| 一、Background:Bronchial asthma (Bronchial asthma, asthma) is a kind of chronic airway inflammatory diseases, which has multiple cytokines involved in regulation, multiple cells (mainly eosinophils) infiltration of.It has three pathological features: chronic airway inflammation, airway remodeling and airway hyperresponsiveness (AHR), airway remodeling is increasingly becoming the focus of people study, this remodeling involves layers of the airway tissue including airway wall thickening and matrix deposition, subepithelial fibrosis, smooth muscle hyperplasia and hypertrophy, myofibroblast proliferation and mucus glands and goblet cell metaplasia and hyperplasia, subepithelial reticular layer thickening angiogenesis,etc. The current study suggests that airway remodeling associated with chronic inflammation of the airways, inflammation, injury, repair, remodeling is a continuous progress of the pathological basis, eventually leading to airway wall thickening, promote airway narrowing and airflow limitation. The view was also expressed that airway remodeling and airway hyperresponsiveness, lung function decline is closely related to, and can form a refractory asthma. The traditional view is that airway smooth muscle cells (airway smooth muscle cell, ASMC) is a passive cells only in the stimulation of inflammatory mediators play a regulating airway contraction and relaxation, research and more focus on inflammatory mediators or neurotransmitters on airway smooth muscle contraction and endogenous or exogenous bronchodilator substances relaxant effect on bronchial smooth muscle. However, recent research evidence that ASMC phenotypic change can occur, own synthesis and secretion of a variety of cytokines, chemokines and inflammatory mediators involved in the asthmatic airway inflammation occurs, the whole process of development, and thus increase airway remodeling and AHR. Some scholars have even raised the target should ASMC as asthma research, by its phenotypic transformation and in-depth study of the role of the synthesis, secretion, to provide a new method for the treatment of asthma。Recent studies have found that renin-angiotensin system (RAS) in addition to participate in in the development of a variety of cardiovascular and renal disease, also is associated with lung disease outcome and prognosis. In recent years, studies have found that angiotensin Ⅱ, the important effect substances in the RAS, except the regulation of vascular tone, blood flow and promote cell growth, proliferation, and also has a proinflammatory role it can play in regulating cytokines by binding to specific receptors on the cell membrane, chemotaxisof factors and adhesion molecule expression of a variety of inflammatory mediators involved in the occurrence and development of inflammatory diseases. It has been found that four kinds of Ang Ⅱ receptor subtypes that of ATIR of angiotensin Ⅱ type2receptor of angiotensin Ⅱ type2receptor, AT4R AT1R and AT2R mainly in people and rodents of angiotensin II type2receptor and AT4R present in the tissues of fish and poultry, like human without any related. Some scholars also detected in the lung tissue of the AT1receptor. And in recent years a large number of studies have shown that angiotensin II with AT1receptor binding play its classic role. Studies have shown that, AngⅡ promotes mesangial cells, cardiac cells, vascular smooth muscle cells, and other cells to secrete a variety of extracellular matrix (collagen, fibronectin, laminin, hyaluronic acid, etc.), the participating organizations remodeling, and this effect can be blocked by the AT1receptor blockers [11,12,13]. Type Ⅰ collagen is the major ECM proteins in normal and diseased airway wall. Carol [13] using [3H]-proline incorporation release assay detected AngⅡ(10-7mol/L) stimulation of vascular smooth muscle cell collagen synthesis increased significantly, and the AT1receptor antagonist losartan intervention collagen synthesis was significantly reduced. Of AngⅡ primarily through AT1receptors are involved in the rat asthmatic airway remodeling process, specific angiotensin AT receptor blockers can reduce the formation of airway remodeling in animal models of asthma [13]. Liu Cuili [14] by rats trial confirmed asthma rat lung tissue local RAS activation, AngⅡ generate increased. Promote ASM proliferation and hypertrophy and airway wall collagen deposition increased local increase of AngⅡ AT1receptor antagonist irbesartan the rat ASM the thickness and COLIII in the deposition of the airway wall, can reduce the incidence of airway remodeling, and lung function improvement。