| Bronchial asthma is a serious threat to public health, the most common chronic lung disease, clinical manifestations with recurrent episodes of shortness of breath, chest tightness, coughing as the main symptom, often at night and (or) early morning attack, if it can cause long-term recurrent airway remodeling causing the airways to thicken and narrow, becoming obstructive pulmonary emphysema. At present the pathogenesis of asthma has not yet completely clear, studies have shown that bronchial epithelial cells is involved in the initial part of asthma[1],bronchial epithelial cell loss and injury is the pathology of asthma. Perform (PFP) and granzyme B(GzmB) is an important factor secreted by cytotoxic T cell (CTL) cells and natural killer cells (NK cells), It is very important by mediating apoptosis, both in the lung tissue of asthmatic rats expression and the induction of apoptosis in bronchial epithelial cells is unclear. Recombinant human growth hormone (rhGH) is a synthetic peptide, which can be inhibited by some mechanism to achieve the appropriate treatment of the apoptosis effect. In this study, rat asthma model and apply rhGH intervention, observation PFP and GzmB expression, apoptosis of epithelial cells and their systems in order to provide a theoretical basis for the clinical treatment of asthma. ObjectiveIn this study, through the establishment of asthmatic rat model to investigate the expression of PFP, GzmB in the lung tissue of asthmatic rats and the intervention of rhGH.Materials and methods1 Animal groups and asthma model makand4-6 week old male SD 30 rats were randomly divided into normal control group, asthma group and the rhGH treatment group, n=10. No.1,8,15 days, the intervention group and asthma group were applied to 10% of freshly prepared ovalbumin aluminum hydroxide suspension in 1 ml (OVA aluminum hydroxide 100 mg+100 mg) by intraperitoneal injection, induced 3 times, the control group received normal saline 1 mL intraperitoneal injection. The 16th day, the asthma group and intervention group were applied 1% ovalbumin-5 ml suspension of aluminum hydroxide inhalation 30 min, every other day for a total of 20 times; intervention group,11 rats Times 30 min before inhalation suspension for subcutaneous injection of recombinant human growth hormone (1.33 KU/Kg), were injected 10 times; the control group inhaled normal saline per 5 ml 30min,1 every other day, a total of 20 times.2 Preparation of lung tissueThe rats were killed by intraperitoneal injection of pentobarbital 100 mg after the last inhalation. To place the right lung in the refrigerant tube and quickly frozen in liquid nitrogen filling to save, to do with the RT-PCR. Application of sterile saline and 4% formaldehyde for perfusion and flushing of the left lung, formalin-fixed, paraffin-embedded. Respectively perpendicular and parallel to the direction derived airway sections for HE staining and TUNEL in situ apoptosis detection.3 Results of in situ apoptosis The results in the light microscope:5 randomly selected high power microscope field (×400 and contains the same level of bronchial) of the total number of bronchial epithelial cells and the number of positive staining (brown staining), according to the formula:number of positive cells/total cells×100% of epithelial cell apoptosis index.4 RT-PCR analysisThe frozen lung tissue was extracted using Trizol total RNA, reverse transcription after the application designed for the cDNA amplified fragment PFP, GzmB and GAPDH. After agarose gel electrophoresis, with Band Scan 5.0 gel analysis software were measured PFP, GzmB and the gray value of GAPDH, and the ratio of each of the two and as a representative value of the rats.5 Statistical MethodsUsing SPSS 17.0 statistical package for statistical analysis of the data, experimental data±s, multiple samples were used to compare between the number of single-factor analysis of variance between groups using LSD-t test; correlation line by pearson correlation test; Toα=0.05 level for the test.Results1 Pathological changesMorphology of the normal control group, normal lung tissue. HE staining showed bronchial lumen with mucus plug formation in the Lung tissue of asthmatic model group, bronchial epithelial edema, loss, irregular structure; goblet cells increased, submucosa and smooth muscle thickening; airway wall of a large number of interstitial lung Infiltration of inflammatory cells to eosinophils and lymphocyte predominant; rhGH intervention group compared with asthma to reduce inflammation, goblet cell reduction, thickening of submucosa and smooth muscle in asthma group reduced. 2 Changes in epithelial cellsLess bronchial epithelial apoptosis in the control group (TUNEL in situ cell apoptosis detection brown staining); asthmatic bronchial epithelial apoptosis more than the normal control group, the difference was statistically significant (P<0.05); rhGH intervention in bronchial epithelial cells compared with normal control group is higher, but significantly reduced compared with asthma group, the difference was statistically significant (P<0.05).3 RT-PCR resuandPFP and GzmB expression increased in asthma group and the intervention group compared with control group, the difference was statistically significant (P<0.05),. Both were less the expressed in rhGH intervention group compared with asthma group, the difference was statistically significant (P<0.05).4 Correlation AnalysisPFP expression was positively correlated with bronchial epithelial cell apoptosis index (r=0.800, P<0.05). GzmB expression was positively correlated with bronchial epithelial apoptotic index (r=0.806, P<0.05).Conclusion(1) PFP and GzmB by promoting apoptosis of bronchial epithelial cells involved in the occurrence of asthma and the development process.(2) rhGH by reducing the PFP and GzmB expression in relief of airway inflammation and airway remodeling. |
PDF Full Text Request