Cloning And Characterization Of Differentially Expressed Genes In Peripheral Blood Eosinophils From Patients During Asthma Attacks

BACKGROUNDBronchial asthma is a common chronic disease in the world, It has been widely accepted that asthma is a chronic disorder characterized by chronic airway inflammation, mucus hypersecretion, airway hyperresponsiveness, airway remolding,variable airway obstruction and elevated concentrations of serum immunoglobulin-E (Ig-E). The heterogeneous nature of the clinical manifestations and therapeutic responses of asthma in both adult and pediatric patients indicate that it may be more of a syndrome rather than a specific disease entity. The increasing incidence and prevalence of asthma in many parts of the world continue to make it a global health concern. So the basic research of asthma is ongoing worldwide and are involved in several thematic areas: Genetics of asthma, environmental influences, immunology (including antigen presentation, early-life events, Thl/Th2 imbalance) and immunotherapy, airway inflammation and anti-inflammation treatment, airway remodeling and anti-remodeling and et al. Molecular genetics has identified that asthma is a complicated multifactorial disease, Genetics provides the basis for the host response to a variety of environmental factors that can play a role in the generation of complex genetic diseases.So understanding the mechanisms of interactions between genes and environment represents a major challenge for pulmonary researchers.The research on inflammation mechanism is the base for medicine development and gene therapy toward asthma presently, which is also the major target for asthma therapeutic intervention. But atopic inflammation of asthma depends on regulation by many genes, so it is not very easy to identify specific genes that relate to occurrence and development of asthma. It has been widely recognized that infiltration of the lung with increased number of EOSs is a hallmark of this disease. Airway eosinophilic inflammation has been recognized for nearly 100 years in asthmatic lung tissue. EOSs play a critical role in airway inflammation, airway remolding and immunoregulation of lung microenvironment. The number of peripheral blood EOSs is greatly associated with asthma symptoms,FEVl and airway reactivity, regardless of patients' Atopic state(Ectogenesis or endogenesis),which can reflect airway EOSs inflammation. The increasing of peripheral blood EOSs are significantly and independently related to severe airflow obstruction, especially in persistent and fatal asthma attacks, the airway wall of fatal asthmatics is thickened and the airway lumen is occluded by mucus plugs with large numbers of EOSs, which may also well explain the severe airflow obstruction in fatal asthma attacks.Aggregation and degranulation of EOSs in airway is an important factor to induce airway hyperreactivity. EOSs are known to produce, store and release many proinflammatory cytokines and mediators, such as major basic protein, EOSs cationic protein(ECP)and cysteinyl leukotrienes (Cys-cls), PAF^ PGD2, reactive oxygen intermediates, which are considered to play a critical role in the pathophysiology of asthma. Strong correlations between baseline airway EOSs and airway hyperresponsiveness as well as FEVl have also been reported in patients with asthma, especially mild asthma. EOSs are also recognized important immunoregulative cells capable of presenting antigens and secreting both Thl and Th2 cytokines such as IL-3. IL-^ IL-8 , GM-CSF, TGF- P l,IL-13,interleukins associated with acute-phase responses, TNF- a and several chemokines, amplifying or perpetuating TH2 inflammatory processes at sites of disease, which may play an important role in maintaining and developing eosinophils inflammation. EOSs are an important source of TGF-a, TGF-0 1, Vascular endothelial growth factor (VEGF), fibroblast growth factor-2(FGF-2), Plasminogen activator inhibitor-1 (PAI-1) ,PAI-2> IL-13 , IL-17, Cycles, matrix metalloprotease-9 (MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1) directed attention to a critical role of these cells in airway remolding. Recently Humbles AA and colleagues observed Adbl GATA mice were significantly protected from peribronchiolar collagen deposition and increases in airway smooth muscle. Lee JJ and colleagues found that EOSs were required for pulmonary mucus accumulation and the airway hyperresponsiveness associated with asthma after allergen challenge of PHIL mice,Th2 cytokines production are suppressed during experimental asthma in PHIL mice. Both studies suggest that EOSs are an integral part of experimental allergic asthma, but apart from this common finding they report divergent results, its difference also indicated that asthma is a very complex disease and EOSs are at least a promise target for a subset of asthma. A number of human diseases including allergic disorders (asthma, allergic rhinitis, allergic dermatitis), parasite infections, and tumorigenesis and many fibrosis related diseases, have been reported associated with EOSs.Recently many studies in the asthma fields have focused on analysis of EOSs and their roles in asthma mechanism, such as release of lipid Medium, Granules protein, active oxygen-derived free radicals et al, but its real mechanism including cell's migration, adhesion, apoptosis, exocytosis and degranulation remain largely to be further identified. So systematically study genes expression of EOSs in asthmatics may help clarify asthma pathophysiology and lay theoretic foundation for future targeting therapy. So we screen and identify differentially expressed genes in peripheral blood EOSs from patients with asthma before and after treatment to explore their molecule mechanism during asthma attacks. OBJECTIVEwe first acquired high purity EOSs by discontinuous density gradient method, then amplify high quality