inhibition tumor cell apoptosis etc., new problem is proposed to medical investigator.To prevent about retardance or antagonism G-CSF or being reported in of disease therapy does not domesticly have especially.

In sum, G-CSF antagonist is suitably utilized to stop the infiltration of MDSC cell will become effective prevention means in the symptom that reduces inflammation, prevention and therapy inflammation related neoplasms.

Summary of the invention

The object of this invention is to provide the application of a kind of G-CSF antagonist in treatment chronic inflammatory disease, prevention of inflammation related neoplasms.

The invention provides a kind of test kit preventing and/or treating inflammation or tumor, this test kit comprises the antagonist of granulocyte colony-stimulating factor;

Described antagonist is specially the antibody of granulocyte colony-stimulating factor;

The T suppression cell (MDSC) that described inflammation is embodied in inflammatory tissue local and/or peripheral blood the marrow being invaded by pathogen sample source is raised in a large number, and the content of granulocyte colony-stimulating factor (G-CSF) raises;

Described inflammation is specially digestive tract chronic inflammatory disease.

In mentioned reagent box, described tumor is the tumor that inflammation is relevant;

The tumor that described inflammation is correlated with is the tumor from diseases associated with inflammation development.

In above-mentioned arbitrary described test kit, described inflammation is alimentary canal inflammation;

Described tumor is digestive tract tumor.

In above-mentioned arbitrary described test kit, described digestive tract is intestinal.

In above-mentioned arbitrary described test kit, described intestinal is Colon and rectum.

In above-mentioned arbitrary described test kit, described tumor is colitis dependency colorectal cancer.

The antagonist of mentioned reagent box or granulocyte colony-stimulating factor suppresses the application in the propagation of T suppression cell in marrow sample source and/or the product of chemotactic also to belong to protection scope of the present invention in preparation;

Described chemotactic is that the antagonist of granulocyte colony-stimulating factor suppresses the T suppression cell in marrow sample source to move to the direction of granulocyte colony-stimulating factor.

The application of antagonist in the product of preparation treatment chronic inflammatory disease, prevention of inflammation related neoplasms of mentioned reagent box or granulocyte colony-stimulating factor also belongs to protection scope of the present invention.

In above-mentioned application, described tumor is the tumor that inflammation is relevant;

The tumor that described inflammation is correlated with is the tumor from diseases associated with inflammation development.

In above-mentioned arbitrary described application, described inflammation is alimentary canal inflammation;

Described tumor is digestive tract tumor;

Described digestive tract is specially intestinal;

Described intestinal is specially Colon and rectum;

Described digestive tract tumor is specially colitis dependency colorectal cancer.

G-CSF antagonist, as the effective prevention means of tumor, can be applied in the preventing and/or treating of inflammation or tumor.

Accompanying drawing explanation

Fig. 1 is the expression of mouse intestinal epithelial cell G-CSF.

Fig. 2 is G-CSF expression in clinical tissue specimen.

Fig. 3 is normal mice and AD mouse peripheral blood MDSC ratio.

Fig. 4 is normal mice and AD mice digestive tract MDSC ratio.

Fig. 5 is MDSC original cuiture proliferative conditions.

Fig. 6 is that G-CSF induces the external migration experiment of MDSC.

Fig. 7 is the impact of G-CSF Antybody therapy on MDSC ratio in mouse bone marrow cells.

Fig. 8 is the impact of G-CSF Antybody therapy on MDSC ratio in mouse peripheral blood.

Fig. 9 is the impact of G-CSF Antybody therapy on the MDSC ratio in mouse Colon.

Figure 10 is that the proinflammatory inflammation factor of MDSC emiocytosis detects.

Figure 11 is G-CSF Antybody therapy postcolon MDSC Infiltrating.

Figure 12 is that G-CSF Antybody therapy postcolon organizes iNOS to express.

Figure 13 is mice Colon and rectum general condition and length statistics after Antybody therapy.

Figure 14 is mice colorectal carcinoma tuberosity quantity statistics result.

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Azoxymethane (Azoxymethane, AOM) purchased from American Sigma company, catalog number is A5486.

