CN1420358A - Use of chemotaxin-oid factor-1 in inflammatory damage of organs - Google Patents

Abstract

The use of chemotactic factor-1 in detecting the pathological state is inflammatory injury of sample, preparing its animal model, and preparing the CKLF-1 antibody and antigon of inflammatory injury, in particular, for respiratory tract injury and lung fiberosis is disclosed.

Description

Use of chemokine-like factor-1 in inflammatory injury of organs

Technical Field

The invention relates to application of a chemotactic factor in organ inflammatory injury. In particular to the application of the chemokine-like factor-1 in detecting the pathological state of inflammatory injury of a sample, manufacturing an animal model of inflammatory injury of an organ and preparing CKLF-1 antibody and antagonist for treating the inflammatory injury of the organ, in particular to the application in the airway injury and remodeling of bronchial asthma and pulmonary fibrosis.

Background

Bronchial asthma is a chronic disease that seriously harms human health, and its incidence rate is increasing worldwide. At present, the number of the diseases in the United states reaches 1500 thousands, almost 1/7 children in the United kingdom suffer from asthma, and about tens of millions of patients exist in China. Therefore, the study and prevention of asthma is an urgent and difficult task.

During the past decade, asthma research has focused on studying the mechanisms that trigger inflammatory infiltration of airway mucosa, such as inflammatory cell adhesion, rolling, chemotaxis, migration, local infiltration, inflammatory injury caused by various inflammatory mediators such as histamine, lipid mediators and reactive oxygen, which are released by respiratory burst and released by various pro-inflammatory factors secreted by immunocompetent cells. Bronchial asthma is clearly defined in the compendial document "global asthma control strategy" made by the WHO in 1995 together with the american heart-lung-blood study as: "chronic airway inflammation involving a variety of cells, particularly eosinophils, mast cells, lymphocytes, and thus leading to airway hyperresponsiveness and airway obstruction". The document also states that "such airway obstruction can be partially reversed or self-reversed by treatment. "(Global protocol for analysis and prediction NHLBI/WHO workhop report: the definition of analysis in Global, 1995, 6-7).

However, a large body of clinical data suggests: the clinical manifestations of some asthmatic patients cannot be explained by simple airway inflammation, and some patients have an irreversible airway obstruction even after a normative and reasonable anti-inflammatory treatment. It is now clear that those refractory cases are due to a marked reduction in tracheal diameter following an inflammatory injury to the airway mucosa as a result of an incomplete or excessive repair, including epithelial cell detachment, mucus cell proliferation, submucosal collagen fiber deposition, hypertrophic smooth muscle proliferation, and the like. When inflammatory secretion increases, airway smooth muscle contracts and mucus gland secretion increases, airway obstruction is aggravated, so that clinical symptoms such as continuous dyspnea appear, and the corticosteroid cannot reverse the clinical symptoms. This pathological change in airway structure is known as "airway remodeling".

Bronchial mucosa biopsy and death case autopsy of patients with bronchial asthma also confirmed that a significant structural change of the airways is a pathological feature of bronchial asthma (Reidogton A.E., et al. Thorax, 1997; 52: 310-. For many years, people have tried to copy animal models of structural changes of asthma airways, but all models are inflammatory lesions of bronchial mucosa. For example, allergic asthma models, infectious asthma models, occupational asthma models, etc. all show only obvious inflammatory infiltrates (such as aggregation and infiltration of eosinophils, lymphocytes, macrophages, neutrophils, etc. around bronchi), but lack lesions such as severe damage of airway epithelium, subepithelial collagen deposition, smooth muscle hyperplasia, etc., and thus cannot truly reflect pathological features of asthma.

Some studies have also suggested that certain immunocompetent cells and cytokines are involved in the pathogenesis of asthma, as the expression of multiple cytokines is detected in asthmatic diseased tissues. For example, high levels of cytokines and their receptors (e.g., EGF, TGF- β, GM-CSF, PDGF, ET-1, EGFR) are detected in peripheral blood mononuclear cells, alveolar lavage fluid, and bronchial mucosa biopsies from patients with bronchial asthma. However, due to the limitation of in vivo experiments, the direct relationship between cytokines and airway inflammation and repair, especially the relationship between a single factor and pathological changes of lung bronchi, is rarely reported. Which is clearly important to elucidate the pathogenesis of asthma.

Chemokine-like factor-1 (Chemokine 1, CKLF-1) is a newly discovered Chemokine with GenBank accession AF 096895. CKLF-1 has the characteristics of C-C subfamily chemokines, i.e., the first and second cysteines (Cys) at the N-terminus of the amino acid sequence are closely linked in the Cys-Cys arrangement, i.e., the CC structure. It has been found that a number of tumour cells express CKLF-1, a factor which is chemotactic for neutrophils, monocytes, lymphocytes and which promotes proliferation and colony formation of human low density bone marrow cells (see PCT application PCT/CN 00/00026).

Summary of The Invention

The inventor selects the chemotactic factor-1 from a plurality of cytokines according to clinical data and experimental data accumulated for many years and carries out the research of bronchial asthma. After long-term research and a large number of experiments, the inventor firstly discloses the important function of CKLF-1 on organ inflammatory injury, especially on bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis, and successfully establishes an animal model of airway inflammatory injury and remodeling and pulmonary fibrosis.

The invention discloses application of chemokine-like factor-1 in organ inflammatory injury, especially in bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis.

The invention relates to an in vitro detection method for determining a sample as inflammatory damaged tissue by detecting the over-expression of CKLF-1 in the sample.

In another aspect, the invention discloses a method for preparing an animal model of organ inflammatory injury by using chemokine-like factor-1, in particular to a method for preparing an animal model of bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis.

In another aspect, the invention discloses the use of chemokine-like factor-1 in the preparation of antibodies and antagonists with the function of treating organ inflammatory injury.

According to one aspect of the present invention, the effects of chemokine-like factor-1 on inflammatory injury of organs, particularly on inflammatory injury and remodeling of bronchial asthma airways and pulmonary fibrosis are disclosed.

In one embodiment of the invention, the introduction of CKLF-1 directly mediates airway injury and remodelling-like changes in mice. For example, 2 weeks after CKLF-1 introduction, the mouse airway epithelial cells were significantly hyperemic and edematous, bronchial deformed, and exfoliated epithelium and exudate were visible in the lumen. Subsequently epithelial cilia disappear and goblet cells proliferate (characteristic of altered bronchial asthma airway epithelial phenotype). After 6 weeks, the arrangement of the epithelial structure of the mouse airway is seriously disordered, obvious mucosal folds appear, a large number of epithelial cells are taken out of the bronchial cavity, the basement membrane is thickened, the collagen deposition under the epithelium is carried out, and the smooth muscle is proliferated.

