【摘要】：目的探討沙眼衣原體(Chlamydia trachomatis,Ct)質(zhì)粒蛋白pORF5對HeLa細(xì)胞自噬的影響,為進(jìn)一步闡明Ct致病機(jī)制提供實(shí)驗(yàn)依據(jù)。方法 PCR擴(kuò)增Ct pORF5質(zhì)粒蛋白基因,克隆入PLenO-DCE慢病毒質(zhì)粒,構(gòu)建慢病毒重組表達(dá)載體。慢病毒重組表達(dá)載體經(jīng)雙酶切及測序鑒定后與輔助質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,制備慢病毒。收集慢病毒,再感染HeLa細(xì)胞,流式細(xì)胞儀分選獲得pORF5基因穩(wěn)定轉(zhuǎn)染細(xì)胞株(PLenO-DCE/pORF5-HeLa)。實(shí)驗(yàn)同時建立對照細(xì)胞株(PLenO-DCE-HeLa)。血清饑餓處理兩組細(xì)胞24h,Real-time PCR和Western blot檢測微管相關(guān)蛋白1輕鏈3(LC3)、Becin1的蛋白和mRNA表達(dá)水平,計(jì)算LC3-II/LC3I比率;采用間接免疫熒光檢測自噬熒光斑點(diǎn)。結(jié)果 PLenO-DCE/pORF5-HeLa和PLenO-DCE-HeLa細(xì)胞饑餓處理后均出現(xiàn)LC3紅色熒光斑點(diǎn),斑點(diǎn)數(shù)分別為(97.6±12.1)個/細(xì)胞和(34.0±2.6)個/細(xì)胞,差異有統(tǒng)計(jì)學(xué)意義(t=45.36;P0.05);饑餓處理后PLenO-DCE-HeLa和PLenO-DCE/pORF5-HeLa細(xì)胞中LC3、Beclin1的mRNA表達(dá)水平均顯著高于未處理組,其中PLenO-DCE/pORF5-HeLa細(xì)胞中LC3、Beclin1mRNA的表達(dá)水平較未處理組增加3.10倍(t=95.25;P0.01)和0.85倍(t=16.56;P0.05),較PLenO-DCE-HeLa細(xì)胞分別增加1.95倍(t=79.12;P0.01)和1.57倍(t=23.95;P0.05);PLenO-DCE/pORF5-HeLa經(jīng)饑餓處理24h后LC3-Ⅱ/LC3-Ⅰ比率和Beclin1蛋白較未處理組分別增加52.17%和76.00%(t值分別是15.13,57.24;P均0.05),較PLenO-DCE-HeLa細(xì)胞組分別增加1.05倍(t=35.21;P0.05)和4.34倍(t=112.23;P0.01)。結(jié)論 pORF5質(zhì)粒蛋白可誘導(dǎo)HeLa細(xì)胞自噬,可能在Ct致病過程中發(fā)揮重要作用。
[Abstract]:Objective to investigate the effect of Chlamydia trachomatis (Chlamydia trachomatis,Ct) plasmid pORF5 on autophagy of HeLa cells, and to provide experimental evidence for further elucidation of the pathogenesis of chlamydia trachomatis (Chlamydia trachomatis,Ct). Methods Ct pORF5 plasmid protein gene was amplified by PCR and cloned into PLenO-DCE lentivirus plasmid. The recombinant expression vector of lentivirus was identified by double enzyme digestion and sequencing. The recombinant vector was co-transfected into 293T cells to prepare lentivirus. The lentivirus was collected and reinfected with HeLa cells. The stable transfection cell line (PLenO-DCE/pORF5-HeLa) of pORF5 gene was obtained by flow cytometry. The control cell line (PLenO-DCE-HeLa) was also established. Real-time PCR and Western blot were used to detect the protein and mRNA expression of microtubule-associated protein 1 (LC3), Becin1 and LC3-II/LC3I, and indirect immunofluorescence was used to detect the fluorescent spots of autophagy. Results the number of LC3 red fluorescent spots in PLenO-DCE/pORF5-HeLa and PLenO-DCE-HeLa cells was (97.6 鹵12.1) / cell and (34.0 鹵2.6) / cell, respectively. The difference was statistically significant (t = 45.36). P0.05); The expression of LC3,Beclin1 mRNA in PLenO-DCE-HeLa and PLenO-DCE/pORF5-HeLa cells after starvation was significantly higher than that in the untreated group, and the expression of LC3,Beclin1mRNA in PLenO-DCE/pORF5-HeLa cells was 3.10-fold higher than that in the untreated group (tb95.25). P0.01) and 0.85 times (tampon 16.56%), 1.95 times than PLenO-DCE-HeLa cells (79.12% P0.01) and 1.57 times (tF23.95% P0.01); The ratio of LC3- 鈪，

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