Dextran sulfate sodium (Dextran sodium sulfate, DSS) purchased from American MP Biomedicals company, catalog number is 160110.

IL-4 purchased from American PeproTech company, catalog number is AF-214-14.

Recombined small-mouse G-CSF purchased from American PeproTech company, catalog number is AF-250-05.

G-CSF antibody purchased from American PeproTech company, catalog number is 500-P69.

Rabbit anteserum IgG purchased from American Invitrogen company, catalog number is PLN5001.

Mouse lymphocyte separating medium (Ficoll) is purchased from Tianjin Hao ocean biological product science and technology limited Company, and catalog number is LTS1092P-100.

70um BD nylon film is purchased from BD Bioscience, and catalog number is REF 352350.

Anti-CD16/32 blocking antibody purchased from American ebioscience, catalog number is 14-0161.

Anti-mouse Gr1-FITC antibody purchased from American ebioscience, catalog number is 11-5931.

Anti-mouse CD11b-APC antibody purchased from American ebioscience, catalog number is 17-0112.

Normal mouse in following embodiment is C57B/L6 mice, purchased from Beijing HFK Bio-Technology Co., Ltd..

In following embodiment, AD refers to the process of AOM-DSS induction, comprises AOM and injects the process that rear three cycles drink DSS, and AD1, AD2, AD3 refer to respectively 1,2,3 DSS drink after mice.IBD refers to inflammatory bowel disease inflammatory bowel, the human colorectal chronic inflammation diseases of the mankind, include Crohn disease (Crohn is sick) and ulcerative colitis, from the colorectal cancer that inflammatory bowel develops, the colorectal cancer of difference general significance, be referred to as colitis dependency colorectal cancer (CAC, colitis-associatedcancer).AD model mice digestive tract becomes the process of tumor to be intestinal mucosa from inflammation to atypical hyperplasia developing deeply to be a process of tumor, simulate the mankind develop into colorectal cancer process from inflammatory bowel (IBD) well, simulate the scorching dependency colorectal cancer of human colon to develop into relevant colorectal cancer CAC process from inflammatory bowel IBD at mouse model from the process nature that AD1 develops into AD3, so IBD period of the corresponding mice of AD1 in following embodiment, the CAC period of the corresponding mice of AD3.

Embodiment 1, gastrointestinal epithelial cells secretion G-CSF

One, in order to the generation development course of the digestive tract tumor that is correlated with to inflammation is carried out systematic research and proposes therapeutic strategy targetedly, first must set up can the animal model of the simulating human course of disease.

The foundation of animal model: adopt carcinogen azoxymethane (Azoxymethane, AOM) induce, be aided with 3 proinflammatory agent dextran sulfate sodium (Dextran sodium sulfate simultaneously, the course of disease of the injury repairing repeatedly of discontinuity continued stimulus simulating human intestinal mucosa DSS), method is as follows:

Select more than 18g C57BL/6 mice (female), lumbar injection AOM (prepare by sterilized water for injection, injected dose is 12.5mg/kg body weight), feed containing 2.5g/100mlDSS drinking water to mice after 10 days, normal water is recovered after continuous 5 days DSS drinking-water, mice drinks two circulations again after having a rest 14 days (each circulation recovers normal water after DSS drinking-water in continuous 5 days, mice has a rest 14 days again) DSS water can complete CAC (Colitis-associatedcancer, colitis dependency colorectal cancer) induction of model, C57BL/6 mice is made to develop into colorectal cancer in 53 days.The mice obtained after first time circulation, second time circulation and third time circulation DSS induction is called AD1, AD2 and AD3 mice.

Pathological examination confirms, AD1 mice shows as master with the inflammation at Colon and rectum position; AD2 mice is based on precancerous lesions such as the atypical hyperplasia at Colon and rectum position and adenomas; AD3 mice is based on the cancer in situ at Colon and rectum position.

Two, the digestive tract tissue getting mice AD1, AD2 and AD3 of each pathologic stage carries out protein chip detection, and result is as shown in table 1.

Table 1 mice digestive tract histone chip detection result

Table 1 shows, to suffer from the mice digestive tract tissue of alimentary canal inflammation and tumor granulocyte colony-stimulating factor (G-CSF) expression all higher than normal mouse.