In embodiments, introduction of CKLF-1 also causes pulmonary fibrotic lesions in mice. For example, the lung small blood vessel abnormality, the thickening of the alveolar wall, the accumulation and proliferation of alveolar epithelial cells, neutrophils, macrophages and fibroblasts, and the lung interstitial collagen deposition are caused. Therefore, CKLF-1 is widely involved in the inflammatory injury and repair process of bronchopulmonary, and may play an important role in the pathogenesis of chronic obstructive pulmonary disease and acute respiratory failure.

In addition to pulmonary effects, CKLF-1 has been found to cause degenerative swelling and structural changes in tissues such as liver, spleen, kidney, testis, etc. Therefore, it is reasonable to speculate that CKLF-1 also has pathophysiological effects on other tissues and organs, namely CKLF-1 has the function of causing organ inflammatory injury.

Inflammatory injury refers to tissue cell damage caused by inflammatory cell accumulation activation and release of a large amount of inflammatory mediators, such as capillary permeability increase, plasma exudation, cell swelling and degeneration, function obstruction and the like. Inflammatory lesions occur in different tissues and organs with both a general manifestation of inflammation and different properties.

Accordingly, the present invention relates to an in vitro assay comprising a method for determining the inflammatory injury status of a sample by detecting overexpression of CKLF-1 in the sample relative to a normal control. The sample may be tissue or cells from an organism, such as lung surgically excised tissue, lung bronchial mucosa tissue, peripheral blood mononuclear cells, and the like. The test target may be a CKLF-1 nucleic acid coding sequence or a CKLF-1 protein. Assays for detecting altered levels of CKLF-1 in a sample from a host are well known to those skilled in the art. For example, at the DNA level, nucleic acids for diagnosis can be obtained from sample cells, genomic DNA can be used directly for detection, or PCR amplification can be performed with primers complementary to CKLF-1 nucleic acid sequences prior to analysis. Analytical methods at the protein level include radioimmunoassays, competitive binding assays, Western blot analysis, ELASA assays, and the like.

In a preferred embodiment of the invention, PCR amplification using specific primers for CKLF-1 detected significantly higher CKLF-1 expression in asthmatic PBMCs than in non-asthmatic patients.

In accordance with yet another aspect of the present invention, a method of using CKLF-1 to make an animal model of inflammatory injury, in particular an animal model of asthma airway injury and remodeling and pulmonary fibrosis, is disclosed.

CKLF-1 can be introduced into animals by various known methods to create pathological models of inflammatory lesions. For example, nasal inhalation, oral administration, intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intraarticular injection and infusion methods can be used to establish a pathological model of bronchial asthma airway injury. The injection and inhalation routes are preferred, and the aerosol inhalation route is more preferred.

Aerosol inhalation refers to inhalation of solutes into the bronchi of the lungs by means of an aerosol device, causing the solutes to act directly on the local lung. Since airway inhalation of antigens is the starting route for most asthma, the aerosol inhalation route is very close to the real situation where asthma occurs.

CKLF-1 introduced into animals can be formulated with pharmaceutically acceptable carriers or excipients into compositions for injection or infusion, including sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and powders for constitution in sterile injectable solutions or dispersions just prior to use. For use in compositions suitable for inhalation, CKLF-1 may be compressed or contained in a compressed gas such as nitrogen or a liquefied gas propellant.

The animal used for making the pathological model may be a mammal other than a human, such as a mouse, a rat, a rabbit, and the like. The animals used meet the national standards for laboratory animals.

In a preferred embodiment, the inventor successfully reproduces a mouse animal model similar to bronchial asthma airway injury and remodeling by introducing a eukaryotic expression plasmid pcDI-CKLF1 into a mouse body by adopting an electric pulse naked gene intramuscular injection method, wherein the mouse animal model shows a large amount of lesions such as airway epithelial abscission, goblet cell proliferation, basement membrane thickening, subepithelial collagen deposition, smooth muscle proliferation and lung small blood vessel abnormality. The inventor establishes a pathological model of the pulmonary fibrosis of the mice by the method.

According to another aspect of the invention, the use of CKLF-1 in preparing antibodies and antagonists for treating inflammatory injury of organs is disclosed.

According to the invention, CKLF-1 can be used for preparing and screening antibodies and antagonists with the function of treating organ inflammatory injury. The antibody may be monoclonal or polyclonal. Antagonists include oligopeptides or compounds that bind to CKLF-1 under certain conditions. Antagonists may bind to CKLF-1 and block its activity at the receptor site, or alternatively, may bind to CKLF-1 receptor, thereby preventing CKLF-1 binding to the receptor. Various methods known in the art can be used to prepare antibodies and antagonists to CKLF-1. For example, CKLF-1 protein is injected into animals to obtain CKLF-1 antiserum. The CKLF-1 monoclonal antibody is prepared by a human B cell-hybridoma technology, an EB virus-hybridoma technology and the like.

The advantages and features of the present invention are summarized as follows:

firstly, the important role of CKLF-1 in organ inflammatory injury, especially bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis is disclosed for the first time. The invention not only provides a novel method for detecting organ inflammatory injury, but also provides experimental basis for the deep research of the diseases.

Secondly, the invention successfully establishes a mouse airway inflammatory injury and remodeling model. The model overcomes the defects of the traditional asthma research animal model, has pathological changes such as airway epithelial exfoliation and the like, and can truly reflect the pathological characteristics of asthma, thereby providing a beneficial tool for clarifying the pathogenesis of asthma, researching and preventing asthma.

Third, the experimental data and results disclosed by the invention have profound significance in the study of asthma and related diseases. For example, the present inventors found that (1) human airway epithelial cells and Peripheral Blood Mononuclear Cells (PBMCs) express CKLF-1 in a basal state; (2) the proinflammatory factor TNF-alpha can up-regulate the expression of human airway epithelial cells CKLF-1; (3) eosinophils are not necessarily an essential factor in asthma airway injury and remodeling; (4) aggregation activation of neutrophils is of great significance in asthmatic-like airway injury and remodeling.

Brief Description of Drawings

FIG. 1 shows the expression of human airway epithelial 16HBE cells and peripheral blood mononuclear cells CKLF-1.