and high purity double-strand cDNA from small amount of total RNA from a few EOSs by Super SMART cDNA synthesis technology. Then we primarily screen and identify genes related to asthma by SSH technology, and to illustrate the molecular mechanism of EOSs for its role in bronchial asthma; if further study the genes' functions, we may provide some useful clues for early diagnosis of asthma and new treatment strategy towards inflammatory cells. METHODSEOSs were isolated from the asthmatic patient at the time of exacerbation andafter improvement by discontinuous density gradient method, the cDNAs were synthesized by Super SMART cDNA synthesis technology, Suppression subtractive hybridization was performed between peripheral blood EOSs before and after treatment, which originated from the same patient. Two subtracted cDNA libraries for asthma attacks genes or asthma suppressor genes of human EOSs had been then constructed. About 260 bacterial PCR fragments were picked out randomly to obtain the differentially expressed cDNA fragments by differential screening (DS) method. Then the differentially expressed cDNA fragments were sequenced. After that, expression of partial differentially expressed cDNA fragments was validated by Virtual Northern blot. Finally BLAST searching and literature review were done to analyze their characters and illustrate the possible mechanisms of EOSs in the process of bronchial asthma. RESULTS7.155 [ig and 6.568 ng double-strand cDNA were obtained successfully from 20 ng total RNA respectively, the electrophoresis indicated cDNA acquired had high quality and purity.-, Two subtracted cDNA libraries for asthma attacks genes or asthma suppressor genes of human EOSs had been then successfullyconstructed.by SSH technology, totally 45 differentially expressed cDNA clones were obtained and were then sequenced and Blast analysis showed that 38 differentially expressed gene fragments were obtained. There were 27 known genes, among which 25 genes were differentially expressed higher in EOSs at the time of exacerbation, 2 genes were at the time of remission, which were charcot-Leyden crystal protein(Galectin-10,CLC) and myosin regulatory light chain interacting protein,in addition we acquired 11 unknown ESTs.The genes can be divided into six groups according to their potential molecular mechanism playing in asthma. l.The group associated with eosinophils cytoskeleton remolding, they include slingshot homolog 2 (Drosophila)> Homo sapiens nonfunctional glycosyl transferase-like protein\DOCK-8, protein tyrosine phosphatase, non-receptor type6 (PTPN6), PTPN12),MIR and et al which may play important roles in maintaining cell shape and polarity, leukocyte adhsion\movement\chemotaxis and gene expression; 2.The group associated with eosinophils migration, adhsion and immunoregulation of lung microenviroment. They include galectin-lOs IL-8> putative pre-mRNA splicing regulator female-lethal(2D), Aquaporin 9(AQP9), SSH-2L, protein phosphatase 1, catalytic subunit, beta isoform (PPPlc P ), helicase with zinc finger domain (HELZ)> beta-2-microglobulin ( P -MG ), Homo sapiens DEADH (Asp-Glu-Ala-Asp-His) box polypeptide (DDX26, DBI-1,DICE1), beta-globin and go on; 3.The group associated with eosinophils differentiation, survival and apoptosis.They include human SNF-1 related kinase (hSRK), cell division cycle 10 homolog (S. cerevisiae) (CDC-10), transmembrane protein BRI/TTM2B, Fem-K Cystatin Ax thioredoxin interacting protein (TXNIP); 4.The group associated with eosinophils degranuation and oxidative stress. They include golgi phosphoprotein 3 (GPP34) , SSH-2L, TXNIP, human ras -related protein (Rap 1, Krev-1 )and a mitochondrion related gene.; 5.The group associated with eosinophils intracellular signal transduction. They include mesoderm induction early response 1 Nl-beta 1 (MI-ER1), glucocorticoid receptor (GR) interaction protein 1 (GRIP-1) \ transcriptional intermediary factor 2 (TIF-2), dual specificity phosphatase 1 (DUSP-l/MKPl/CL-100) , high-mobihty group box 2 (HGMB-2) ; 6.11unknown expression sequence tags(ESTs).the unknown genes were found to be located on chromosome 1, 2, 6, 7, 10, 11, 12, 13, 17.,which may be novel genes associated with asthma. CONCLUSION1. The occurrence and progress of asthma results from a series of cellular and molecular events, attributing to many genes associated with asthma whose expression go out of control;2.SSH is a technology for gene expression research, with high sensitivity, high efficiency, and low false-positive rate, If combined with Super SMART cDNA synthesis technology, it may be used to study gene expression from a very few total RNA, especially useful for those only limited clinical sample can be obtained;3.We acquired many differentially expressed genes by SSH and differential screening method, which are probably involved in progression and exacerbation of asthma through prolonging EOSs survival, recruiting EOSs to lung, increasing release of inflammatory mediators and oxygen free radials and immunoregulating lung microenvirnment. They also may be genes resulting in susceptibility to asthma, and provide a valuable opportunity to define new pathways involved in the pathogenesis of allergic airway inflammation. .Further study may isolate all genes differentiallyexpressed in EOS and systematically clarify their molecular mechanism in asthma, also may provide some clues for early diagnosis of asthma,And develop new therapeutics including both EOSs-specific and EOSs-selective therapeutic targets towards asthma; we can acquire full-span new genes from unknwn ESTs, and find out new asthma related proteins4.. It is also useful to diagnose and treat allergic disorders (allergic rhinitis, allergic dermatitis), parasite infections, and tumorigenesis and many fibrosis related diseases associated with EOSs after we got to know how do eosinophils play in asthma..