| [BACKGROUND] Up to now, there is no effective treatment and prevent project for bronchial asthma. Asthma is caused by many cells and the inflammatory media produced by them. Particularly, airway epithelium is the first interface between outside stimulators and internal environment. Epithelium is exposed to continuous damage and abnormal plerosis in asthma, and can release a lot of inflammatory media that can affect structure and function of circumferencial tissues and inflammatory cells. Therefore, airway epithelium has been considered as one of initiating organs for asthma. Recent researches showed that the enhancement of expression and/or activity of a sort of calcium-activated chloride channel (CLCA), which specifically expressed in the goblet cells among epithelia, play a critical role in asthma. At present, the known CLCAs include mCLCA3 (mouse) and hCLCAl (human). In a word, CLCA is a functional unit of goblet cell involving asthma and a potential target for prevention and therapy of asthma.[AIM] Compared with chemical substance, peptide and gene interference, antibody would be more accurate and specific in the role of inhibiting CLCA. We aimed to (1)observe whether DNA vaccine based on xenogeneichomologous hCLCA1 could block mCLCA3 through inducing the crossing antibody for mCLCA3 in asthmatic mice, (2)study the mechanism of vaccine in murine asthma model in vivo and goblet cells model in vitro.[METHODS] (1) The DNA vaccine was constructed by inserting hCLCAl gene into pSecTag2B, and BALB/c mice were vaccinated by i.m. once every two weeks. Sera of mice were collected and vital signs were observed. Genes of three extracellular domains (EDs) of mCLCA3, N-ED, C-ED and M-ED were amplifying with PCR and reconstructed into pRSET-A/N-ED, pRSET-A/C-ED and pGEX-4T-l/M-ED, then three EDs were expressed in E.coli BL21(DE3) and purified. The binding activities of immune sera to three EDs were examined with ELISA and Western blotting analysis. (2) Asthma model was induced with ovalbumin in mice of vaccine group, control vector group and asthma group. Airway pressure-time index (APTI) and number of cells in BALF were checked. The quantity of goblet cells in bronchiole and the level of MUC5AC mRNA in lung were investigated by PAS staining and RT-PCR assay. The levels of protein and mRNA of GM-CSF were observed with immunohitochemical staining and RT-PCR analysis. (3)The NCI-H292/ hCLCAl cell line that stably expressed hCLCA1 was established in vitro. The changes in proliferation of cells, levels of protein and mRNA of MUC5AC and GM-CSF were compared when exposed to normal sera and immune sera.[RESULTS]一 Construction of hCLCAl DNA vaccine and its immunologic characteristics1. Sequences of hCLCAl and mCLCA3 were in accordance with NO.AF039400 and NO.NM-017474 in GeneBank, respectively. pSecTag2-B/hCLCAl was restructured and then injected into mice to collect immune sera.2. SDS-PAGE analysis showed that 6His-N-ED, GST-M-ED and 6His-C-ED were expressed successfully and their MrX 103 were 25.21, 33.94 and 29.31, respectively. The productions existed in form of inclusion bodies and were purified.3. The serum anbodies of vaccinated mice could bind to three EDs of mCLCA3, and tites of sera showed the increasing trend with time. The binding tites to 6His-N-ED and 6His-C-ED were higher than those of GST-M-ED.4. The organism reliability of DNA vaccine was good. The quantity of goblet cells and the synthesis of mucin in intestinal tract were nomal.二 Preventive effect of hCLCA1 DNA vaccine on asthma in mice1. Compared with normal group, the levels of APTI and numbers of eosinophils in asthma group, control vector group and DNA vaccine group were significantly higher. However, the above indexes in DNA vaccine group were decreased remarkbaly than asthma group and vector group.2. There appeared a lot of goblet cells in bronchiole epithela in asthma group and control vector group by PAS staining and the levels of lung MUC5AC mRNA also increased. The number of goblet cells and MUC5AC...
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