人鼻黏膜上皮细胞的培养及hBD-2的诱导
发布时间：2018-11-05 07:34
【摘要】：目的： 以正常鼻咽上皮细胞NP69作参照,建立人鼻黏膜原代上皮细胞模型,探索脂多糖(LPS)和肿瘤坏死因子a(TNF-a)对人鼻黏膜上皮细胞诱导hBD-2的可表达性和差异性,以及不同浓度与诱导hBD-2表达的强弱和持续时间的关系。 方法： 1.采用人下鼻甲黏膜,以工型胶原酶加胰酶消化法分离上皮细胞,用添加了生长因子和牛下丘脑提取物的Keratinocyte-SFM培基,在体外培养原代鼻黏膜上皮细胞,并通过免疫细胞化学法对培养的鼻黏膜上皮细胞和复苏的NP69细胞进行细胞角蛋白鉴定。2.应用PCR和免疫细胞化学技术检测脂多糖(LPS)和肿瘤坏死因子a(TNF-a)诱导鼻黏膜上皮细胞和NP69细胞中hBD-2的表达量。 结果： 1.体外培养的人鼻黏膜原代细胞经细胞角蛋白AE1/AE3鉴定为上皮细胞,阳性率高达92.97%,较NP69细胞高； 2.不同质量浓度的LPS(1、10mg/L)、TNF-a(0.1、1、10、100ng/ml)刺激人鼻黏膜上皮细胞4h后,hBD-2mRNA表达呈剂量依赖性升高,两两比较差异显著(p0.05)。 3.不同质量浓度的LPS(0.01、0.1、1、10mg/L)、TNF-a(0.1、1、10、100ng/ml)刺激人鼻咽上皮细胞(NP69)4h后,hBD-2mRNA表达均较对照组(未刺激组)降低,两两比较差异显著(p0.05)。 4.一定浓度的LPS(10ug/ml)刺激培养的鼻黏膜上皮细胞,hBD-2mRNA的表达量0-4h逐渐升高,4-24h逐渐下降,两两比较差异显著(p0.05)；一定浓度的LPS(10ug-ml)刺激培养的NP69不同时间后,hBD-2mRNA的表达量无明显增加,两两比较无明显差异(p0.05) 5.一定浓度的LPS(10ug/ml).TNF-a(100ng/ml)刺激培养的鼻黏膜上皮细胞和NP69细胞4h后,免疫细胞化学证实鼻黏膜上皮细胞胞浆中hBD-2蛋白表达；NP69细胞胞浆基本无hBD-2蛋白表达。 结论： 1.本实验用K-SFM培基成功培养了人鼻黏膜上皮细胞。 2.脂多糖(LPS)和肿瘤坏死因子a(TNF-a)两种不同的诱导因素均可诱导人鼻黏膜上皮细胞中hBD-2的表达,这种表达不仅具有差异性,而且具有剂量依赖性和时间依赖性。 3.相同剂量的LPS诱导鼻黏膜上皮细胞和NP69细胞两种不同细胞hBD-2的表达时,其时效性不同。
[Abstract]:Objective: to establish a primary epithelial cell model of human nasal mucosa using normal nasopharyngeal epithelial cells (NP69) as reference. To explore the expression of hBD-2 induced by lipopolysaccharide (LPS) and tumor necrosis factor a (TNF-a) in human nasal epithelial cells, and the relationship between different concentrations and the intensity and duration of hBD-2 expression. Methods: 1. Human inferior turbinate mucosa was used to isolate epithelial cells by collagenase and trypsin digestion. Primary nasal mucosal epithelial cells were cultured in vitro by Keratinocyte-SFM culture, which was supplemented with growth factor and bovine hypothalamus extract. Cytokeratin was identified by immunocytochemistry in cultured nasal epithelial cells and resuscitated NP69 cells. 2. 2. The expression of hBD-2 in nasal epithelial cells and NP69 cells induced by lipopolysaccharide (LPS) and tumor necrosis factor a (TNF-a) was detected by PCR and immunocytochemistry. Results: 1. The primary cells of human nasal mucosa were identified as epithelial cells by cytokeratin AE1/AE3 in vitro, and the positive rate was 92.97%, which was higher than that of NP69 cells. 2. The expression of hBD-2mRNA in human nasal epithelial cells was increased in a dose-dependent manner with different concentrations of LPS (110 mg / L) and TNF-a (0.1 mg / ml) in a dose-dependent manner (p0.05). 3. The expression of hBD-2mRNA in human nasopharyngeal epithelial cells (NP69) stimulated with different concentrations of LPS (0.01g / L) and TNF-a (0.1 mg / L) for 4 h was lower than that in control group (unstimulated group). The difference was significant (p0.05). 4. In nasal epithelial cells stimulated by certain concentration of LPS (10ug/ml), the expression of hBD-2mRNA increased gradually from 0-4 h to 24 h, and decreased gradually from 4 to 24 h, the difference was significant (p0.05). The expression of hBD-2mRNA in NP69 stimulated by LPS (10ug-ml) at a certain concentration did not increase significantly at different time, but there was no significant difference between two groups (p0.05) 5. After a certain concentration of LPS (10ug/ml), TNF-a (100ng/ml) stimulated cultured nasal epithelial cells and NP69 cells for 4 h, the expression of hBD-2 protein in the cytoplasm of nasal epithelial cells was confirmed by immunocytochemistry. There was no expression of hBD-2 protein in cytoplasm of NP69 cells. Conclusion: 1. In this study, human nasal epithelial cells were successfully cultured with K-SFM. 2. Lipopolysaccharide (LPS) and tumor necrosis factor a (TNF-a) can induce the expression of hBD-2 in human nasal epithelial cells in a dose-dependent and time-dependent manner. 3. When the same dose of LPS induces the expression of hBD-2 in nasal epithelial cells and NP69 cells, its timeliness is different.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R765