FIG. 2 shows the effect of TNF- α on CKLF-1 expression in 16HBE cells.

FIGS. 3A-6B show the effect of CKLF-1 on airway epithelium in mice. Wherein,

FIG. 3A shows the result of hematoxylin-eosin (HE) staining of airway epithelial cells of mice (control) 2 weeks after introduction of pcDI empty plasmid (20X);

FIG. 3B shows HE staining of airway epithelial cells (20X) in mice (experimental group) 2 weeks after introduction of pcDI-CKLF1 plasmid;

FIG. 4 shows HE staining of airway epithelial cells (20X) in mice (experimental group) 3 weeks after introduction of pcDI-CKLF1 plasmid, showing airway epithelial goblet cell proliferation;

FIG. 5A shows HE staining of airway epithelial cells (20X) in mice (experimental group) 6 weeks after introduction of pcDI-CKLF1 plasmid;

FIG. 5B shows HE staining of airway epithelial cells (20X) in mice (experimental) 6 weeks after introduction of pcDI-CKLF1 plasmid, showing evidence of airway mucosal folding;

FIG. 6A shows that mice (control) were stained with Jimsa for a small number of epithelial cells (20X) in bronchoalveolar lavage fluid (BALF) 8 weeks after the introduction of pcDI empty plasmid;

FIG. 6B shows that 8 weeks after introduction of pcDI-CKLF1 plasmid, mice (experimental group) were stained with Giemsa for epithelial cells that shed as colonies in bronchoalveolar lavage fluid (BALF) (20X);

FIG. 7 shows the effect of CKLF-1 on mouse airway basement membrane (HE staining, 20X).

FIG. 8 shows the effect of CKLF-1 on mouse airway smooth muscle (20X).

FIGS. 9A-10B show the effect of CKLF-1 on mouse lung tissue. Wherein,

FIG. 9A shows HE staining of lung tissue of mice (control) 2 weeks after introduction of pcDI empty plasmid (20X);

FIG. 9B shows HE staining of lung tissue of mice (experimental group) (10X) 2 weeks after introduction of pcDI-CKLF1 plasmid;

FIG. 10A shows the results of Mallory modified staining of lung tissue in mice (control) 4 weeks after introduction of pcDI empty plasmid (40X);

FIG. 10B shows the results of Mallory modified staining of lung tissue in mice (experimental) 4 weeks after introduction of pcDI-CKLF1 plasmid (40X), showing collagen fibers.

FIGS. 11A-11B show the effect of CKLF-1 on blood vessels in mouse lung tissue. Wherein,

FIG. 11A shows HE staining of blood vessels in lung tissue of mice (control) 4 weeks after introduction of pcDI empty plasmid (20X);

FIG. 11B shows HE staining of blood vessels of lung tissue of mice (experimental group) 4 weeks after introduction of pcDI-CKLF1 plasmid (20X).

FIGS. 12A-12B show the effect of CKLF-1 on mouse lung tissue macrophages. Wherein,

FIG. 12A shows lung tissue sections (20X) from control mice following trypan blue in vivo injection;

FIG. 12B shows lung tissue sections (20X) from experimental mice after trypan blue in vivo injection.

FIGS. 13A-13B show immunohistochemical detection of CKLF-1 expression in lung tissue. Wherein,

FIG. 13A is an immunohistochemical assay (40X) of CKLF-1 expression in lung tissue of control mice;

FIG. 13B is an immunohistochemical detection of CKLF-1 expression in lung tissue of experimental mice (40X).

FIG. 14 shows CKLF-1 expression in peripheral blood mononuclear cells of normal control group and asthma group.

FIGS. 15A-15B show immunohistochemical detection of bronchial mucosa CKLF-1 protein expression in normal controls and asthmatic patients. Wherein,

FIG. 15A is an immunohistochemical assay of normal control bronchial mucosa CKLF-1 protein expression (hematoxylin counterstain, 40 ×);

FIG. 15B is an immunohistochemical assay of CKLF-1 protein expression in bronchial mucosa from asthmatics (hematoxylin counterstain, 40X).

In order that the invention may be more clearly understood, reference will now be made to the following examples. The examples are not intended to limit the scope of the invention in any way.Description of the preferred embodimentsExample 1 expression of CKLF-1 in human airway epithelial cells and peripheral blood mononuclear cells

And the Effect of TNF alpha on CKLF-1 expression in human airway epithelial cells

To detect whether CKLF-1 is involved in the development of bronchial asthma, it was determined whether CKLF-1 is expressed in inflammatory cells and airway structural cells, and whether CKLF-1 expression is altered under pro-inflammatory factor stimulation.

In this example (1), CKLF-1 expression was detected using normal human peripheral blood mononuclear cells as inflammatory cells and normal human airway epithelial cells 16HBE as airway structural cells. (2) The effect of pro-inflammatory factor TNF-alpha on the expression of CKLF-1 in airway epithelial cells was examined.

1. Culture of normal human airway epithelial cell line 16HBE

Taking 1 tube of a normal human airway epithelial cell strain 16HBE (Southampton), quickly freezing and thawing in a water bath at 37 ℃, and adding 1640 culture solution containing 10% fetal calf serum, 100u/ml penicillin and 100u/ml streptomycin. At 1 × 10⁵Cells/ml seeded at 25cm²Culturing in a culture flask at 37 deg.C in a 5% carbon dioxide incubator. The liquid was changed every 2 days, and the cells were passaged at 1: 3 when they had grown to a substantially confluent state. The cells were digested with 0.125% trypsin for 2 minutes at passage time, and the cells were suspended by blowing with serum-containing medium and centrifuged. At 1 × 10⁵The cells were inoculated in 12-well plates and cultured in DMEM medium containing 10% fetal bovine serum. When cells were 80% confluent to pieces, the supernatant was aspirated and cultured in serum-free medium. After 12 hours the cells were cultured with 1000U/ml TNF-. alpha.stimulation.

2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

Peripheral venous blood was drawn from healthy subjects in 3-4 ml and anticoagulated with heparin. The fresh blood was diluted 1-fold with 4 ℃ pre-chilled physiological saline and mixed well. The diluted blood was added to 2ml of a lymphocyte separation medium (specific gravity 1.077), centrifuged at 2000 rpm for 20 minutes. The middle mononuclear cells were aspirated and added to another tube. The mononuclear cells were washed with 2ml of physiological saline. Centrifuge at 1000 rpm for 10 minutes at 4 ℃. The supernatant was discarded, and 1ml of physiological saline was added for repeated washing. Centrifuge at 2000 rpm for 5 minutes at 4 ℃. And discarding the supernatant, and obtaining the precipitate as the PBMCs.

