【摘要】：目的：觀(guān)察不同濃度肺炎支原體脂質(zhì)相關(guān)膜蛋白(Mycoplasma pneumoniae lipid-associated membrane proteins,MP-LAMPs)在不同時(shí)間點(diǎn)對(duì)人支氣管上皮細(xì)胞(HBECs)短的上腭、肺及鼻咽上皮克隆1(Short Palate Lung and Nasal epithelium Clone1, SPLUNC1)表達(dá)的影響,探討SPLUNC1在呼吸道感染中的作用及其表達(dá)與肺炎支原體感染的時(shí)效關(guān)系和量效關(guān)系。 方法：①通過(guò)間接免疫熒光法觀(guān)察SPLUNC1蛋白在HBECs細(xì)胞內(nèi)定位；②不同濃度MP-LAMPs (0.10.5、1.0、5.0μg/mL)刺激HBECs后,通過(guò)實(shí)時(shí)熒光定量FQ-PCR法檢測(cè)不同時(shí)間點(diǎn)(2hμ4hμ8hμ16h及24h) SPLUNC1基因表達(dá)；同時(shí)用0.5μg/mL MP-LAMPs誘導(dǎo)HBECs不同時(shí)間點(diǎn)通過(guò)Western-blotting法檢測(cè)SPLUNC1蛋白的表達(dá)。③MTT法檢測(cè)不同濃度的MP-LAMPs和LPS對(duì)HBECs細(xì)胞增殖的影響,找出對(duì)HBECs細(xì)胞增殖影響相似的MP-LAMPs與LPS濃度,比較MP-LAMPs與LPS刺激HBECs后,SPLUNC1基因表達(dá)的差異。 結(jié)果：①SPLUNC1蛋白主要分布于細(xì)胞胞漿中。②在相同時(shí)間點(diǎn),不同濃度的MP-LAMPs能誘導(dǎo)SPLUNC1表達(dá)上調(diào),其中0.5μg/mL MP-LAMPs濃度組SPLUNC1基因表達(dá)量最顯著,不同濃度組間兩兩比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)；在同一濃度組,MP-LAMPs刺激細(xì)胞2h后SPLUNC1基因及蛋白表達(dá)量開(kāi)始上調(diào),并在4h時(shí)達(dá)高峰,隨著時(shí)間延長(zhǎng)逐漸下降,不同時(shí)間組兩兩比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)。③與MP-LAMPs刺激細(xì)胞后24h內(nèi)SPLUNC1基因表達(dá)先增高后降低不同,LPS刺激細(xì)胞后同時(shí)間內(nèi)SPLUNC1基因表達(dá)呈持續(xù)增高趨勢(shì)。 結(jié)論：①M(fèi)P-LAMPs能誘導(dǎo)HBECs中的SPLUNC1基因和蛋白表達(dá)上調(diào),并存在一定的時(shí)間和劑量依耐性,其表達(dá)量最佳濃度和時(shí)間點(diǎn)為0.5μg/mL MP-LAMPs刺激HBECs后4h。②不同濃度的MP-LAMPs LPS刺激細(xì)胞后,24h內(nèi)SPLUNC1基因表達(dá)變化趨勢(shì)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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R563.1

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