本文選題：谷胱甘肽S轉(zhuǎn)移酶mu + 重組慢病毒表達質(zhì)粒��； 參考：《中國呼吸與危重監(jiān)護雜志》2017年05期

【摘要】：目的構(gòu)建穩(wěn)定表達谷胱甘肽S轉(zhuǎn)移酶mu5(GSTM5)的人支氣管上皮16HBE細胞株,探討誘導GSTM5核轉(zhuǎn)位的關(guān)鍵因素。方法構(gòu)建表達GSTM5基因的重組慢病毒載體(PLVX-puro-GSTM5-SBP-3*flag-N、PLVX-puro-3*flag-SBP-GSTM5-C)并制備出相應的慢病毒,感染人支氣管上皮16HBE細胞后,經(jīng)嘌呤霉素篩選獲得細胞克隆;實時熒光定量PCR和蛋白印跡法(Western blot)分別檢測細胞株中GSTM5的mRNA、蛋白表達水平;以腫瘤壞死因子-α(TNF-α;10ng/ml)刺激穩(wěn)定轉(zhuǎn)染細胞株(穩(wěn)轉(zhuǎn)株)0.5h,共聚焦熒光顯微鏡檢測GSTM5核轉(zhuǎn)位。結(jié)果成功構(gòu)建穩(wěn)定表達GSTM5人支氣管上皮細胞株(16HBE-GSTM5-SBP-3*flag-N、16HBE-3*flagSBP-GSTM5-C);熒光共聚焦顯微鏡顯示穩(wěn)轉(zhuǎn)株在TNF-α刺激誘導后,GSTM5由胞漿轉(zhuǎn)入胞核,以16HBEGSTM5-SBP-3*flag-N穩(wěn)轉(zhuǎn)株更顯著。結(jié)論 TNF-α刺激可誘導GSTM5人支氣管上皮細胞株中GSTM5發(fā)生核轉(zhuǎn)位,可能與其N端含有非經(jīng)典核定位信號密切相關(guān)。
[Abstract]:Objective to construct a human bronchial epithelial 16HBE cell line stably expressing glutathione S transferase mu5 (GSTM5) and to explore the key factors of inducing GSTM5 nuclear translocation. Methods Recombinant lentivirus vector PLVX-puro-GSTM5-SBP-3 flag-nban-PLVX-puro-3ban-SBP-GSTM5-C) expressing GSTM5 gene was constructed, and the corresponding lentivirus was prepared. Human bronchial epithelial 16HBE cells were infected with PLVX-puro-GSTM5-SBP-3 flag-SBP-GSTM5-C5. The clones were obtained by purine mycin screening. The expression of GSTM5 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot respectively, and the nuclear translocation of GSTM5 was detected by confocal fluorescence microscope (confocal fluorescence microscope) after the tumor necrosis factor- 偽 TNF- 偽 10 ng / ml was stimulated to stably transfect the cell line (stable strain 0. 5 h). Results stable expression of GSTM5 in human bronchial epithelial cell line (16HBE-GSTM5-SBP-3) was successfully constructed, and fluorescent confocal microscopy showed that the stable transformed strain was transferred from cytoplasm to nucleus after TNF- 偽 stimulation. Conclusion TNF- 偽 stimulation can induce nuclear translocation of GSTM5 in GSTM5 human bronchial epithelial cell line, which may be closely related to the presence of non-classical nuclear localization signal in the N-terminal of TNF- 偽.
【作者單位】： 南方醫(yī)科大學研究生學院;廣州軍區(qū)廣州總醫(yī)院呼吸內(nèi)科;廣州軍區(qū)廣州總醫(yī)院MICU科;
【基金】：國家自然科學基金面上項目(81370173);國家自然科學基金面上項目(81070003) 廣東省老年感染與器官功能支持重點實驗室 廣州市老年感染與臟器功能支持重點實驗室資金
【分類號】：R563.8
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本文編號：1921770