Three, under normal circumstances, G-CSF mainly acts on the propagation of neutrophil series hematopoietic cell, differentiation and activation, and its important function stimulates granulocyte ripe, promotes that mature cell discharges to peripheral blood, and can promote the several functions of neutrophilic granulocyte.

In order to study the source of G-CSF, detected from gene level by the G-CSF in AD1, AD2 and AD3 mice of RT-PCR technology convection type sorting gained, normal mouse (normal) intestinal epithelial cell, step is as follows:

Total serum IgE is extracted respectively from Normal, AD1, AD2 and AD3 mice Colon and rectum, and reverse transcription synthesis cDNA, be template respectively with cDNA, with G-CSF-F and G-CSF-R for primer, carry out realtime-PCR amplifying target genes, detect the gene expression dose of Normal, AD1, AD2 and AD3 mice Colon and rectum G-CSF.

G-CSF-F：5’-TGCTTAAGTCCCTGGAGCAA-3’；(SEQ ID No.1)

G-CSF-R：5’-AGCTTGTAGGTGGCACACAA-3’。(SEQ ID No.2)

Result as shown in Figure 1.

Fig. 1 shows, in AD1, AD2 and AD3 mice, G-CSF is compared to normal mouse (normal) and raises 2.14 times, 5.54 times and 2.59 times of (n=4 respectively, P<0.01), this result points out impaired intestinal epithelial cell to secrete G-CSF.

Four, ((colon's specimen of normal person and colorectal cancer patients is provided by Cancer Hospital of Chinese Academy of Medical Sciences colon's specimen (cancer) of colon's specimen (normal) of 30 routine normal persons, colon's specimen (IBD) of 30 routine patients with inflammatory bowel disease, 30 routine colorectal cancer patients to adopt Immunohistochemical Method to detect 90 routine clinical samples, patients with inflammatory bowel disease colon specimen is provided by Beijing Friendship Hospital) expression of G-CSF is studied, and result is as shown in Fig. 2 and table 2.

Fig. 2 shows, the colon of colon's specimen of normal person has no G-CSF dyeing; The colon G-CSF of colon's specimen of patients with inflammatory bowel disease is mainly expressed in mucous membrane of colon epithelium position; The colon G-CSF of colon's specimen of colorectal cancer patients dyes as some positive (often organizing specimen n=30).

The detection of expression of G-CSF in table 2 clinical samples

Table 2 shows, colon's specimen of all normal persons is G-CSF feminine gender; The positive ratio of G-CSF of colon's specimen of patients with inflammatory bowel disease is 53.3% (16/30); The positive ratio of G-CSF of colon's specimen of colorectal cancer patients is 80% (24/30).

The data of clinical samples support the conclusion on mouse model further, and namely impaired intestinal epithelial cell can secrete G-CSF.

The T suppression cell (MDSC cell) in embodiment 2, marrow sample source is at the proportion grading of peripheral blood and colon

Detect AD1 mice and normal mouse peripheral blood and digestive tract tissue infiltration CD11b ⁺gr1 ⁺the ratio of MDSC.Step is as follows:

One, the preparation of mouse peripheral blood single cell suspension and detection

1) gather centrifugal 20 minutes of mice fresh peripheral blood 2500rpm, draw upper plasma;

2) with PBS or normal saline resuspended lower floor hemocyte, re-suspended cell liquid is obtained,

3) add Ficoll in centrifuge tube, upper strata adds re-suspended cell liquid slowly, and the volume ratio of Ficoll and re-suspended cell liquid is 1:2;

4) slowly to accelerate and centrifugal 20 minutes of slow deceleration 2000rpm;

5) zigzag draws PBMC layering;

6) PBS or normal saline clean cell three times and count;

7) by resuspended for cell PBS, grouping adds in streaming pipe, often pipe 100ul;

8) anti-CD16/32 blocking antibody is added, 4 DEG C of reaction 10min, the unspecific staining of removing mouse immune cell surface;

9) in streaming pipe, antibody is added according to antibody preparation volume, common vast scale cell can a marker detection antibody, and several situation needs to prepare blanc cell pipe, Isotype control pipe simultaneously, detects antibody pipe and detect below: cell proportion content is low, cell surface marker expresses new antibodies that is more weak, that use first, with needing often kind of labelling to prepare all separately monospecific antibody labelling pipe during tense marker multiclass mark.

