细胞内酸化对自噬的影响

摘要

Objective To explore the influence of intracellular acidification on autophagy. Methods Using the pAd-miR-NHEl virus levitation liquid to infect SH-SY5Y cells, for the preparation of cell model of low NHE-1 gene, observing the formation of autophagy, changes of mitochondrial form and lysosome number of negative control group, the group after the succesful transfection 0.5, 2, 4, 8 h of adenovirus levitation liquid by transmission electron microscopy, testing the ex-pression of autophagy marker-LC3 by Western blotting. Results Through the transmission electron microscopy, the normal mitochondrial form and lysosome number were shown in negative control group. After the succesful transfection 0.5-8 h of adenovirus levitation liquid, swelling mitochondria, the lysosome populations, autophagy vesicles increased, the autophagy body formed. After 8 hours, apoptotic body appeared, mitochondria numbers decreased, no autophagy body, a bit lyso-somes. There was a low LC-3 expression in negative control group, after the succesful transfection 0.5 h, the expression of LC-3 rose (P <0.01) , then reached the peak in 4 h (P <0.01). In 8 h, the expression of LC-3 was lowest, it was lower than that in negative control group (P<0.01). Conclusions Cells acidification could activate the happening of autoph-agy.%目的 探讨细胞内酸化对自噬的影响.方法 利用pAd-miR-NHE-1病毒悬液转染人源性神经母细胞瘤细胞株,制备NHE-1基因敲低细胞模型.在透射电镜下观察阴性对照组及腺病毒转组转染成功后0.5、2、4、8h自噬体的形成、线粒体形态及溶酶体数目的变化；Western blotting法检测自噬标记物LC3Ⅱ的表达.结果 透射电镜下,阴性对照组线粒体形态及溶酶体数目正常,腺病毒转染成功后0.5～4 h可见线粒体肿胀、变形,溶酶体数目增多,自噬囊泡增多,自噬小体形成.8h可见凋亡小体出现,线粒体数目减少,未见自噬小体,可见少量溶酶体.Western blotting显示,阴性对照组LC3Ⅱ表达较低,腺病毒转染成功后0.5 h LC3Ⅱ表达升高(P＜0.01),4h达高峰(P＜0.01),8h表达降低,且低于阴性对照组(P＜0.01).结论 细胞内酸化可以激活自噬的发生.