Construction Of Mouse IL-33 Rnai Retroviral Vector And It's Effects On Cigarette Smoke-induced Mouse Lung Inflammation

IL-33, as one member of IL-1 family, binds to it's special receptor ST2. IL-33/ST2 signalling is involved in Th2 associated immune responses. It plays important roles in autoimmune diseases, allergic diseases, cardiovascular diseases and neurodegenerative diseases. Chronic obstructive pulmonary diseases are the co-occurrence of chronic bronchitis and emphysema, a pair of commonly co-existing diseases of the lungs in which the airways become narrowed. This leads to a limitation of the flow of air to and from the lungs. The primary risk factor for COPD is chronic tobacco smoking.To investigate the relationships between the IL-33 and the lung inflammation, we used RNA inteference to reduce the expression of IL-33 mRNA and protein. The main results of this paper are as follows:1) Two highly specific siRNA candidates targeting mouse IL-33 gene were disigned through GenScript's design center siRNA Target Finder.2) The chemosynthetic shRNAs of targeting mouse IL-33 gene with the retroviral vector pRNATin-H1.4 were ligated. The sequencing analysis showed that the mouse IL-33 RNA inteference retroviral vectors were successfully constructed.3) The recombinant vectors were transfected into RetroPack PT67 cells through Lipofectamine2000, hygromycin resistant gene was used to select for stably tranduced cells. The empty vector was used for negative control. The stably transduced cells were selected by using hygromycin resistant gene.4) In animal experiments, the levels of IL-33 mRNA and protein expression were detected by RT-PCR and Immunohistochemistry respectively. The results showed that the recombinded retroviral vectors can effectively knocked-down the expressions of IL-33 mRNA and protein in mouse lung tissue. HE staining indicated that the decrease of IL-33 could improve the pathology of smoke-induced mouse lung inflammation.