10) anti-mouse Gr1-FITC antibody, anti-mouse CD11b-APC antibody is added, 4 DEG C of reaction 30min;

11) 3ml 1 × PBS cleans once, as needed sorting, directly goes up machine afterwards with PBS is resuspended; As needed to analyze, detect after the formaldehyde PBS with 1% is fixing, fluorescence staining retains 48h at most, stream data FlowJo software analysis.

Two, the preparation of colon's single cell suspension and detection

1) get mice colorectal carcinoma, cut open along vertical diameter shears, PBS cleaning foreign material wherein, and clean up.

2) colorectal carcinoma cleaned up is put into 4ml tubule, add about 2-3ml DMEM culture medium, be cut into rotten shape.

3) colorectal carcinoma shredded is transferred in 50ml beaker, adds DMEM culture medium, make cumulative volume be 10ml.Add Collagenase A, final concentration is 1mg/ml; DNase I, final concentration is 0.2mg/ml, 37 DEG C of stirring at low speed 30min.

4) the colorectal carcinoma screen filtration will be stirred, discards foreign material, liquid hold-up, proceeds in 50ml centrifuge tube.

5) the centrifugal 6min of 1200r, retains precipitation.

6) PBS cleans 2 times, and with 70um BD nylon membrane filtration once, remove larger tissue.

7) precipitation PBS is resuspended, grouping adds in streaming pipe, often pipe 100ul.

8) anti-CD16/32 blocking antibody is added, 4 DEG C of reaction 10min, the unspecific staining of removing mouse immune cell surface.

9) lucifuge adds the antibody that needs detect, and adds anti-mouse Gr1-FITC antibody, anti-mouseCD11b-APC antibody, 4 DEG C of reaction 30min.

10) 3ml 1 × PBS cleans once, as needed sorting, directly goes up machine afterwards with PBS is resuspended; As needed to analyze, detect after the formaldehyde PBS with 1% is fixing, fluorescence staining retains 48h at most, stream data FlowJo software analysis.

The cell surface marker that the MDSC in different germline source expresses is different, and it is CD11b that the MDSC in Mice Body has surface marker ⁺gr1 ⁺.

In peripheral blood, the ratio testing result of MDSC cell as shown in Figure 3.

In Fig. 3 and Fig. 4, normal represents normal mouse.

Fig. 3 shows, compared with normal mouse, when the IBD of AD mice falls ill, peripheral blood MDSC ratio raises by 0.37% ± 0.031% is 2.567% ± 0.182% (n=3, P<0.01).

In colon, MDSC ratio testing result as shown in Figure 4.

Fig. 4 shows, compared with normal mouse, the MDSC ratio that the colon site of AD mice infiltrates is 2.057% ± 0.046% (n=3, P<0.01) by 0.1% ± 0.012% rising.

Above result shows, suffers from inflammatory bowel bone marrow cells in mice impaired development and is raised out bone marrow in advance, cause the immature MDSC occurring higher proportion in peripheral blood, and finally raised to mouse Colon site of pathological change.

Embodiment 3, G-CSF affect propagation and the chemotactic of MDSC

Embodiment 1 and embodiment 2 show, (process from AD1 to AD3 is referred to in CAC evolution at IBD, essence is the whole Induction Process of model mice), colon epithelial cell secretion G-CSF, the ratio that MDSC cell infiltrates at peripheral blood and colon simultaneously increases, whether there is mutual relation for the above phenomenon occurred with disease to design following G-CSF and stimulate the in-vitro multiplication of MDSC cell to verify, concrete steps are as follows:

One, G-CSF is on the impact of the propagation of MDSC

Utilize fluidic cell separating method from the bone marrow of C57B/L6 mice, obtain the CD11b of purification ⁺gr1 ⁺mDSC is also divided into two groups, is respectively experimental group and matched group, processes as follows:

Experimental group: be 5x10 in concentration ⁵the CD11b of/ml ⁺gr1 ⁺the recombined small-mouse G-CSF that IL-4 that final concentration is 10ng/ml and final concentration are 100ng/ml is added in MDSC.

