Human Nasal Mucosa Epithelial Cells Culture And Induction Of Human β-defensin-2(HBD-2)

Objective:To develop a primary culture system of human nasal mucosa epithelial cell contrasting to the normal nasopharynx epithelial cell NP69; to observe the difference of expression of hBD-2after the stimulation of LPS and TNF-a in nasal mucosa epithelial cells; to analyze the relationship between intensity and duration of expression and various concentrations of LPS and TNF-a.Methods:1. Type I collagenase and pancreatic enzyme were used to digest the inferior turbinate mucosa in order to separate epithelial cells; to cultivate the nasal mucosal epithelium cells in vitro with Keratinocyte-SFM culture added growth factors and cow hypothalamus extracts; make cell keratin identification in nasal mucosal epithelium cells and NP69cells by immunocytochemi stry.2. PCR and immunocytochemistry were employed to observe the expression of hBD-2in the human nasal mucosa epithelial cells and NP69cells induced by LPS and TNF-a.Results:1. The nasal mucosa cells cultured in vitro were identified as epithelial cells by keratin AE1/AE3,which positive rate was as high as92.97%, more than NP69cells;2. The hBD-2mRNA was detected at4h after stimulation of LPS(lug/ml-lOug/ml)and TNF-a(0.1ng/ml-100ng/ml) in the nasal mucosal epithelium cells. The expression of hBD-2mRNA was a dose-dependent manner induced by LPS and TNF-a,and the differences were significant in two samples intercomparison(p<0.05).3. The hBD-2mRNA was detected at4h after LPS(0.01ug/ml-10ug/ml)and TNF-a(0.1ng/ml-100ng/ml) stimulate NP69. The expressions of hBD-2mRNA of various stimulant groups were fewer than the control group,and the differences were significant in two samples intercomparison(p<0.05).4. The expression of hBD-2mRNA raised gradually from Oh to4h, and then declined gradually from4h to24h after the nasal mucosal epithelium cells were stimulated by a certain concentration of LPS (10ug/ml). The differences were significant in two samples intercomparison(p<0.05); After NP69were stimulated by a certain concentration of LPS (10ug/ml),the expression of hBD-2mRNA at different times did not present obvious increase.The differences were not significant in two samples intercomparison(p>0.05).5. After the nasal mucosal epithelium cells and NP69cells were stimulated4h by a certain concentration of LPS (10ug/ml) and TNF-a (100ng/ml),The hBD-2protein was detected in cytoplasm of nasal mucosa epithelial cells by immunocytochemistry,but weakly be detected in cytoplasm of NP69cells.Conclusions:1. The nasal mucosal epithelium cells were successfully cultured in the serum-free of Keratinocyte-SFM culture.2. The hBD-2could be induced by different factors such as LPS and TNF-a in the nasal mucosal epithelium cells. The expression not only show variability,but also present dose and time-dependent characteristics.3. The chronergys of expression of hBD-2were different in nasal mucosal epithelium cells and NP69cells induced by LPS with the same dose.