本文编号：2311358
[Abstract]:Objective: to establish a primary epithelial cell model of human nasal mucosa using normal nasopharyngeal epithelial cells (NP69) as reference. To explore the expression of hBD-2 induced by lipopolysaccharide (LPS) and tumor necrosis factor a (TNF-a) in human nasal epithelial cells, and the relationship between different concentrations and the intensity and duration of hBD-2 expression. Methods: 1. Human inferior turbinate mucosa was used to isolate epithelial cells by collagenase and trypsin digestion. Primary nasal mucosal epithelial cells were cultured in vitro by Keratinocyte-SFM culture, which was supplemented with growth factor and bovine hypothalamus extract. Cytokeratin was identified by immunocytochemistry in cultured nasal epithelial cells and resuscitated NP69 cells. 2. 2. The expression of hBD-2 in nasal epithelial cells and NP69 cells induced by lipopolysaccharide (LPS) and tumor necrosis factor a (TNF-a) was detected by PCR and immunocytochemistry. Results: 1. The primary cells of human nasal mucosa were identified as epithelial cells by cytokeratin AE1/AE3 in vitro, and the positive rate was 92.97%, which was higher than that of NP69 cells. 2. The expression of hBD-2mRNA in human nasal epithelial cells was increased in a dose-dependent manner with different concentrations of LPS (110 mg / L) and TNF-a (0.1 mg / ml) in a dose-dependent manner (p0.05). 3. The expression of hBD-2mRNA in human nasopharyngeal epithelial cells (NP69) stimulated with different concentrations of LPS (0.01g / L) and TNF-a (0.1 mg / L) for 4 h was lower than that in control group (unstimulated group). The difference was significant (p0.05). 4. In nasal epithelial cells stimulated by certain concentration of LPS (10ug/ml), the expression of hBD-2mRNA increased gradually from 0-4 h to 24 h, and decreased gradually from 4 to 24 h, the difference was significant (p0.05). The expression of hBD-2mRNA in NP69 stimulated by LPS (10ug-ml) at a certain concentration did not increase significantly at different time, but there was no significant difference between two groups (p0.05) 5. After a certain concentration of LPS (10ug/ml), TNF-a (100ng/ml) stimulated cultured nasal epithelial cells and NP69 cells for 4 h, the expression of hBD-2 protein in the cytoplasm of nasal epithelial cells was confirmed by immunocytochemistry. There was no expression of hBD-2 protein in cytoplasm of NP69 cells. Conclusion: 1. In this study, human nasal epithelial cells were successfully cultured with K-SFM. 2. Lipopolysaccharide (LPS) and tumor necrosis factor a (TNF-a) can induce the expression of hBD-2 in human nasal epithelial cells in a dose-dependent and time-dependent manner. 3. When the same dose of LPS induces the expression of hBD-2 in nasal epithelial cells and NP69 cells, its timeliness is different.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R765
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