Matched group: in concentration for being 5x10 ⁵the CD11b of/ml ⁺gr1 ⁺the IL-4 that final concentration is 10ng/ml is added in MDSC.

The cell of experimental group and matched group is cultivated 24 hours, 48 hours, 72 hours in DMEM culture medium, and to cell counting, add up the proliferation times of two groups of MDSC cells, result as shown in Figure 5.

In Fig. 5, IL-4 represents matched group; IL-4 & G-CSF represents experimental group.

Fig. 5 shows, cellular control unit does not increase, and experimental group MDSC cell under the condition of culture having G-CSF there occurs propagation (n=3, P<0.01).

Two, G-CSF is on the impact of the external chemotactic of MDSC

Utilize fluidic cell separating method from the bone marrow of C57B/L6 mice, obtain the CD11b of purification ⁺gr1 ⁺mDSC is also divided into two groups, is respectively experimental group and matched group, processes as follows:

To be 5x10 ⁵the experimental group of/ml and cellular control unit are all placed in TRANSWELL cell upper strata, the TRANSWELL cell lower floor of experimental group is the DMEM culture medium containing 10ng/ml IL-4 and 100ng/ml recombined small-mouse G-CSF, the TRANSWELL cell lower floor of matched group is the DMEM culture medium containing 10ng/ml IL-4, cultivate the TRANSWELL cell lower floor cell counting to experimental group and matched group after 4 hours, result as shown in Figure 6.

In Fig. 6, IL-4 represents matched group; IL-4 & G-CSF represents experimental group.

Fig. 6 shows, G-CSF can carry out effective chemotactic (n=3, P<0.01) to MDSC cell.

The present embodiment confirms, G-CSF not only has proliferation to MDSC, also has chemotaxis simultaneously.

Embodiment 4, G-CSF Antybody therapy are on the impact of the MDSC ratio of AD mice

In order to verify the G-CSF IBD course of disease in vivo complex environment in can produce same mechanism to MDSC, contrived experiment G-CSF antibody neutralizes the G-CSF in AD Mice Body, and detects the index of correlation of MDSC, and concrete steps are as follows:

Select more than 18g C57BL/6 mice (female), mice once, is divided into two groups after 10 days by lumbar injection AOM (sterilized water for injection is prepared, and injected dose is 12.5mg/kg body weight), is respectively experimental group and matched group, processes as follows:

Experimental group: mice feeds 2.5g/100ml DSS water, the G-CSF antibody prepared by tail vein injection sterilized water for injection according to the dosage of 10ng/kg Mouse Weight for first 2 hours, drinks and within 5 days, counts 120 hours with proinflammatory agent DSS water;

Matched group: control group mice is only fed 2.5g/100ml DSS water and counted 120 hours for 5 days.

After each group of mice drinks proinflammatory agent DSS water, by flow cytometry test experience group, control group mice and normal mouse group (without any process) bone marrow (position, source of MDSC), the ratio of the middle MDSC cell of peripheral blood (the intermediate transportation link of MDSC) and colon's (final aggregate site of MDSC), method is with embodiment 2.

The mice of experimental group, control group mice and normal mouse group is 3.

In each group of mouse bone marrow cells, MDSC ratio as shown in Figure 7.

In Fig. 7, normal represents normal mouse; AD1 represents matched group; AD1-GCSF represents experimental group.

Fig. 7 shows, MDSC cell proportion zero difference in experimental group, control group mice and normal mouse bone marrow, illustrates that G-CSF antibody does not disturb the generation of MDSC.

In each group of mouse peripheral blood, MDSC ratio as shown in Figure 8.

In Fig. 8, normal represents normal mouse; AD1 represents matched group; AD1-GCSF represents experimental group.

Fig. 8 shows, compared with control group mice, experimental mice peripheral blood MDSC cell proportion obviously reduces, and immature medullary cell is blocked by the trend of raising into blood.

