【摘要】：目的： 以正常鼻咽上皮細(xì)胞NP69作參照,建立人鼻黏膜原代上皮細(xì)胞模型,探索脂多糖(LPS)和腫瘤壞死因子a(TNF-a)對人鼻黏膜上皮細(xì)胞誘導(dǎo)hBD-2的可表達(dá)性和差異性,以及不同濃度與誘導(dǎo)hBD-2表達(dá)的強(qiáng)弱和持續(xù)時(shí)間的關(guān)系。 方法： 1.采用人下鼻甲黏膜,以工型膠原酶加胰酶消化法分離上皮細(xì)胞,用添加了生長因子和牛下丘腦提取物的Keratinocyte-SFM培基,在體外培養(yǎng)原代鼻黏膜上皮細(xì)胞,并通過免疫細(xì)胞化學(xué)法對培養(yǎng)的鼻黏膜上皮細(xì)胞和復(fù)蘇的NP69細(xì)胞進(jìn)行細(xì)胞角蛋白鑒定。2.應(yīng)用PCR和免疫細(xì)胞化學(xué)技術(shù)檢測脂多糖(LPS)和腫瘤壞死因子a(TNF-a)誘導(dǎo)鼻黏膜上皮細(xì)胞和NP69細(xì)胞中hBD-2的表達(dá)量。 結(jié)果： 1.體外培養(yǎng)的人鼻黏膜原代細(xì)胞經(jīng)細(xì)胞角蛋白AE1/AE3鑒定為上皮細(xì)胞,陽性率高達(dá)92.97%,較NP69細(xì)胞高； 2.不同質(zhì)量濃度的LPS(1、10mg/L)、TNF-a(0.1、1、10、100ng/ml)刺激人鼻黏膜上皮細(xì)胞4h后,hBD-2mRNA表達(dá)呈劑量依賴性升高,兩兩比較差異顯著(p0.05)。 3.不同質(zhì)量濃度的LPS(0.01、0.1、1、10mg/L)、TNF-a(0.1、1、10、100ng/ml)刺激人鼻咽上皮細(xì)胞(NP69)4h后,hBD-2mRNA表達(dá)均較對照組(未刺激組)降低,兩兩比較差異顯著(p0.05)。 4.一定濃度的LPS(10ug/ml)刺激培養(yǎng)的鼻黏膜上皮細(xì)胞,hBD-2mRNA的表達(dá)量0-4h逐漸升高,4-24h逐漸下降,兩兩比較差異顯著(p0.05)；一定濃度的LPS(10ug-ml)刺激培養(yǎng)的NP69不同時(shí)間后,hBD-2mRNA的表達(dá)量無明顯增加,兩兩比較無明顯差異(p0.05) 5.一定濃度的LPS(10ug/ml).TNF-a(100ng/ml)刺激培養(yǎng)的鼻黏膜上皮細(xì)胞和NP69細(xì)胞4h后,免疫細(xì)胞化學(xué)證實(shí)鼻黏膜上皮細(xì)胞胞漿中hBD-2蛋白表達(dá)；NP69細(xì)胞胞漿基本無hBD-2蛋白表達(dá)。 結(jié)論： 1.本實(shí)驗(yàn)用K-SFM培基成功培養(yǎng)了人鼻黏膜上皮細(xì)胞。 2.脂多糖(LPS)和腫瘤壞死因子a(TNF-a)兩種不同的誘導(dǎo)因素均可誘導(dǎo)人鼻黏膜上皮細(xì)胞中hBD-2的表達(dá),這種表達(dá)不僅具有差異性,而且具有劑量依賴性和時(shí)間依賴性。 3.相同劑量的LPS誘導(dǎo)鼻黏膜上皮細(xì)胞和NP69細(xì)胞兩種不同細(xì)胞hBD-2的表達(dá)時(shí),其時(shí)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R765

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