3. Total RNA extraction and cDNA Synthesis

16HBE and PBMCs total RNA were extracted and cDNA was synthesized by reverse transcriptase (Promega corporation) according to the methods of Gene manipulation technology (first edition) (ed by Wangshen five, published by Beijing university of medicine, and medical university Combined Press, 1991; 46-48) and molecular cloning (second edition) (published by scientific Press, Sunsylvania, J. Sampuk et al) for detecting CKLF-1 expression in normal airway epithelial cells and peripheral blood mononuclear cells.

After TNF-alpha stimulates 16HBE cells for 8 hours, 12 hours and 20 hours, total RNA of the cells is respectively extracted and subjected to RT-PCR (reverse transcription-polymerase chain reaction) to detect the influence of TNF-alpha on the CKLF-1 expression of airway epithelial cells. Only DMEM serum-free culture solution is added to the experimental control group.

PCR amplification of CKLF-1 fragment

(1) Primer and method for producing the same

CKLF-1 amplification primer

An upstream primer: 5'-ATG GAT AAC GTG CAG CCG AAA AT-3'

A downstream primer: 5'-CCG CTC GAG TTA CAA AAC TTC TTT TTT TTC-3'

Beta-actin amplification primer

An upstream primer: 5- "ATG TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3',

a downstream primer: 5-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'.

(2) Reaction system

PCR amplification was performed using cDNAs reverse transcribed from mRNA 8, 12, and 20 hours after stimulation of 16HBE cells, peripheral blood mononuclear cells, and TNF-. alpha.stimulating 16HBE cells, respectively, as templates.

The reaction system comprises: 10 XPCR buffer 2.5ul, 1.25mM dNTP 4ul, upstream, downstream primer each 50pmol, template 2ul, Taq DNA polymerase 1U, adding sterile double distilled water to 25 ul.

Beta-actin amplification was used as a control for the PCR reaction. The fragment amplified from plasmid pcDI-CKLF1 (containing the CKLF-1 open reading frame, constructed below) was used as a control for CKLF-1.

(3) Reaction parameters

Pre-denaturation: denaturation at 94 ℃ for 8 min, adding 0.8. mu.l of Taq enzyme, and denaturation at 94 ℃ for 2 min;

and (3) circulation: denaturation at 94 ℃ for 15 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds,

35 cycles;

extension: 7 minutes at 72 ℃.

PCR product identification and quantitative analysis

After the reaction, the PCR reaction tube was placed in a refrigerator at 4 ℃ for 10 minutes. Subsequently, 6. mu.l of the final reaction product was electrophoresed in 2% agarose gel (containing 0.5. mu.g/ml ethidium bromide) at a constant pressure of 6V/cm for 30 minutes. The gel was observed under an ultraviolet lamp and photographed and measured for fluorescence intensity and relative quantitative analysis on the gel on a UVP dark box type ultraviolet gel image analyzer.

Results

Expression of CKLF-1 in normal human airway epithelial cells and peripheral blood mononuclear cells

As shown in FIG. 1, 838bp of beta-actin was amplified from the reaction tube used as a PCR control. A300 bp fragment (band B) was amplified from plasmid pcDI-CKLF1 as CKLF-1 control using CKLF-1 specific primers; a300 bp fragment was also amplified from 16HBE and PBMCs (band C, D, E). As can be seen, CKLF-1 is highly expressed in human airway epithelial cells, and is also expressed at a certain level in human peripheral blood mononuclear cells.

The airway epithelial cells not only have the functions of defense protection and physical barrier, but also have the function of mediating inflammatory reaction. For example, under the stimulation of environmental factors, airway epithelial cells are activated to secrete a large amount of adhesion molecules and cytokines, and the substances act on airway epithelia to trigger a series of characteristic asthma airway inflammatory responses. The expression of CKLF-1 in airway epithelial cells suggests that this factor may be involved in the inflammatory response of the airways.

In addition, it has been confirmed that asthma occurs with the involvement of inflammatory cells, and bioactive substances secreted from inflammatory cells may mediate the occurrence of inflammation. CKLF-1 expression in inflammatory cells (e.g., peripheral blood mononuclear cells) suggests: when asthma occurs and inflammatory cells gather in airway mucosa, the inflammatory cells may express and release CKLF-1 and participate in the generation of asthma inflammation.

2. Effect of pro-inflammatory factor TNF- α on CKLF-1 expression in airway epithelial cells.

As shown in figure 2, after 8 hours and 12 hours of stimulation of proinflammatory factor TNF-alpha, CKLF-1 expression of human airway epithelial cells is not obviously changed; after 20 hours of stimulation, the CKLF-1 expression level of the human airway epithelial cells is obviously increased.

Activation of TNF- α is one of the important features of asthma pathogenesis. TNF-alpha activation triggers the secretion of a series of pro-inflammatory factors, which form a cytokine network with complex action and wide effect, and finally lead to the characteristic reaction of inflammatory injury and remodeling of asthma. The experiment shows that the airway epithelial cell CKLF-1 can be activated by TNF-alpha, so that the CKLF-1 can participate in airway inflammatory reaction of asthma through a cytokine network. The inventors have also found that CKLF-1 expression is not immediately enhanced following TNF- α stimulation by the pro-inflammatory factor. Indicating that the up-regulation of CKLF-1 expression depends on the sustained action of TNF-alpha, or that its expression is enhanced by the action of other cytokines that are up-regulated upon stimulation with TNF-alpha, i.e., the response of CKLF-1 to TNF-alpha needs to go through a certain period of time.

In combination with the above results, CKLF-1 may be involved in asthma airway inflammation and remodeling lesions.

Example 2 CKLF-1 mediated Lung bronchopathy in mice

CKLF-1 is introduced into mice by using an electric pulse naked gene injection method. Pathological changes in the lung bronchi of mice were observed 2 to 8 weeks after CKLF-1 injection to detect the direct pathological effects of the single factor CKLF-1 on the lung bronchi.

1. Plasmids

(1) Plasmid construction

Plasmid pcDI: the BglI-kpnI fragment from pcDNA3 (Invitrogen) was replaced with the BglI-kpnI fragment from PcI (Promega) to give a pcDI eukaryotic expression plasmid.