MDSC ratio in each group of mouse Colon as shown in Figure 9.

In Fig. 9, normal represents normal mouse; AD1 represents matched group; AD1-GCSF represents experimental group.

Fig. 9 shows, compared with control group mice, the MDSC cell proportion that colon infiltrates also is down to normal level.

The above results shows, after having neutralized G-CSF, MDSC cell reduces at colon local infiltration, reduce owing to raising trend on the one hand, because the MDSC multiplication capacity having infiltrated colon local reduces on the other hand, this experiment in vivo conclusion is consistent with in vitro study, oppositely demonstrates G-CSF has short propagation effect to MDSC.

The MDSC of embodiment 5, colon site finally causes epithelial vicious transformation

Under the stimulation of Th17 cell, colon epithelial cell can be raised MDSC by secretion G-CSF, and a large amount of results of study shows, the MDSC cell infiltrated in tumor tissues can play negative immune regulatory function to the lethal immunocyte of multiple antitumor.

Recent studies have found that, the proinflammatory inflammation factors such as IL-1 β, IL-6 and iNOS can maintain inflammatory microenvironment in body, and normal cell vicious transformation can occur under the long term of these proinflammatory inflammation factors.Therefore, MDSC cell is raised the vicious transformation that can take part in normal colon epithelial cells to colon site by secreting proinflammatory inflammation factor.In order to verify that this supposes, design is tested as follows:

One, CD11b is obtained by fluidic cell separating method purification from normal mouse bone marrow ⁺gr1 ⁺mDSC cell, arranges experimental group and matched group, and the process of each group is as follows:

Experimental group: cultivate CD11b in the DMEM culture medium containing 10ng/ml IL-4 and 100ng/ml recombined small-mouse G-CSF ⁺gr1 ⁺(concentration is 5x10 to MDSC ⁵/ ml);

Matched group: cultivate CD11b in the DMEM culture medium containing 10ng/ml IL-4 ⁺gr1 ⁺(concentration is 5x10 to MDSC ⁵/ ml).

Two, the cell culture of step one was collected after 48 hours, extract the total serum IgE of each group of cell, and reverse transcription is cDNA, take cDNA as template, the primer of answering with IL-1 β, IL-6 or iNOS gene pairs is for primer, carry out RT-PCR, detect the expression of IL-1 β, IL-6 and iNOS from gene level, with β-actin for reference gene.

Primer sequence is as follows:

IL1β-F：5’-GCCCATCCTCTGTGACTCAT-3’；(SEQ ID No.3)

IL1β-R：5’-AGGCCACAGGTATTTTGTCG-3’；(SEQ ID No.4)

IL6-F：5’-GCTAAGGACCAAGACCATCCAAT-3’；(SEQ ID No.5)

IL6-R：5’-GGCATAACGCACTAGGTTTGC-3’；(SEQ ID No.6)

iNOS-F：5’-CACCTTGGAGTTCACCCAGT-3’；(SEQ ID No.7)

iNOS-R：5’-ACCACTCGTACTTGGGATGC-3’；(SEQ ID No.8)

β-actin-F：5’-AGCCATGTACGTAGCCATCC-3’；(SEQ ID No.9)

β-actin-R：5’-CTCTCAGCTGTGGTGGTGGA-3’。(SEQ ID No.10)

The relative expression quantity testing result of proinflammatory inflammation factor IL-1 β, IL-6 and iNOS of experimental group and matched group as shown in Figure 10.

In Figure 10, IL-4 represents matched group; IL-4 & G-CSF represents experimental group.

Figure 10 shows, compared with matched group, the ability to express of IL-6 and iNOS significantly improves (n=4, P<0.01).

This result support is supposed above, and namely MDSC cell STAT-3 path under the stimulation of G-CSF is activated, and proinflammatory inflammation factor IL-6 and iNOS of long-term secretion acts on colon epithelial cell, lures that vicious transformation occurs for it into.

