CAJ | 학술논문

目的：研究中脑星形胶质细胞来源的神经营养因子( mesencephalic astrocyte-derived neurotrophic factor, MANF )对糖氧剥夺/再灌注( oxygen-glucose deprivation /reperfusion, OGD/R)诱导的人神经母细胞瘤细胞( SH-SY5Y)损伤的保护作用。方法体外培养SH-SY5Y细胞,对细胞进行糖氧剥夺(OGD)6 h,再灌注(R)12 h。在再灌注时期,根据是否给予重组人MANF蛋白处理(2μmol·L-1,12 h),将细胞分为正常对照组( NC)、NC+MANF组、OGD/R组和OGD/R+MANF组。随后光学显微镜下观察SH-SY5Y细胞形态的改变,MTT法检测细胞存活率, PI染色法检测细胞死亡率,免疫印迹法检测内源性MANF蛋白、内质网( ER)应激相关蛋白 GRP78/BiP、p-IRE1、p-eIF2α及促凋亡蛋白 cleaved caspase-3和 CHOP 的表达。结果光学显微镜下可见OGD/R组SH-SY5Y细胞胞体变小、变圆,突起缩短或消失。进一步研究发现, MANF 能明显改善 OGD/R 诱导的 SH-SY5Y细胞存活率下降和死亡率增加。免疫印迹法检测发现：OGD/R组细胞内源性MANF蛋白表达增高；ER应激相关蛋白GRP78/BiP、p-IRE1、p-eIF2α表达增高；促凋亡蛋白CHOP及cleaved caspase-3的表达明显高于NC组。给予重组人 MANF 蛋白可降低 OGD/R 组 GRP78/BiP、CHOP 及cleaved caspase-3的表达水平。结论 OGD/R 可诱导凋亡性ER应激；MANF蛋白可通过抑制OGD/R诱导的凋亡性ER应激而对SH-SY5Y细胞具有保护作用。
목적：연구중뇌성형효질세포래원적신경영양인자( mesencephalic astrocyte-derived neurotrophic factor, MANF )대당양박탈/재관주( oxygen-glucose deprivation /reperfusion, OGD/R)유도적인신경모세포류세포( SH-SY5Y)손상적보호작용。방법체외배양SH-SY5Y세포,대세포진행당양박탈(OGD)6 h,재관주(R)12 h。재재관주시기,근거시부급여중조인MANF단백처리(2μmol·L-1,12 h),장세포분위정상대조조( NC)、NC+MANF조、OGD/R조화OGD/R+MANF조。수후광학현미경하관찰SH-SY5Y세포형태적개변,MTT법검측세포존활솔, PI염색법검측세포사망솔,면역인적법검측내원성MANF단백、내질망( ER)응격상관단백 GRP78/BiP、p-IRE1、p-eIF2α급촉조망단백 cleaved caspase-3화 CHOP 적표체。결과광학현미경하가견OGD/R조SH-SY5Y세포포체변소、변원,돌기축단혹소실。진일보연구발현, MANF 능명현개선 OGD/R 유도적 SH-SY5Y세포존활솔하강화사망솔증가。면역인적법검측발현：OGD/R조세포내원성MANF단백표체증고；ER응격상관단백GRP78/BiP、p-IRE1、p-eIF2α표체증고；촉조망단백CHOP급cleaved caspase-3적표체명현고우NC조。급여중조인 MANF 단백가강저 OGD/R 조 GRP78/BiP、CHOP 급cleaved caspase-3적표체수평。결론 OGD/R 가유도조망성ER응격；MANF단백가통과억제OGD/R유도적조망성ER응격이대SH-SY5Y세포구유보호작용。
Aim To investigate the protective effects of MANF on human neuroblastoma SH-SY5 Y cells suf-fering from oxygen-glucose deprivation/reperfusion ( OGD/R) and the underlying mechanism. Methods SH-SY5Y cells were treated with OGD for 6 h, fol-lowed by reperfusion for 12 h. Meanwhile, the cells were incubated with 2 μmol · L-1 recombinant human protein MANF for 12 h during reperfusion. The cell morphology was observed under an optical microscope. The cell viability was determined by MTT assay. PI <br> staining was performed to detect the number of dead cells. Western blot was performed to determine the protein levels of endogenous MANF, glucose-related protein 78 ( GRP78/BiP) , phosphorylated inositol re-quiring enzyme 1 ( p-IRE1 ) , phosphorylated eukaryot-ic translation initiator factor 2α ( p-eIF2α) , cleaved caspase-3, and C/EBP-homologous protein (CHOP). Results The cells exposed to OGD/R became smaller and round, and the neurites of the cells were shortened or disappeared . Recombinant human protein MANF <br> improved the survival rate ( P <0. 05 ) and decreased the death rate ( P <0. 05 ) of SH-SY5 Y cells treated with by OGD/R. Western blot assay showed that the endoplasmic reticulum ( ER) stress-associated proteins GRP78/BiP, p-IRE1, p-eIF2α, and MANF were in-creased significantly after OGD/R treatment, compared with the untreated controls. However, the increases of secretion levels of apoptosis-associated proteins CHOP <br> and cleaved caspase-3 in SH-SY5 Y cells induced by OGD/R were significantly suppressed by MANF. Con-clusion OGD/R up-regulates the ER stress-associated proteins and causes apoptosis. MANF inhibits OGD/R-induced cell death, which may be related to attenua-ting ER stress-induced apoptosis.