Electrophysiological Study On HCO₃-secretion By Distal Airway Epithelium

The electrolytes transport and regulated epithelial surface liquid transport by airway epithelium is the important aspect in non-specific immune function of respiratory system. Some common diseases such as chronic obstruction pulmonary disease, asthma and hereditary Cystic Fibrosis (CF) lung disease arise from this distal (small) airway. The formation of epithelial surface liquid and its transport properties in small airway play an important role in the physiology and pathology of these diseases. However, little is known about the ion transport and the pathogenesis of many related respiratory diseases in small airway epithelia. Therefore, further studies on ion transport mechanisms of distal airway will reveal the physiological and pathological characteristics of respiratory tract and provide theoretical basis for the defence and treatment of respiratory diseases. Recent researches indicate that HCO₃^- secretion by epithelial cells is important for regulating pH of airway surface liquid and host defense of respiratory system. To date the HCO₃^- transport properties of the distal airway epithelia has not been clearly explained.In this study, the ion transport properties of freshly isolated intact epithelium of pig distal airway were electrophysiologocally investigated using short-circuit current technology. The effects of the agonists such as cAMP-evoking agents forskolin, phosphodiesterase inhibitor IBMX and a Chinese medicinal formula, Huoxiang-zhengqi liquid (HZL) on HCO₃^- secretion by intact porcine distal airway epithelium were detected. The results suggested that HCO₃^- could be secreted by porcine distal airway epithelium under basal and stimulated conditions. HCO₃^- secretion was induced by HZL but did not affected by pretreating tissues with forskolin/IBMX, suggesting that there may be independent of the cAMP-dependent pathway. The stimulatory effect of HZL on HCO₃^- secretion in the distal airway epithelium showed HZL to be a hopeful new agonist for distal airway HCO₃^- secretion that could be of therapeutic significance.In this thesis, primary culture of small airway epithelial cells in vitro was studied. The culture condition was optimized and the gas-liquid interface primary culture system was initially established. During primary culture, the effect of antibiotics, the conditions of enzyme digestion and the types of medium were chosen. The results showed that the increased concentration of penicillin/streptomycin in PBS and the addition of gentamicin into enzyme and medium could effectively avoid bacterial contamination. Our results also showed that the proteinase digestion overnight at 4oC, and medium containing 10% serum replaced with low serum could achieve better cell cultured results. The mechanism of ion transport by the monolayer epithelial cells was also studied using short-circuit current technology. The stimulating effect of forskolin/IBMX and HZL on HCO₃^- secretory Isc were examined. The results showed that in cultured epithelia, forskolin/IBMX and HZL could also induce HCO₃^- secretion, consistent with the results from natural epithelia, which further testify the effect of forskolin/IBMX and HZL on HCO₃^- secretion at cellular level.In summary, the effect of forskolin/IBMX and huoxiang zhengqi liquid on HCO₃^- secretion by natural epithelium and cultured epithelium were investigated in this thesis. The regulatory effect on HCO₃^- secretion stimulated by above agonists were studied at tissue and cellular level, which provided experimental basis for related physiological and pathological mechanism. Also, the established porcine small airway epithelial culture system provided a good experimental model to be widely used in studies of respiratory function, pathological changes and immune regulation mechanism.