Plasmid pcDI-CKLF-1: EcoRI is used for digesting PGEM-T-Easy-CKLF-1 plasmid, ORF fragment of CKLF-1 is released, the plasmid is connected to pcDI plasmid which is digested by EcoRI and treated by CIP, and eukaryotic expression plasmid pcDI-CKLF-1 of CKLF-1 which is inserted in the forward direction is obtained through digestion and identification.

(2) Mass extraction and purification of plasmids

Positive strains were streaked and incubated overnight at 37 ℃. The monoclonal colonies were picked and placed in 3ml LB medium and antibiotics at 37 ℃ for 8 hours with shaking at 200rpm, and the bacterial liquid was transferred to 2000ml LB medium containing antibiotics with shaking at 37 ℃ for overnight at 200 rpm. The procedures and procedures for Plasmid purification and endotoxin treatment were carried out strictly according to the EndoFree Plasmid Giga Kit (IAGEM-tip10000, Hilden, Germany) protocol.

2. Laboratory animal

A total of 140 BALB/c healthy mice (purchased from laboratory animals of the university of Beijing university's basic medical college) aged 4-6 weeks, half male and female, were randomly divided into 5 groups (see Table 1). Half of each group was experimental mice and half was control mice. The experimental mice are 2w, 3w, 4w, 6w and 8w after being introduced into pcDI-CKLF-1 and corresponding control mice respectively. The test results are abbreviated as 2w group, 3w group, 4w group, 6w group, 8w group and control group.

TABLE 1 CKLF-1 transgenic laboratory mice group (only)

Group pcDI-CKLF1 pcDI control

2w groups 1010

3w group 2020

4w groups 2020

6w groups 1010

8w groups 1010

In vivo transfer of CKLF-1 Gene

The plasmid after sterile treatment is dissolved in physiological saline, the concentration is adjusted to 1 mug/mug, 100 mug of plasmid solution is injected into the femoral four-head muscle of the thigh on one side of A laboratory mouse by A micro-injector, 100ul of pcDI empty plasmid (100ug) is injected into the same part of A control mouse, two electrodes are inserted into the injection part immediately after injection, the electrodes are made of stainless steel needles, the diameter is 0.08mm, the length is 10mm, the insertion depth is 5mm, the electrode spacing is 5mm, the electrodes are connected to A Thomson gene therapeutic apparatus (TS-A), and direct current square wave stimulation is given, wherein the parameters are field intensity of 200v/cm, pulse wave width is 40ms, stimulation frequency is 1Hz, and action time is 6 seconds. Then cervical dislocation is killed by stages and batches. 4-6 of 10 mice per group were used for morphology observation, the remainder were bronchoalveolar lavages.

4. Detection of physiological indicators in animals following introduction of CKLF-1

(1) Observation of animal respiratory rate, body weight and lung coefficient

Recording the respiratory frequency of 3, 4W experimental mice and control mice, weighing the body weight, picking the lung to weigh the total weight, and calculating the lung coefficient. The lung coefficient is: lung weight g/body weight g × 100.

(2) Trypan blue in vivo injection

Weighing appropriate amount of trypan blue powder, dissolving in normal saline to prepare 1% trypan blue water solution, boiling for 10 min, and sterilizing. Selecting mice 3-4 w after CKLF-1 introduction, injecting sterilized trypan blue solution into abdominal cavity of mice at a dose of 2-5ml/kg by aseptic operation method, and repeating the injection once after 48 hours. After another 24 hours, the mice were sacrificed and lung tissue was removed and immediately fixed.

(3) Preparation of pathological light microscope specimen

Fresh lung tissue was immediately placed into the Bouin solution for more than 48 hours, dehydrated with 70%, 80%, 90%, 95% ethanol for 12 hours, and the solution was changed 3 times during each dehydration. Then dehydrated for 2 hours by using absolute ethyl alcohol, and the liquid is changed for 2 times in the period. The solution was then cleared with xylene for 45-60 minutes, during which time the solution was changed 1 time. Wax dipping is carried out for one hour at 65 ℃. The tissues after being waxed are embedded in paraffin at 65 ℃ and are sliced continuously by 4 mm. Staining with HE and modified Mallory, and microscopic observation.

(4) Preparation of Electron microscopy specimen

Fresh lung tissue is immediately fixed in a newly prepared 2.5% glutaraldehyde solution at 4 ℃, the air in the lung is evacuated in vacuum, and the specimen is prepared according to the conventional electron microscope. And positioning the semi-thin slice and then performing ultrathin slicing. The double staining of uranium acetate and lead citrate is carried out, and the observation is carried out under a Hitachi H-600 transmission electron microscope.

(5) Bronchoalveolar lavage

The animals were sacrificed by cervical dislocation grouped at 3, 4, 6, 8 weeks, tracheotomy, and lavage with 1640 culture solution for 3 times each time, 0.8 ml. Lavage recovery > 80% and cell counts are performed. The pellet smear was HE stained or giemsa stained.

(6) Preparation of lavage fluid cell smear, cell counting and sorting

Precipitating the cells in BALF obtained by centrifugation, diluting the precipitated cells with 1ml of physiological saline, taking a part of the precipitated cells for counting, calculating the number of the cells per milliliter, and further calculating the total number of the cells; and meanwhile, smearing, namely immediately and quickly drying the slide by using a cold air baffle of an electric air blowing cylinder, and carrying out HE (high intensity hematoxylin) staining or giemsa staining after the cells are dried and fixed. And (4) classifying the cells under an optical microscope, counting 500 cells, calculating the percentage of the cells, and multiplying the total number of the cells by the percentage to obtain the absolute number of the cells.

Results

CKLF-1 introduced by electric pulse naked gene intramuscular injection successfully obtains expression in local injection to generate corresponding protein, and CKLF-1 protein reaches lung along with blood circulation, thereby causing a series of pathological changes of lung bronchus (see details below). Immunohistochemistry showed that CKLF-1 was predominantly expressed in airway epithelial cells and sub-epithelial mesenchymal cells, also confirming the success of in vivo transfer of CKLF-1.

1. Respiratory rate and weight changes in mice introduced with CKLF-1

As shown in table 2, the mice introduced with CKLF-13 at 4 weeks had increased activity in the cages, increased respiratory rate and decreased body weight compared to the control.