Embodiment 6, G-CSF antibody can be used as the targeted therapy means of colitis, CAC (colitis dependency colorectal cancer)

The above result of study of comprehensive analysis, can release and stop IBD to be interrupt the vicious cycle of colonic epithelia tissue inflammation to the key that CAC develops.Th17 cell is belonged to the comparatively earliest events of the IBD course of disease by raising colon, and the most patients being diagnosed as IBD in actual clinical has the infiltration of a large amount of Th17 cell, therefore selects to block comparatively difficulty to Th17 cell.MDSC cell is the direct factor causing colon epithelial cell generation vicious transformation, is also the event in comparatively late period in IBD development, is easy to control, so select to block MDSC cell.In conjunction with result, select to neutralize G-CSF, block inflammatory phase colon raising MDSC, reduce the MDSC ability of cell proliferation and proinflammatory inflammation factor secretion capacity that have infiltrated.

One, more than 18g C57BL/6 mice (female) is selected, mice once, is divided into two groups after 10 days by lumbar injection AOM (sterilized water for injection is prepared, and injected dose is 12.5mg/kg body weight), be respectively experimental group and matched group, process as follows:

Experimental group: mice feeds 2.5g/100ml DSS water, the G-CSF antibody prepared by tail vein injection sterilized water for injection according to the dosage of 10ng/kg Mouse Weight for first 2 hours, drinks proinflammatory agent DSS water and counts 120 hours for 5 days.Recover normal water after DSS drinking-water, mice drinks the DSS water of two circulations (each circulation recovers normal water after DSS drinking-water in continuous 5 days, then mice has a rest 14 days) again after having a rest 14 days.

Matched group: control group mice only feeds 2.5g/100ml DSS water, 5 days meters 120 hours.Recover normal water after DSS drinking-water, mice drinks the DSS water of two circulations (each circulation recovers normal water after DSS drinking-water in continuous 5 days, then mice has a rest 14 days) again after having a rest 14 days.

Often organize each 5 of mice.

Complete after first round DSS drinks water and mouse Colon tissue inflammation level is assessed.

First round DSS rear employing Immunohistochemical Method of having drunk water detects the infiltration ratio (Gr1 labelling) of G-CSF Antibody on Mouse MDSC and the impact of the expression of proinflammatory inflammation factor iNOS in colon.

G-CSF Antybody therapy postcolon MDSC Infiltrating testing result as shown in figure 11.

In Figure 11, A is MDSC Infiltrating under control group mice AD1 Colon and rectum mirror, and B is MDSC Infiltrating under experimental mice AD1 Colon and rectum mirror.

Figure 11 shows, after G-CSF Antybody therapy, MDSC obviously reduces in the infiltration of colon.

G-CSF Antybody therapy postcolon organizes iNOS expression testing result as shown in figure 12.

In Figure 12, A is iNOS expression under control group mice AD1 Colon and rectum mirror, and B is iNOS expression under experimental mice AD1 Colon and rectum mirror.

Figure 12 shows, after G-CSF Antybody therapy, iNOS also significantly reduces in the expression of colon, illustrates that degree of inflammation alleviates.

Three, more than 18g C57BL/6 mice (female) is selected, lumbar injection AOM (prepare by sterilized water for injection, injected dose is 12.5mg/kg body weight) once, after 10 days, mice is divided into two groups, be respectively AD3-anti-GCSF, AD3-IgG and matched group (AD3 group), process as follows:

AD3-anti-GCSF group: mice feeds 2.5g/100ml DSS water, the G-CSF antibody prepared by tail vein injection sterilized water for injection according to the dosage of 10ng/kg Mouse Weight for first 2 hours, drinks proinflammatory agent DSS water and counts 120 hours for 5 days.Normal water is recovered after DSS drinking-water, mice drinks two DSS water circulated again after having a rest 14 days (each circulation drank DSS water before 2 hours by former dosage tail vein injection G-CSF antibody, recover normal water after continuous 5 days DSS drinking-water, mice has a rest 14 days again).

AD3-IgG group: mice feeds 2.5g/100ml DSS water, the rabbit anteserum IgG prepared by tail vein injection sterilized water for injection according to the dosage of 10ng/kg Mouse Weight for first 2 hours, drinks proinflammatory agent DSS water and counts 120 hours for 5 days.Normal water is recovered after DSS drinking-water, mice drinks two DSS water circulated again after having a rest 14 days (each circulation drank DSS water before 2 hours by former dosage tail vein injection rabbit anteserum IgG, recover normal water after continuous 5 days DSS drinking-water, mice has a rest 14 days again).

