CAJ | 학술논문

目的：观察α1抗胰蛋白酶（α1-antitrypsin，AAT）Z型突变体（α1-antitrypsin Z variant，ATZ）表达对细胞自噬水平的影响，探讨ATZ细胞毒作用的可能机制。方法体外培养人胚肾细胞株HEK 293T并转染ATZ真核表达质粒，利用Western blot法检测自噬相关蛋白LC3和p62的表达，免疫荧光染色观察LC3的细胞定位，real-time PCR法检测自噬相关基因Atg5、Atg12和Beclin1的变化，DAB染色观察ATZ过表达对细胞形态的影响。结果与转染空质粒对照组相比，
목적：관찰α1항이단백매（α1-antitrypsin，AAT）Z형돌변체（α1-antitrypsin Z variant，ATZ）표체대세포자서수평적영향，탐토ATZ세포독작용적가능궤제。방법체외배양인배신세포주HEK 293T병전염ATZ진핵표체질립，이용Western blot법검측자서상관단백LC3화p62적표체，면역형광염색관찰LC3적세포정위，real-time PCR법검측자서상관기인Atg5、Atg12화Beclin1적변화，DAB염색관찰ATZ과표체대세포형태적영향。결과여전염공질립대조조상비，
Aim To investigate the effect of α1-anti-trypsin Z variant (ATZ)overexpression on cell autoph-agy.Methods HEK 293T cells were transfected with pcDNA3.1 zeo+/ATM or pcDNA3.1 zeo+/ATZ,e-qual amount of empty vector was used as control.Cells were treated with NH4Cl for 4 hours and processed for detecting ATZ,LC3 and p62 by immunoblot.Mean-while ,expression and intracellular localization of ATZ, LC3 in 293 T cells were observed with double labeled immunofluorescence.The mRNA levels of autophagy-related genes were measured by real-time PCR.Immu-nohistochemistry was used to observe the morphology of ATZ-positive cells.Results Compared with the control,higher LC3Ⅱ levels and LC3 puncta were observed in ATZ transfected cells.Meanwhile,the levelsof p62 were decreased in ATZ transfected cells,andreversed by NH4 Cl (25 mmol·L -1 )treatment.Overexpression of ATZ increased the mRNA levels of Atg5and Atg12,but had no obvious influence on Beclin1.ATZoverexpressing cells presented abnormal morphologies.The nuclei became reduced,condensed,and even disappeared in ATZpositive cells.Conclusion ATZ overexpression increases autophagy activity whichmay be related to increasing Atg5 and Atg12 levels.