TABLE 2 changes in respiratory frequency and body weight of mice 3 and 4 weeks after CKLF-1 introduction

Group breathing frequency (b/min) n 8 body weight (g) n 32

107.8 +/-20.7 of an experimental group is in electric mark of 22.93 +/-1.88

Control group 81.7 + -15.826.09 + -1.57

Compared with a control group, the color is as follows: p is less than 0.05

Effect of CKLF-1 on mouse Lung tissue weight

As shown in Table 3, the lung volume of the mice was enlarged and some lung tissues were significantly congested compared with the control at 3 or 4 weeks after introduction of CKLF-1 by visual observation. The lung coefficient is increased by 39.2 percent compared with the control group.

TABLE 3 Effect of CKLF-1 on mouse Lung tissue weight

Lung coefficient of each group

The experimental group n is 160.78 +/-0.10 in color

Control group n-160.56 + -0.07

Compared with a control group, the color is as follows: p is less than 0.05

Effect of CKLF-1 on cells in mouse bronchoalveolar lavage fluid (BALF)

As shown in table 4, although the total number of BALF cells in the mice in the control group was higher than that in other data, the total number of BALF cells in each experimental group was significantly higher than that in the control group, which was more than 200% higher than that in the control group, and the total number of BALF cells was maintained for at least 8 weeks.

TABLE 4 change in total BALF cell count after CKLF-1 introduction into mice (× 10)⁶)

Group control group experiment group

3w 4.3±2.2 11.6±3.8_※

4w 4.5±2.9 10.7±3.5_※

6w 3.8±2.6 9.9±2.9_※

8w 3.3±1.7 9.5±3.2_※

Compared with the control group: p is less than 0.01, and the difference among experimental groups has no statistical significance.

As shown in Table 5, the classification of the cells in BALF of the experimental group showed that the increase of the epithelial cells was significant in a large number of cells, which was 8.1 times that of the control group. Lymphocytes and neutrophils were also significantly increased compared to the control group. Eosinophils (Eos) were not seen in both groups.

TABLE 5 cytological changes in mouse BALF 3 weeks after CKLF-1 introduction (× 10)⁶)

Total group cell count Eos epithelial cells L + N

Control group 4.3 + -2.200.8 + -0.200.7 + -0.2

11.6 +/-3.8 of the experimental group is in the form of 06.5 +/-2.6 of the auxiliary color, 2.3 +/-0.7 of the auxiliary color

Note: l: lymphocyte N: neutrophils

Compared with the control group: p is less than 0.01

4. Effect on mouse airway epithelium

(1) Congestion, edema and exfoliation of airway epithelial cells in mice 2 weeks after CKL-1 introduction

As shown in FIG. 3A, the airway epithelial cells of the control mice (injected with pcDI empty plasmid) were well-aligned and free from shedding, and there was no significant abnormality in the surrounding alveolar structure and pulmonary vessels. Mice in the experimental group (injected with pcDI-CKLF1) had congestion and edema of airway epithelial cells, bronchial deformity, and exfoliated epithelium and exudate were visible in the lumen, and congestion and edema of peripheral lung tissue were evident (FIG. 3B).

(2) 3 weeks after CKLF-1 introduction, mouse airway epithelial goblet cell proliferated

As shown in fig. 4, mouse airway epithelial goblet cells proliferated (characteristic of altered bronchial asthma airway epithelial phenotype), and a large amount of secretions, exudates, and lumen were significantly narrowed. The proliferating goblet cells were enlarged at 40 Xfield and the goblet cells secreted mucinous particles were visualized. The examination by electron microscope shows that epithelial cell cilia are almost extinct.

(3) 6 weeks after CKLF-1 introduction, airway epithelium is severely damaged and structurally arranged disorganized

As shown in fig. 5A, 3 bronchi arranged in parallel had no normal morphology, cell disorganization, cell junction destruction, and a large number of epithelial cells separated from the basement membrane and fell off the lumen, almost blocking the lumen. As shown in FIG. 5B, the parallel bronchial epithelial cells were disorganized and exfoliated. Obvious folds of the airway mucosa (an important feature of asthmatic airway lesions) are visible.

(4) 2-8 weeks after CKLF-1 introduction, airway epithelial cells in the alveolar lavage fluid are exfoliated

2 weeks after CKLF-1 introduction, large numbers of exfoliated airway epithelial cells were detected in alveolar lavage fluid. After 3 weeks the shed cells appeared as sheets or colonies until week 8, with the shed cells being dominated by columnar epithelial cells. FIG. 6A shows a small number of epithelial cells in BALF 8 weeks after introduction of pcDI (control); FIG. 6B shows epithelial cells shed as colonies in BALF after introduction of pcDI-CKLF 18 weeks.

Effect of CKLF-1 on mouse airway basement membrane

Normally, basement membranes are difficult to see by light microscopic observation of the mouse airway. However, 6 weeks after CKLF-1 transfer, the airway basement membrane of mice was seen to be thickened. Figure 7 shows a deformed bronchus, severely damaged epithelium. The strip-shaped pink uniform structure close to the epithelium is a basement membrane.

Effect of CKLF-1 on mouse airway smooth muscle

Under light microscope observation, the small bronchus of normal guinea pig has obvious smooth muscle structure around the bronchus, and the structure is arranged in a ring shape around the bronchus, while the smooth muscle structure is difficult to be seen in normal mice. Furthermore, 4 weeks after CKLF-1 introduction, mouse bronchial smooth muscle cells proliferated. FIG. 8 shows a deformed bronchus, with coarse folds of the tracheal mucosa. The tissue that is significantly thickened under the mucosa is proliferating smooth muscle cells.

Effect of CKLF-1 on mouse Lung tissue

2 weeks after pcDI introduction, there was no significant abnormality in alveolar architecture in control mice (FIG. 9A); 2 weeks after the introduction of pcDI-CKLF1, normal alveolar structures around the lungs of the mice disappeared, alveolar walls became thicker, alveolar septal cells accumulated and proliferated, and pulmonary interstitial congestion and edema were observed in the experimental group (FIG. 9B).

3 weeks after CKLF-1 introduction, the alveolar wall of the mouse is obviously thickened, a large number of immunocompetent cells are gathered and proliferated, and the increased cells comprise alveolar epithelial cells, fibroblasts, macrophages and neutrophils.

Mallory modified staining (collagen fibers can be visualized) shows: compared to the control group (FIG. 10A), the mouse lung interstitium proliferated much collagen fibers 4 weeks after CKLF-1 introduction (FIG. 10B).

Effect of KLF1 on pulmonary tissue vessels in mice

Bronchial epithelium was damaged and basement membrane was thickened in mice 4 weeks after CKLF-1 introduction compared to the control group (fig. 11A). The small tracheal tube wall was significantly thickened, collagen was deposited, and the hyaline degeneration occurred (FIG. 11B).