Matched group (AD3 group): control group mice only feeds 2.5g/100ml DSS water, 5 days meters 120 hours.Recover normal water after DSS drinking-water, mice drinks the DSS water (each circulation recovers normal water after DSS drinking-water in continuous 5 days, and mice has a rest 14 days again) of two circulations again after having a rest 14 days.

Often organize each 3 of mice.

Detect the colon situation of each group of mice after completing third round DSS drinking-water, each group mouse Colon gross anatomy result as shown in figure 13.

In Figure 13, A is each group of mice Colon and rectum general condition, and B is each group of mice Colon and rectum length statistics.

In Figure 13 A, from top to bottom, Article 1 is do not make the Colon and rectum of the normal mouse of any process (Normal, C57BL/6 mice (female)); Article 2 is the Colon and rectum of AD3-anti-GCSF group mice, the visible a small amount of tumor tuberosity of enteric cavity; Article 3 is the Colon and rectum of AD3-IgG group mice, and naked eyes visual tumors tuberosity is comparatively large, and quantity is more, and intestinal wall thickens, contraction in length, and mucosal fold is uneven; Article 4 is the Colon and rectum of AD3 group mice, and enteric cavity changes and changes similar (n=3, P<0.01) to IgG group mice enteric cavity.

Figure 13 B shows, from angle of statistics, AD3-IgG group and AD3 group mice Colon and rectum contraction in length, AD3-anti-GCSF group mice Colon and rectum length has recovery, and there is statistical significance (n=3, P<0.01), illustrate that AD3-anti-GCSF group mice degree of inflammation alleviates, tumor is formed suppressed.

Each group of mice colorectal carcinoma tuberosity quantity statistics result as shown in figure 14.

Figure 14 shows, from angle of statistics, AD3-IgG group and AD3 group mice colorectal carcinoma tuberosity quantity more than AD3-anti-GCSF group mice, and have statistical significance (n=3, P<0.01), illustrate that anti-GCSF treatment can be formed by Tumor suppression.

Claims (10)

1. prevent and/or treat a test kit for inflammation or tumor, this test kit comprises the antagonist of granulocyte colony-stimulating factor.

2. test kit according to claim 1, is characterized in that: described tumor is the tumor that inflammation is relevant;

The tumor that described inflammation is correlated with is the tumor from diseases associated with inflammation development.

3. test kit according to claim 1 and 2, is characterized in that: described inflammation is alimentary canal inflammation;

Described tumor is digestive tract tumor.

4., according to the arbitrary described test kit of claim 1-3, it is characterized in that: described digestive tract is intestinal.

5., according to the arbitrary described test kit of claim 1-4, it is characterized in that: described intestinal is Colon and rectum.

6., according to the arbitrary described test kit of claim 1-5, it is characterized in that: described tumor is colitis dependency colorectal cancer.

7. the application of antagonist in the propagation of T suppression cell and/or the product of chemotactic in preparation suppression marrow sample source of the arbitrary described test kit of claim 1-6 or granulocyte colony-stimulating factor;

Described chemotactic is that the antagonist of granulocyte colony-stimulating factor suppresses the T suppression cell in marrow sample source to move to the direction of granulocyte colony-stimulating factor.

8. the application of antagonist in the product preparing treatment chronic inflammatory disease or prevention of inflammation related neoplasms of the arbitrary described test kit of claim 1-6 or granulocyte colony-stimulating factor.

9. application according to claim 8, is characterized in that: described tumor is the tumor that inflammation is relevant;

The tumor that described inflammation is correlated with is the tumor from diseases associated with inflammation development.

10. application according to claim 8 or claim 9, is characterized in that: described inflammation is alimentary canal inflammation;

Described tumor is digestive tract tumor;

Described digestive tract is specially intestinal;

Described intestinal is specially Colon and rectum;

Described digestive tract tumor is specially colitis dependency colorectal cancer.