肺炎支原体脂质相关膜蛋白诱导人支气管上皮细胞SPLUNC1表达的变化

发布时间：2019-06-03 13:44

【摘要】：目的：观察不同浓度肺炎支原体脂质相关膜蛋白(Mycoplasma pneumoniae lipid-associated membrane proteins,MP-LAMPs)在不同时间点对人支气管上皮细胞(HBECs)短的上腭、肺及鼻咽上皮克隆1(Short Palate Lung and Nasal epithelium Clone1, SPLUNC1)表达的影响,探讨SPLUNC1在呼吸道感染中的作用及其表达与肺炎支原体感染的时效关系和量效关系。 方法：①通过间接免疫荧光法观察SPLUNC1蛋白在HBECs细胞内定位；②不同浓度MP-LAMPs (0.10.5、1.0、5.0μg/mL)刺激HBECs后,通过实时荧光定量FQ-PCR法检测不同时间点(2hμ4hμ8hμ16h及24h) SPLUNC1基因表达；同时用0.5μg/mL MP-LAMPs诱导HBECs不同时间点通过Western-blotting法检测SPLUNC1蛋白的表达。③MTT法检测不同浓度的MP-LAMPs和LPS对HBECs细胞增殖的影响,找出对HBECs细胞增殖影响相似的MP-LAMPs与LPS浓度,比较MP-LAMPs与LPS刺激HBECs后,SPLUNC1基因表达的差异。 结果：①SPLUNC1蛋白主要分布于细胞胞浆中。②在相同时间点,不同浓度的MP-LAMPs能诱导SPLUNC1表达上调,其中0.5μg/mL MP-LAMPs浓度组SPLUNC1基因表达量最显著,不同浓度组间两两比较差异有统计学意义(P0.01)；在同一浓度组,MP-LAMPs刺激细胞2h后SPLUNC1基因及蛋白表达量开始上调,并在4h时达高峰,随着时间延长逐渐下降,不同时间组两两比较差异有统计学意义(P0.01)。③与MP-LAMPs刺激细胞后24h内SPLUNC1基因表达先增高后降低不同,LPS刺激细胞后同时间内SPLUNC1基因表达呈持续增高趋势。 结论：①MP-LAMPs能诱导HBECs中的SPLUNC1基因和蛋白表达上调,并存在一定的时间和剂量依耐性,其表达量最佳浓度和时间点为0.5μg/mL MP-LAMPs刺激HBECs后4h。②不同浓度的MP-LAMPs LPS刺激细胞后,24h内SPLUNC1基因表达变化趋势不同。
[Abstract]:Objective: to observe the effect of different concentrations of mycoplasma pneumoniae lipid related membrane protein (Mycoplasma pneumoniae lipid-associated membrane proteins,MP-LAMPs) on the cloning of 1 (Short Palate Lung and Nasal epithelium Clone1, in the upper palatal, lung and nasopharyngeal epithelial cells of human bronchial epithelial cells at different time points. The effect of SPLUNC1) on the expression of SPLUNC1 in respiratory tract infection and its relationship with the time-effect and dose-effect relationship of Mycoplasma pneumoniae infection. Methods: 1 the localization of SPLUNC1 protein in HBECs cells was observed by indirect immunofluorescence. 2After different concentrations of MP-LAMPs (0.10.5, 1.0, 5.0 渭 g / mL) stimulated HBECs, the expression of SPLUNC1 gene at different time points (2 h 渭 4 h 渭 8 h 渭 16 h and 24 h) was detected by real-time fluorescence quantitative FQ-PCR. At the same time, the expression of SPLUNC1 protein was detected by Western-blotting assay at different time points in HBECs induced by 0.5 渭 g / mL MP-LAMPs. 3 the effects of different concentrations of MP-LAMPs and LPS on the proliferation of HBECs cells were detected by HBECs assay. The similar concentrations of MP-LAMPs and LPS on the proliferation of HBECs cells were found, and the difference of SPLUNC1 gene expression between MP-LAMPs and LPS stimulated HBECs was compared. Results: 1SPLUNC1 protein was mainly distributed in the cytoplasm. 2 at the same time point, different concentrations of MP-LAMPs could up-regulate the expression of SPLUNC1, and the expression of SPLUNC1 gene was the most significant in 0.5 渭 g / mL MP-LAMPs group. There was significant difference in pairwise between different concentration groups (P 0.01). In the same concentration group, the expression of SPLUNC1 gene and protein began to up-regulate after 2 hours of MP-LAMPs stimulation, and reached the peak at 4 hours, and decreased gradually with the prolongation of time. There was significant difference in pairwise comparison between different time groups (P 0.01). 3 the expression of SPLUNC1 gene increased at first and then decreased within 24 hours after MP-LAMPs stimulation, and the expression of SPLUNC1 gene continued to increase at the same time after LPS stimulation. Conclusion: 1MP-LAMPs can induce the up-regulation of SPLUNC1 gene and protein expression in HBECs, and there is a certain time and dose-dependent tolerance. The optimal concentration and time point of SPLUNC1 gene expression were 0.5 渭 g / mL MP-LAMPs, and the change trend of SPLUNC1 gene expression was different within 24 hours after 4h.2 stimulation with different concentrations of MP-LAMPs LPS.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.1

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