The extracellular matrix (extracellular matrix, ECM) secreted by the cells to the extracellular matrix macromolecules to form a complex network structure, support, and connect organizational structure, regulation of tissue and cell physiological activities. Mainly of collagen, elastin, proteoglycans and glycoproteins. The study shows that the ECM may influence cell differentiation, proliferation, adhesion, morphogenesis, and phenotypic expression and other biological processes. In asthma research found that in recent years, there is excessive ECM deposition in airway remodeling in asthma, and compared with non-asthmatics, the the ECM protein component and the component ratio larger changes in vitro experiments confirmed that ECM proteins can affect HASMC proliferation, migration, and so on。Extracellular signal-regulated kinase1/2(extracellular signalregulated kinase, ERK1/2) signaling pathway is important in intracellular signal transduction pathways involved in asthma chronic airway inflammation, airway hyperresponsiveness and airway smooth muscle proliferation and other pathophysiological process. Foreign studies based on airway smooth muscle cells cultured in vitro, pay attention to the role of the ERK signaling pathway in airway remodeling of asthma. Mainly domestic studies focused on ERK signaling pathway for smooth muscle cell proliferation, migration studies in animal models of asthma research. There is few papers on ERK signaling pathway in the secretion of airway smooth muscle cells secrete in vitro and in vivo。 Because airway smooth muscle cells of asthma patients are difficult to obtain, most of the researchers at home and abroad choose to use asthma serum passively sensitized airway smooth muscle cells. And abroad study confirmed that, compared to normal serum treated cells, passively sensitized airway smooth muscle cells, the secretion of ECM protein composition and content were significantly different. Therefore, adopting asthma serum passively sensitized airway smooth muscle cells for research is helpful to understand the position of the progress of asthma smooth muscle cells and main function.二、purposesThrough collection and separation blood of acute asthma patients, and use serum passively sensitized human airway smooth muscle cells (airway smooth muscle ASMC), use angiotensin Ⅱ (AngⅡ) and angiotensin Ⅱ AT1receptor antagonist losartan treated passive sensitization of smooth muscle cells, to only plus asthma serum group as a control, preliminary research AngⅡ passively sensitized ASMC the secretion of type Ⅰ collagen and other extracellular matrix proteins, and the above the cell culture supernatant stimulation of airway smooth muscle cells, a preliminary understanding of the extracellular matrix protein content of different cell culture supernatants of airway smooth muscle cell proliferation role, provide a new target for the prevention and treatment of asthma airway remodeling。三、Materials and methods1. Human airway smooth muscle cells (HASMC) separation, cultivation and identification:sterile state, take our hospital thoracic surgery in patients with lung cancer lobectomy postoperative tumor adjacent distal normal bronchial tissue. According to the related literature and reports and our research team has mastered, the tissue block adherent culture method is adopted for generations of airway smooth muscle cells in primary culture, take3-5generations of airway smooth muscle cells used in experiments. Cell growth and morphology was observed under an inverted microscope monoclonal antibody and and a-anti smooth muscle actin monoclonal antibody (a-SM actin) immunocytochemistry staining identified human airway smooth muscle cells.2. Experimental groups:the experimental cell is divided into the following six groups:①:asthma serum passively sensitized group:Add DMEM containing10%of asthma serum; The passively sensitized+Ang Ⅱ group:DMEM containing10%of asthma serum+Ang Ⅱ (a final concentration of10-7mol/1);③) The passively sensitized+Losartan group:DMEM containing10%of asthma serum+Losartan (final concentration of10"6mol/l);④passively sensitized+Ang Ⅱ+Lostarn groups: DMEM containing10%of asthma serum+Ang Ⅱ (final concentration of10-7mol/l)+Lostarn (final concentration of10"6mol/l);⑤The passively sensitized+PD98059group:DMEM containing10%of asthma serum+PD98059(final concentration of10-5mol/l);⑥passively sensitized+Ang Ⅱ+PD98059groups:DMEM containing10%of asthma serum+Ang Ⅱ (final concentration of10-7mol/l)+PD98059(final concentration of10"5mol/l)。