| Part 1 Significance of Human chemokine-like factor 1(h-CKLF1)expression in human allergic rhinitis(AR)Objective: To investigate significance of h-CKLF1 expression in human AR.Methods: Sixty patients enrolled were divided to three groups, including 20 cases of allergic rhinitis,20 cases of nasal polyps and 20 cases of deviated nasal septum(DNS). Serum samples were collected before surgery. Interleukin (IL)-4, interferon (IFN)-γ, immunoglobulin (Ig)E levels in the serum were examined by ELISA. Eosinophils(Eos) number were measured in nasal secretions and IFNerior turbinate mucosa smear by Wright staining. Specimens of IFNerior turbinate mucosa and Nasal polyps were respectively divided to two groups, one group was fixed by 4% formaldehyde, the number of total IFNlammatory cells and Eos number were measured in the Specimens by HE staining. The expression of h-CKLF1 and the CCR4 was detected by immunohistochemical staining. The second group without 4% formaldehyde fixation h-CKLF1 mRNA in the IFNerior turbinate mucosa and nasal polyps was detected by semi-quantitative RT-PCR. The results were compared among three groups.Results:1 The percentage of Eos counts to IFNlammatory cell in nasal secretions and IFNerior turbinate mucosa smear in AR group (respectively, 13.40±1.23, 7.25±1.99) were significantly higher than the nasal polyps group (3.9±1.12,1.75±0.72) and the control group (0.65±0.93,0.25±0.44), p<0.05.2 The contents of IL-4 and IgE in AR group (907.50+226.13IU.ml-1, 81.55 +23.68 pg.ml-1) were significantly higher than the nasal polyps group (22.20+10.17,55.45+19.58) and control group (19.05+7.08,39.50+14.61), p<0.05, there was no significant difference between nasal polyps group and DNS group (p>0.05). IFN-γlevels in AR group (21.00+11.69 mg.ml-1) were significantly lower than the nasal polyps group (87.74+30.39) and DNS group (92.55+22.01), p<0.05, there was also no significant difference between nasal polyps group and DNS group, p>0.05.3 Eos counts in the Specimens in AR group (13.30±2.15) were significantly higher than the nasal group (5.15±2.11) and the DNS group (0.90±1.02), p<0.05, there was also no significant difference between nasal polyps group and DNS group, p>0.05.4 The positive findings (brownish yellow, fine granular) of CKLF1 by immunohistochemical analysis under microscope in three groups were found in the cytoplasm of the mucosal epithelial cells, glandular cells and IFNlammatory cells. The percentage of positive area of CKLF1 in the allergic rhinitis group(0.64±0.09) was significantly higher than the nasal polyps group (0.30±0.06) and DNS group (0.07±0.04) p<0.05, there was also no significant difference between nasal polyps group and DNS group p>0.05.The positive finding (brownish yellow, fine granular) of CCR4 were found in the membrane of the IFNlammatory cells mucosal, epithelial cells and glandular cells. The percentage of positive area of CCR4 in the AR group (0.71±0.16) and the nasal polyps group (0.69±0.17) were significantly higher than the DNS group (0.13±0.04), p<0.05, there was no significant difference between AR group and nasal polyps group p>0.05.5 The CKLF1 mRNA expression, relative to internal reference gene GAPDH, in allergic rhinitis (0.71±0.11) was significantly higher than in nasal polyps (0.41±0.06) and DNS group (0.08±0.04) p<0.05, there were also statistically significant difference between nasal polyp and DNS group p<0.05.6 Correlation analysis: The increased mRNA and protein of CKLF1 was in directly proportion with the eosinophils levels in nasal mucosa (r=0.899, p<0.05), and the IL-4 levels (r=0.842, p<0.05), but in inversely proportion with IFN-γlevels (r=-0.819, p<0.05). mRNA and protein expression of CKLF1 (r=0.896, p=0.00) and CCR4 (r=0.734, p=0.00) were directly proportion to each other.Conclusion: Expression upregulation in the mRNA and protein levels of CKLF1 in nasal mucosa plays a key role in Human AR by activation and attraction of Th2 cells and Eos.Part 2 Establishment and evaluation of the SD rat AR modelObjective: To investigate establishment method and evaluation system of the SD rat AR model and use it in next step for AR treatment with C19 and C27.Methods: To establish SD rats AR model by ovalbumin (OVA), 