CAJ | 학술논문

目的：探讨血管内皮生长因子B（ vascular endothelial growth factor-B，VEGF-B）对氯化钴（ cobalt chloride hexahydrate，CoCl2）诱导的大鼠视网膜神经节细胞株（retinal ganglion cells-5，RGC-5）缺氧损伤的保护作用及其机制。方法 CoCl2孵育RGC-5细胞24h诱导缺氧模型。将RGC-5细胞分为正常组、缺氧组、VEGF-B+缺氧组、PD98059（ ERK1/2阻滞剂，PD）+VEGF-B+缺氧组。倒置显微镜观察细胞的形态变化；CCK-8法检测细胞存活率；流式细胞仪 Annexin V-APC/7-AAD 双染色法检测细胞凋亡率；Western Blot 检测蛋白 Bcl-2、Bax 及 ERK1/2、p-ERK1/2表达水平。结果①缺氧使细胞皱缩变小变圆，正常组及药物组细胞体积大有突起。②与正常组相比，缺氧使细胞存活率减低；与缺氧组相比，药物组细胞存活率均升高。③与正常组相比，缺氧组正常细胞率下降、细胞凋亡率升高；与缺氧组相比，药物组正常细胞率升高、细胞凋亡率下降。④与正常组相比，缺氧组Bcl-2、Bcl-2/Bax比值降低，Bax增多；与缺氧组相比，药物组Bcl-2、Bcl-2/Bax比值增高，Bax减少。四组总ERK1/2表达量不变，p-ERK1/2表达量缺氧组较正常组增多，VEGF-B+缺氧组较缺氧组表达量高，PD+VEGF-B+缺氧组较VEGF-B+缺氧组减少。结论 VEGF-B部分通过ERK1/2信号通路增加Bcl-2表达，降低Bax表达从而抑制CoCl2诱导的RGC-5细胞凋亡。
목적：탐토혈관내피생장인자B（ vascular endothelial growth factor-B，VEGF-B）대록화고（ cobalt chloride hexahydrate，CoCl2）유도적대서시망막신경절세포주（retinal ganglion cells-5，RGC-5）결양손상적보호작용급기궤제。방법 CoCl2부육RGC-5세포24h유도결양모형。장RGC-5세포분위정상조、결양조、VEGF-B+결양조、PD98059（ ERK1/2조체제，PD）+VEGF-B+결양조。도치현미경관찰세포적형태변화；CCK-8법검측세포존활솔；류식세포의 Annexin V-APC/7-AAD 쌍염색법검측세포조망솔；Western Blot 검측단백 Bcl-2、Bax 급 ERK1/2、p-ERK1/2표체수평。결과①결양사세포추축변소변원，정상조급약물조세포체적대유돌기。②여정상조상비，결양사세포존활솔감저；여결양조상비，약물조세포존활솔균승고。③여정상조상비，결양조정상세포솔하강、세포조망솔승고；여결양조상비，약물조정상세포솔승고、세포조망솔하강。④여정상조상비，결양조Bcl-2、Bcl-2/Bax비치강저，Bax증다；여결양조상비，약물조Bcl-2、Bcl-2/Bax비치증고，Bax감소。사조총ERK1/2표체량불변，p-ERK1/2표체량결양조교정상조증다，VEGF-B+결양조교결양조표체량고，PD+VEGF-B+결양조교VEGF-B+결양조감소。결론 VEGF-B부분통과ERK1/2신호통로증가Bcl-2표체，강저Bax표체종이억제CoCl2유도적RGC-5세포조망。
Objective:To expore the protective effects and the underlying mechanism of vascular endothelial growth fac-tor-B(VEGF-B)in retinal ganglion cells line(RGC-5)in rats with hypoxia injury induced by cobalt chloride hexahydrate ( CoCl2 ). Methods:RGC-5 were incubated with CoCl2 to induce hypoxia injury as the apoptosis model. RGC-5 were divided into four groups:normal group,hypoxia group,VEGF-B +Hypoxia group and PD98059( inhibitor of ERK1/2,PD)+VEGF-B+Hypoxia group. The morphology of cells was observed by inverted microscope. The cell viability rate was detec-ted by CCK-8 assay. Annexin-V APC/7-AAD double staining flow cytometry was used to test the normal cells rate and ap-optotic cells rate. The levels of Bcl-2,Bax,EKK1/2 and p-ERK1/2 protein expression were detected by using Western Blot. Results:① The morphology of cells in the normal group and VEGF-B +Hypoxia group were big and with many processes. The cells of the hypoxia group shrinked to be round and smaller. ② The cell viability was decreased by CoCl2 treatment compared with the normal group. VEGF-B treatment significantly increased cell viability compared with the hy-poxia group. ③ The normal cells rate decreased and the apoptotic cells rate increased in the hypoxia group compared with the normal group. VEGF-B treatment significantly increased normal cells rate and decreased apoptotic cells rate compared with the hypoxia group. ④ Compared with the normal group,Bcl-2 protein expression and Bcl-2/Bax ratio decreased and Bax protein expression increased in the hypoxia group. However,VEGF-B treatment up-regulated Bcl-2 protein expression and Bcl-2/Bax ratio and down-regulated Bax protein expression compared with the hypoxia group. Moreover,hypoxia treat-ment up-regulated level of p-ERK1/2 protein compared with Normal group. Level of p-ERK1/2 protein was higher in VEGF-B+Hypoxia group compared with the hypoxia group but there was no change of total ERK1/2 protein expression in the four groups. Conclusion:VEGF-B can inhibit apoptosis induced by CoCl2 in RGC-5 cell line through up-regulating Bcl-2 protein expression and down-regulating Bax protein expression,partially via ERK1/2 signaling pathway.