3.Picrosirius red staining:The collagen molecules rich in basic amino acids, can play a strong reaction with acid dyes. Sirius red as a strong acid dyes, powerful combination with collagen. Ordinary light microscope, picrosirius red staining of collagen. The cells seeded staining, microscopy see the HASMC cytoplasm red。4. Enzyme-linked immunosorbent method to detect the ECM protein concentration in the cell culture supernatant.5.Determined by MTT method to detect cell culture supernatant of HASMC whether have effect on promoting proliferation.6.Test the raw data with mean+/-standard deviation (X+s), according to SPSS13.0statistical analysis software, more mean compared with single factor analysis of variance between groups, P<0.05, said the difference was statistically significant.四、Results1. Inverted microscope at X100magnification observation ASMCs was examined before convergence is not more than fusiform or polygon, part of the region after the confluence cells into bundles arranged, showing the ups and downs like growth, was the typical "peak and valley"-like, a-smooth muscle cell-specific actin (aSM-actin) immunofluorescence staining, x200high magnification visible aSM-actin in airway smooth muscle evenly distributed within the cytoplasm in green fluorescence. The transmitted first3-4generation, smooth muscle cell purity of95%.2picrosirius red stainingThe collagen molecules rich in basic amino acids, can play a strong reaction with acid dyes. Picrosirius red (Sinus red) as a strong acid dyes, powerful combination with collagen. Ordinary light microscope, picrosirius red staining of collagen. The cells seeded staining, microscopy see the HASMC cytoplasm red.3.AngII on passive sensitization the HASMC COLImRNA expression affect Real-time PCR results, Angll (10-7mol/l) can promote passively sensitized HASMCs COLImRNA expression., Compared with the control group, the proliferation rate of1.63±0.96-fold (P<0.01). Circumstances exist in angiotensin AT1R-specific blocker losartan (Losartan), AngⅡ role of passively sensitized HASMCs COLImRNA expression decreased to1.02±0.61-fold (P<0.01). ERK1/2signaling pathway antagonist PD98059pretreatment30min, Angll the induced the HASMCs secretion COLI protein concentration was reduced to1.17±0.46-fold (P <0.01).4. Angll on the passively sensitized HASMC COLI secretagogue roleElisa results, Angll (10-7mol/l) can promote the secretion of passively sensitized HASMCs COLI protein concentration up to86.94±6.25ng/ml (P<0.01). Circumstances exist in the angiotensin AT1receptor specific blocker losartan (ARB) treatment of Angll the HASMCs secretion COLI protein concentration dropped to55.55±3.29ng/ml (P<0.01). ERK1/2signaling pathway antagonist PD98059pretreatment30min, AngⅡ-induced the the HASMCs secretion of COLI protein concentration was reduced to55.92±5.19ng/ml (P<0.01)。5. Cell culture supernatant on HASMCs proliferation. MTT assay showed that passively sensitized asthmatic serum group, passively sensitized+AngⅡ group, passively sensitized+Losartan group passively sensitized+Ang Ⅱ+Lostarn group passively sensitized+PD98059group and passively sensitized+Ang Ⅱ+PD98059group cell culture supernatant processing HASMCs proliferation was not statistically significant (P>0.05).五、ConclusionOf the study from the the passively sensitized HASMC start AngⅡ (10-7mol/l) treated with simple passive sensitized group was significantly different from the result of testing on COLL COL I-α1mRNA expression level and protein content was significantly higher than that of passively sensitized group, AngⅡ (10-7mol/l) to promote passively sensitized HASMC secretion COLI. In order to verify whether the AngⅡ promoting secretion mediated by the AT1receptor, we joined the AT1receptor antagonist losartan (10-6mol/1) intervention.The results show that, compared with passively sensitized Ang Ⅱgroup, passively sensitized the the Ang II Lostarn group COL I-almRNA expression level and protein content decreased, no statistically significant difference compared with the purely passive sensitized group, showed that AngⅡ passivesensitized HASMC secretion COLI role is mainly mediated by ATIR AT1receptor antagonist losartan can block the effect. Groups cell culture supernatant of HASMC proliferation had no significant role in promoting。... |
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