20 cases SD rats were randomly divided into two groups, namely healthy group (10cases) and AR group(10 cases). AR models were sensitized and challenged by OVA. AR rats were induced intraperitoneally by injecting OVA(10mg) and aluminum hydroxide(15mg) 8 times on alternate days and after fifteen days,was boosted by nasal challenge with 50μl physiological saline containing OVA(1%).Healthy group was induced and boosted with 0.9% Nacl.The pathological and praxiology score were observed in AR group during symptoms. IL-4, IFN-γ, IgE levels in the serum were examined by ELISA. According to the behavioral score, it was checked weither nasal histology and content of IL-4, IFN-γ, IgE in serum of AR rat was established successfully or not.Results: Behavioral scores were significantly higher in OVA-challenged rats(6.3±0.67) compared with the healthy group(2.1±0.73) p<0.05. Nasal epithelial goblet cells, eosinophils and lymphocytes in nasal mucosa of AR rats were higher than healthy group.Obvious increase in IL-4, IgE levels and decrease in IFN-γlevels in AR rat was found p<0.05.Conclusion: OVA sensitization of successfully established SD rat model of allergic rhinitis,can continue to the next step experiment.Serum IgE, IFN-γ, IL-4 can be used in objective evaluation of allergic rhinitis animal models.Part 3 The CKLF1 and CCR4 expression in the nasal mucosa of the AR in rats and the effects of C terminal peptide(C19 and C27) of CKLF1 on the treatment of the AR in rats.Objective: To investigate the CKLF1 and CCR4 expression in the nasal mucosa of the AR in rats and the effects of CKLF1 C terminal peptide(C19 and C27) on the treatment of the AR in rats.Methods: 50 cases SD rats were randomly divided into 5 groups, namely heathy group(10 cases), AR group(10 cases), fluticasone propionate group(10 cases), C19 group(10 cases) and C27 group(10 cases). Establish SD rats AR model by ovalbumin(OVA) used the same method as the experiment part 2. In this study, fluticasone propionate group were received the fluticasone propionate(10ug) by bilateral nasal drip before 30minutes of every challenge and C19 group (C19:10ug) and C27 group(C27:10ug) were received test drugs by same method. We observed the symptoms of the rats such as sneezing, nasal rubbing and nasal discharge. IL-4, IFN-γ, IgE levels in serum were examined by ELISA. Immunohistochemical staining was used to examine the CKLF1 and CCR4 in nasal mucosa and CKLF1 mRNA in nasal mucosa was detected by semi-quantitative RT-PCR.Results:The findings of CKLF1 and CCR4 by immunohistochemical analysis and CKLF1 mRNA by semi-quantitative RT-PCR in nasal mucosa in AR animals were significantly increased when compared with the heathy group (p<0.05).C19 significantly reduced behavioral scores and suppressed the increased Eos in the nasal mucosa and bone marrow in OVA-challenged rats compared with C27-treated rats and AR group.IL-4, IgE levels in the C19 administration rats exhibited obvious reduction relative to OVA-challenged while IFN-γlevels exhibited obvious increase. C19 reduced the increased expression of CKLF1&CCR4 after OVA challenge and decreased the enhanced expression of CKLF1 mRNA in nasal mucosa in AR animals. While the C27 group were not observed the similar results. But the above-mentioned results in C19 group compared with the heathy group and fluticasone propionate group, differences were statistically significant (p<0.05). Differences of the above-mentioned results in C19 group compared with the C27 group and AR group were all statistically significant (p<0.05) .while there were not statistically significant between C27 group and AR group(p>0.05) , there were also not statistically significant between heathy group and fluticasone propionate group (p>0.05).Conclusion:1 Expression upregulation in the mRNA and protein levels of CKLF1 in nasal mucosa plays a key role in the pathogenesis of AR rat model.2 Nasal administration of the C19 resulted in the reduction of activation and IFNiltration of Th2 cell and Eos in the Nasal mucosa of rat AR and the suppression of the eosinophilic proliferation in the bone marrow and so may offer a new strategy for the control of AR. While the C27 was not observed the Similar effect.
|
PDF Full Text Request