Effect of CKLF-1 on mouse Lung tissue macrophages

Mice are sacrificed after trypan blue living body is injected for a certain time, lung specimens are taken and immediately fixed, and the mice are routinely sliced and observed under a microscope. The blue particles engulfed by the lung scaffold and macrophages are seen in FIGS. 12A and 12B. Blue particles were significantly increased in the lungs of mice introduced with CKLF-1 compared to the control (fig. 12A), indicating macrophage proliferation and aggregation. The trypan blue particles in the mouse lung were both abundant and bulky, indicating that the lung tissue macrophages were significantly increased in number and enhanced in activity (fig. 12B).

10. Immunohistochemical analysis of lung tissue CKLF-1 expression

Control mice had no specific positive reaction, and the dark brown particles in the vessels were non-specifically stained (fig. 13A); the yellow brown particles in the airway epithelial cells of the mice in the experimental group are strongly positive in CKLF-1 immunoreaction, CKLF-1 is mainly expressed in cytoplasm of the airway epithelial cells and interstitial cells at the lower parts of the epithelium, and the cells have no specific immunoreaction in nucleus and are still blue (figure 13B).

The above results are fully documented: CKLF-1 is introduced into mice, and directly mediates airway injury and remodeling-like changes seen in bronchial asthma airway mucosa biopsy and autopsy. The inventors speculate that the mechanism by which CKLF-1 mediates airway injury and remodeling lies in: (1) directly stimulating the proliferation of airway epithelial cells, smooth muscle cells, and the like (direct action); (2) as a chemokine, it chemotaxis and activates immune cells, and the activated immune cells secrete a large amount of cytokines, thereby causing a series of pathological changes (indirect action).

Besides causing airway injury and remodeling-like change, the CKLF-1 introduced into the body of the mouse also causes pulmonary alveolar congestion and edema, alveolar wall thickening, lung interstitial cells such as fibroblasts and macrophages are aggregated and activated, and the lung interstitial collagen is deposited to form fibrosis-like change. Therefore, CKLF-1 is also involved in the development of pulmonary interstitial fibrosis.

Furthermore, the inventors have found that: (1) eosinophils (Eos) are not necessarily essential factors in the injury and remodeling of the airway of asthma. Previous studies suggest that Eos infiltration is a major feature of asthmatic airway inflammation, and the inventors have seen no shedding of Eos in bronchoalveolar lavage fluid and no mucosal Eos infiltration in bronchopulmonary histopathological sections within 2 to 8 weeks after CKLF-1 was introduced into mice. These results suggest that Eos is not an indispensable factor in airway injury and remodeling, and that treatment for Eos alone may be lopsided. (2) Neutrophils are an important factor in the pathogenesis of asthma. The inventor proves that the chemotactic activity of neutrophils in the lung of CKLF-1 is combined with the lung bronchial lesion caused by CKLF-1, and the neutrophil aggregation activation caused by CKLF-1 is considered to have important significance in CKLF-1 mediated asthma-like airway injury and remodeling.

Example 3 expression of peripheral blood mononuclear cell CKLF-1mRNA in asthmatic patients

And detection of bronchial mucosa CKLF-1 protein

The expression of CKLF-1mRNA of peripheral blood mononuclear cells of the asthmatic patients is observed by an RT-PCR method, and the expression of the protein level of CKLF-1 protein of bronchial mucosa of the asthmatic patients is detected by an immunohistochemical method. To verify whether CKLF-1 is involved in the pathogenesis of asthma by clinical studies.

1. Expression of CKLF-1mRNA from peripheral blood mononuclear cells of asthmatic patients

(1) Test object

Bronchial asthma group: 12 men and 9 women. Mean age 30 ± 7.2 years. And (3) inclusion standard: 1. meets the diagnosis standard of asthma of mild or moderate degree established by the Chinese medical society of 1997. 2. No glucocorticoid or other anti-inflammatory drug was used for three weeks. 3. There was no respiratory infection recently. 4. And in the skin allergen test, more than two allergens are positive. 5 recent wheezing episodes.

Normal control group: 21 volunteer subjects: 11 men and 9 women, age 29 + -8.2 years. It has no asthma, allergic rhinitis, other allergic diseases, tumor and chronic inflammation.

(2) PCR detection of CKLF-1 expression

Peripheral venous blood 3-4 ml and heparin anticoagulation were collected from asthmatic patients and healthy subjects. The method for isolating peripheral blood mononuclear cells is as described above.

Extracting PBMCs cell total RNA, and performing reverse transcription to synthesize cDNA.

And (3) carrying out PCR reaction by using the PBMCs reverse transcription product as a template. The amplification primers, reaction system and parameters were as described above.

Results

As shown in FIG. 14, the expression level of CKLF-1 in PBMCs of asthmatic patients was significantly higher than that of healthy persons. The ratio of CKLF-1/beta-actin in the asthmatic group in the symptom phase is significantly higher than that in the normal people, and is respectively 0.815 +/-0.103 and 0.561 +/-0.0862 (P is less than 0.05) (see table 6).

TABLE 6 CKLF-1/β -actin ratio X + -SD for asthma versus control PBMC

Group number CKLF-1/beta-actin

210.815 +/-0.103 of asthma group

Control group 210.561. + -. 0.0862

Compared with the control group: p is less than 0.05

2. Pathological observation of bronchial mucosa and immunohistochemical analysis of CKLF-1 of asthmatic patients

(1) Test object

Asthma group: 4 men and 4 women, with a mean age of 30 ± 7.2 years. And (3) inclusion standard: 1. meets the diagnosis standard of asthma of moderate or severe state of illness established by the Chinese medical society of 1997. 2. No glucocorticoid or other anti-inflammatory drug was used for three weeks. 3. Recent absence of respiratory tract infections excludes other chronic inflammations. 4. Lung ventilation function and histamine challenge experiments were performed the day before bronchoscopy.

Control group: 4 men and 2 women, age 29 + -8.2 years. Without asthma, allergic rhinitis, other allergic diseases, lung tumor and inflammatory infection diseases. Non-asthmatic patients who need bronchofiberscopy due to local shadows in the lungs.

