天冬氨酸β羟化酶对细胞自噬的调节作用  

Regulation of autophagy by aspartate β - hydroxylase

作　　者：邹婧[1] 何继满[1] Zou Jing;He Jiman(Department of Gastroenterology, Nanfnng Hospital, Southern Medical University, Guangzhou 510515, China)

机构地区：[1]南方医科大学南方医院消化科,广州510515

出　　处：《中华实验外科杂志》2018年第4期642-644,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81170743）

摘　　要：目的观察天冬氨酸β羟化酶（ASPH）在调节细胞自噬中的作用。 方法向OUMS-29、HEK-293T细胞转染ASPH质粒，Western blot检测转染后细胞p62、微管相关蛋白1轻链3（LC3）-Ⅱ的表达；向OUMS-29细胞共转染ASPH、pEGFP-LC3质粒，荧光显微镜下计算绿色荧光蛋白（GFP）-LC3-Ⅱ点状聚集阳性细胞数占GFP阳性细胞数的比率。沉默HuCCT1、SSP25细胞ASPH基因，Western blot检测细胞p62、LC3-Ⅱ的表达，并用噻唑蓝（MTT）法检测细胞自噬诱导剂雷帕霉素（Rapa）或二甲双胍（Met）对细胞生长速率的影响。 结果过表达ASPH使OUMS-29、HEK-293T细胞内LC3-Ⅱ表达下降44%（P＝0.002，P＝0.008），但不改变p62表达量（P＝0.060，P＝0.798）；过表达ASPH使OUMS-29细胞GFP-LC3-Ⅱ点状聚集阳性细胞数占GFP阳性细胞数比率由50.84%降至28.31%（P＝0.002）；ASPH基因沉默使HuCCT1、SSP25细胞内LC3-Ⅱ表达分别上调87%、133%（P＝0.001，P＝0.000），但不影响p62表达（P＝0.677，P＝0.168）；细胞自噬诱导剂雷帕霉素或二甲双胍掩盖ASPH沉默对HuCCT1、SSP25细胞生长速率的影响。 结论ASPH通过抑制LC3-Ⅱ生成抑制细胞自噬；ASPH对细胞自噬的调节可能是其参与细胞生长调控的机制之一。ObjectiveTo investigate the regulation of autophagy by aspartate β-hydroxylase （ASPH）. MethodsASPH plasmid was transfected into OUMS-29 and HEK-293T cells. Then the expression of p62 and microtubule-associated protein 1 light chain 3 （LC3）-Ⅱ was verified by Western blotting. OUMS-29 cells were co-transfected with ASPH and pEGFP-LC3 plasmid. The ratio of green fluorescent protein （GFP）-LC3-Ⅱ punctate positive to GFP positive cells was calculated under fluorescence microscope. ASPH was knocked down in HuCCT1 and SSP25 cells. Western blotting was performed to examine the expression of p62 and LC3-Ⅱ. Then, the effect of autophagy inducer rapamycin （Rapa） or metformin （Met） on cell growth rate was detected by methyl thiazol tetrazolium （MTT） assay. ResultsASPH overexpression could inhibit LC3-Ⅱ expression in OUMS-29 and HEK-293T cells （44% lower than control） （P=0.002, P=0.008）, but did not alter the p62 expression level （P=0.060, P=0.798）. ASPH overexpression could lower the ratio of GFP-LC3-Ⅱ punctate positive cells to GFP positive cells in OUMS-29 cells from 50.84% to 28.31% （P=0.002）. LC3-Ⅱ expression level increased in HuCCT1 and SSP25 cells with ASPH knocked down （87% and 133% higher than control, respectively） （P=0.001, P=0.000）. However, p62 expression level did not change in these cells （P=0.677, P=0.168）. In HuCCT1 and SSP25 cells, Rapa or Met could mask the effect of ASPH knock-down on cell growth. ConclusionASPH suppresses autophagy via inhibiting the generation of LC3-Ⅱ. ASPH might regulate cell growth by modulating autophagy.

关 键 词：天冬氨酸β羟化酶 自噬 细胞生长 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]