| Object: bronchial asthma is a global health problem that results from a complex interplay between genetic and environmental factors. It is a chronic inflammatory disease of the airways, characterized by airway obstruction, airway hyper- responsiveness, IgE production, increase mucus secretion , airway remodeling and an airway infiltrate consisting mainly of eosinophilic granulocytes, mast cells and lymphocytes. Eosinophil response appears to be a critical feature in asthma. Eosinophils play an effector role by release of pro-inflammatory mediators, cytotoxic mediators, and cytokines, resulting in vascular leakage, hypersecretion of mucus, smooth muscle contraction, epithelial shedding, and bronchial hyperresponsiveness. These cells are also involved in the regulation of the airways inflammation and in the initiation of the remodeling process by the release of cytokines and growth factors. Various studies have demonstrated that agents decreasing Th2 cytokines or increasing production of Th1 cytokines can prevent airway inflammation and development of AHR. IL-12 is a key cytokine promoting Th1-type cell-mediated immunity and upregulates production of Th1-type cytokines, such as IFN-γ, IL-2 and TNF, promotes development of Th1 cells and inhibits expression of Th2 cytokines in vivo. In this experiment we investigate the effects and mechanisms of AdCMVIL-12 to airway inflammation and correlated factors in asthmic BALB/c mice model.Methods Adenovirus vectors (AdCMVIL-12, AdCMVLacZ) were amplificated in 293cells and were purificated by CsCl density gradient centrifugation, then titration for use. For determine if carried target gene adenovirus vector transfect pulmonary tissue effectively, the pulmonary tissue of normal BALB/c mice were transfected with AdCMVLacZ by drop nose, frozen section were prepared after 48 hours, then X-gal staining to observe transfect result. 50 female BALB/c mice, 6-8 weeks of age, were divided into 5 groups: asthma model group (A), AdCMVIL-12 treatment group (B). AdCMVLacZ treatment group (C), Pulmicort Respules treatment group (D), and normal control group (E). Mice (except normal control group) were sensitized on days 0 and 14 by intraperitoneal injection of 20μg ovalbumin (Sigma, grade V) emulsified in 2.25 mg of imject alum in a total volume of 200μl, the mice of normal control group were injected saline in same volume. 21th day, mice were anesthetized, then AdCMVIL-12 group were adminisatratd by drop dosage (5×108PFU/100μl),AdCMVLacZ group were administrated same dose AdCMVLacZ, hormone therapy group were administrated Pulmicort Respules(2ml) by atomization for 30minutes before challenge. On days 21-27, mice were challenged with an aerosol challenage of 5% (w/v) OVA in saline (or with saline as a control) using an ultrasonic nebulizer, 30minutes/per for 7 days. Mice were sacrificed by cervical dislocation and the peripheral bloods were obtained at 24 hours after the last aerosol chanllenge, the right lungs were deligated and the left lungs were lavaged, the numbers of inflammatory cell in the mice bronchoaleolar lavage fluids (BALF) were counted with microscope, and the concentration, mRNA levelsof IL-4, IL-5, IL-13, IFN-γ, IL-12 in blood serum and BALF supernatant were also detected by using sandwich ELISA and RT-PCR according to the manufacturer's protocol. Right lung were fixed in 10% formalin/PBS, tissue slides were investigated the eosinophil infiltration in pulmonary tissue after stained with HE and the hyperplasia of goblet cells in the bronchial mucous membrane after PAS. Eosinophil cells of pulmonary tissue in each group were stained by Conge Red staining to observe infiltration degree. The expressions of eotaxin and GATA-3 in pulmonary tissue of each group were compared by immunohistochemistry and RT-PCR techniques. The mechanisms of AdCMVIL-12 for asthma inflammation and correlated facots were investigated,and provide experimential proof for IL-12 asthma gene therapy.Results: the titer of AdCMVIL-12 is 2×1010 PFU/ml by ampliferation in 293 cells and were purified using Cscl density gradient centrifugation. X-gal staining determined AdCMVLacZ could transfected air duct, alveolar epithelium in bronchiole and surrounding pulmonary tissue, transfected cells showed ultra marine blue, so carried target gene adenovirus vectors could transfected bronchus and pulmonary tissue, this result provide experimental data for using AdCMVIL-12 in asthma gene therapy and to investigate its effect on air duct and correlated cytokines. The mice of asthma model group appeared pruritus in head and face, dysphoria, depressed, quiet, aggregation, breath to deepen and fast, bend from the waist,straighten,fore-leg shrink and lift, nutation breathe, cough, breathlessness, urinary and fecal incontinence. Some mice showed breathe rev down, behavior delay, proneness. The weight of mice became loss, hair tarn in continuous provocation, individual mice had limbs twitch. These action determined asthma model were prepared successfully. Compared with asthma model and AdCMVLacZ group, AdCMVIL-12 can depress obviously the numbers of total cells and eosinophils in BALF, airway inflammation and infiltration of eosinophils were obviously inhibited. Pathologicalchanges of pulmonary tissue in each group were scored according the degree of inflammation, the score of asthma model group were higher than those of AdCMVIL-12 group, Pulmicort Respules treatment group and normal control group, but no significant difference compared with AdCMVLacZ group.The levels of IL-12 in serum from each group were detected by ELISA, the IL-12 levels of asthma model group were lower than those of AdCMVIL-12 group, Pulmicort Respules treatment group and normal control group, but no significant difference compared with AdCMVLacZ group. This result determine that AdCMVIL-12 could elevate the levels of IL-12 in vivo, IL-12 may be enhancd cytoimmunity to adjust balance of Th1/Th2 , thus to decrease inflammation of air duct.AdCMVIL-12 can increase the level of Th1 type cytokine (IFN-γ) and downregulate Th2 type cytokine (IL-4, IL-5, IL-13and eoatxin) in BALF and serum by ELISA and RT-PCR technique. PAS staining showed AdCMVIL-12 can repress hyperplasia of airway goblet cells and mucus secretion. The numbers of eosinophils in pulmonary tissue were stained by Conge Red, results showed that the eosinophils numbers of asthma model group and AdCMVLacZ group increased obviously, but those of AdCMVIL-12 group and Pulmicort Respules treatment group decreased obviously. AdCMVIL-12 significantly inhibited the expression of eotaxin and GATA-3 by immunohistochemistry and RT-PCR.Conclusion: AdCMVIL-12 can efficiently inhibit inflammatory cell infiltration of airway and blood vessel by drop dosage, especially decrease the number of eosinophil, and downregulated the expression level of IL-4, IL-5, IL-13, eotaxin in BALF and serum, decrease the hyperplasia of airway goblet cells and eosinophil infiltration. The mechanism of AdCMVIL-12 to asthma may be it increase level of endogenously IL-12, IFN-γ, and downregulate the secretion of Th2 type cytokines, such as IL-4, IL-5, IL-13, regain the balance of Th1/Th2 cell. AdCMVIL-12 also inhibited the expression of eotaxin and GATA-3, its anti-inflammation may be related to eosinophil infiltration and Th2 specific transcription factor (GATA-3) were inhibited, so IL-12 may be as an ideal gene for asthma gene therapy. |
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