(2) Fiberoptic bronchoscopy

The method comprises the steps of advising a subject to take an oral tranquilization 2.5mg and atropine 0.3mg in the morning on an empty stomach half an hour before an operation, 5% lidocaine 2ml and 0.5-1ml of albuterol 0.5-1ml mixed aerosol inhalation before an operation for 30 minutes before an operation, locally atomizing and anaesthetizing the oropharyngeal larynx by 2% lidocaine, treating the nasal cavity by ephedrine and 2% lidocaine before an operation, enabling the subject to lie on the back, inhaling oxygen, monitoring the heart rate of the subject, inserting a bronchofiberscope into the trachea through the nasal cavity according to a conventional operation, then respectively taking 2-3 pieces of mucosal tissue by using forceps of a right upper leafcrest and a right middle leafcrest, and placing the mucosal tissue into 1% paraformaldehyde fixing solution for fixation.

(3) Pathological observation sample preparation

The biopsied lung tissue was immediately fixed in 10% formalin overnight. 70% ethanol for 2 hours → 80% ethanol for 1 hour → 95% ethanol for 1 hour → anhydrous ethanol for 2 hours → anhydrous ethanol for 1.5 hours → anhydrous ethanol for 1.3 hours, and dehydrating. The TO biological clearing agent (Guangxi rosin plant) is treated for 30 minutes, 1 hour and 30 minutes in sequence. And (3) soaking wax at the low temperature of 50 ℃ for 1 hour. The tissues after being waxed were embedded in paraffin wax at 60 ℃. 4mm serial sections. HE staining, microscopic observation and photography.

(4) Immunohistochemistry

2 paraffin-embedded tissue sections were taken and dewaxed 2 times in xylene for 5 minutes each. 90%, 80%, 70% ethanol for 5 minutes each. The mixture was rinsed with tap water for 2 minutes and distilled water for 2 times. Heating in sodium citrate buffer solution at 92-98 deg.C for 1-2 min, and cooling at room temperature. Placing in 3% H₂O₂Rinse for 10 minutes. Add 50. mu.l peroxidase blocker and incubate for 10 min at room temperature. PBS rinse 3 times for 5 minutes. 50 μ l of non-immune animal serum was added. Incubate for 10 minutes at room temperature and aspirate excess fluid.

50 μ l of PBS and CKLF-1 monoclonal antibody were added to 2 slides, incubated at room temperature for 60 minutes, and washed 3 times with PBS. Add 50. mu.l biotin-labeled secondary antibody, incubate for 10 min at room temperature, and wash 3 times with PBS. Mu.l of streptavidin-peroxidase was added, incubated at room temperature for 10 minutes, and washed 3 times with PBS. 100 μ l of the freshly prepared DAB solution was added, and the staining was observed under a microscope and washed with tap water for 5 minutes to terminate the reaction. And (5) performing hematoxylin counterstaining. Washing with tap water, naturally drying, and sealing with neutral gum.

Results

1. Histological observation of bronchial mucosa biopsy

(1) Control group (healthy subjects)

Normal airway epithelium is composed of a variety of cells, such as pseudostratified ciliated columnar epithelial cells, goblet cells, basal cells, and the like. Despite the irregular arrangement of the various cells in the epithelium, all cells lie closely above the basement membrane and remain a monolayer (pseudo-stratified epithelium). Epithelial cells near the apical chamber are tightly connected, forming an epithelial barrier. The basal cells are close to the surface of the basement membrane. Goblet cells are rare.

(2) Experimental group (asthma sufferer)

The fiberbronchoscopy shows that 8 asthma patients have congestion and edema of airway mucosa, severe patients have whitish mucosa and narrow bronchial tube diameter. Bronchoalveolar lavage fluid cell smears of 2 patients showed that a large number of airway epithelial cells were exfoliated. Bronchial mucosa biopsy showed: the airway mucosa of a patient mainly infiltrates lymphocytes and Eos, airway epithelial cells are damaged, and extracellular matrix is obviously increased. In 1 of the patients, the airway mucosa has no obvious Eos infiltration, ciliated cells and goblet cells of the airway epithelial layer are almost completely shed, and only a small amount of basal cells are attached to a basement membrane; the basement membrane is obviously thickened and is transparent; collagen deposition in the interstitium, massive cell aggregation, and smooth muscle proliferation. These are sufficient indications that bronchial asthma is an airway inflammation with significant changes in airway structure.

2. Immunohistochemical analysis of bronchial mucosa CKLF-1 protein expression

(1) Control group (healthy subjects)

The normal control epithelial cilia are aligned to form an intact barrier, and basement membrane structures are not seen. CKLF-1 immunoreaction was negative without specific color development (FIG. 15A).

(2) Experimental group (asthma sufferer)

Protein immunohistochemical analysis showed: CKLF-1mRNA is highly expressed in PBMCs (including mononuclear cells and lymphocytes, mainly lymphocytes) of a patient. In 5 of 8 patients, partial epithelial cells and subepithelial stromal cells showed positive immune responses. The positive response was most pronounced in 1 of these critically ill patients (fig. 15B).

Based on the above results, it is believed that asthma patients have activated lymphocytes, airway epithelial cells, fibroblasts of subepithelial stroma and macrophages at least at the cellular level, thereby increasing the expression level of CKLF-1. The experimental result shows that the airway epithelium and the subepithelial stroma of asthma patients, particularly severe patients have high-level CKLF-1 protein expression, and the CKLF-1 is presumed to have the function of strengthening the proliferation of the stroma and smooth muscle. Epithelial cells as airway portals were first stimulated by environmental factors, thereby activating subepithelial fibroblasts, initiating cascade-like inflammatory chain reactions that are progressively amplified and repair reactions following inflammatory injury.

Claims (10)

1. An in vitro assay comprising detecting over-expression of chemokine-like factor-1 in a sample from a host and determining the pathological state of inflammatory injury in the sample.

2. The method of claim 1, wherein the inflammatory injury is bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis.

3. The method of claim 2, wherein the sample from the host is human peripheral blood cells, lung surgically excised tissue, lung bronchial mucosal tissue.

4. A method of making an animal model of inflammatory injury to an organ comprising introducing chemokine-like factor-1 into a mammal other than a human.

5. The method of claim 4, wherein the inflammatory injury is bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis.

6. The method of claim 5, wherein the chemokine-like factor-1 is introduced into the animal by injection.

7. The method of claim 5, wherein the chemokine-like factor-1 is introduced into the animal by nebulization.

8. The method of any one of claims 4-7, wherein the mammal is a mouse.

9. Use of chemokine-like factor-1 in preparing antibody and antagonist for treating organ inflammatory injury.

10. The method of claim 9, wherein the inflammatory injury is bronchial asthma airway inflammatory injury and remodeling and pulmonary fibrosis.

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