CAJ | 학술논문

目的探讨重组人促红细胞生成素(rhEPO)在预防氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVECs)凋亡过程中对天冬氨酸特异性半胱氨酸蛋白酶3(caspases-3)亚家族的影响.方法体外培养HUVECs,将细胞随机分组,分别加入caspase-3抑制剂DEVD-CHO、caspase-8抑制剂z-IETD-fmk、caspase-9抑制剂z-LEHD-fmk各25 μmol/L预孵育24 h后,再加入100 mg/L ox-LDL处理48 h.平行实验中,加入6.25、25.00、100.00 kU/L的rhEPO预处理HUVECs 24 h,再加入100 mg/L ox-LDL处理48 h.用比色法检测caspase-3、caspase-8、caspase-9活性;用四甲基偶氮唑盐(MTT)比色法检测HUVECs活性;用流式细胞术检测caspase-3阳性表达率和HUVECs凋亡率.结果 caspase-3抑制剂、caspase-8抑制剂分别降低各自对应的caspase活性,caspase-9抑制剂可同时降低caspase-3、caspase-9活性(P均<0.05).加入rhEPO预处理后,ox-LDL诱导内皮细胞caspase-3、caspase-9活性及caspase-3阳性表达率降低,且呈剂量依赖性(P均<0.05),而caspase-8活性无明显变化,HUVECs的细胞凋亡率也呈剂量依赖性降低(P均<0.05).结论 rhEPO可呈剂量依赖性抑制ox-LDL诱导HUVECs 凋亡时caspase-3、caspase-9活性,提示ox-LDL诱导HUVECs 凋亡信号通路涉及caspase-3、caspase-9,而与caspase-8无关.
목적 탐토중조인촉홍세포생성소(rhEPO)재예방양화저밀도지단백(ox-LDL)유도인제정맥내피세포(HUVECs)조망과정중대천동안산특이성반광안산단백매3(caspases-3)아가족적영향.방법 체외배양HUVECs,장세포수궤분조,분별가입caspase-3억제제DEVD-CHO、caspase-8억제제z-IETD-fmk、caspase-9억제제z-LEHD-fmk각25 μmol/L예부육24 h후,재가입100 mg/L ox-LDL처리48 h.평행실험중,가입6.25、25.00、100.00 kU/L적rhEPO예처리HUVECs 24 h,재가입100 mg/L ox-LDL처리48 h.용비색법검측caspase-3、caspase-8、caspase-9활성;용사갑기우담서염(MTT)비색법검측HUVECs활성;용류식세포술검측caspase-3양성표체솔화HUVECs조망솔.결과 caspase-3억제제、caspase-8억제제분별강저각자대응적caspase활성,caspase-9억제제가동시강저caspase-3、caspase-9활성(P균<0.05).가입rhEPO예처리후,ox-LDL유도내피세포caspase-3、caspase-9활성급caspase-3양성표체솔강저,차정제량의뢰성(P균<0.05),이caspase-8활성무명현변화,HUVECs적세포조망솔야정제량의뢰성강저(P균<0.05).결론 rhEPO가정제량의뢰성억제ox-LDL유도HUVECs 조망시caspase-3、caspase-9활성,제시ox-LDL유도HUVECs 조망신호통로섭급caspase-3、caspase-9,이여caspase-8무관.
Objective To explore effect of erythropoietin on the caspase-3 subfamily in preventing apoptosis of human umbilical vein endothelial cells (HUVECs) induced by oxidized-low density lipoprotein (ox-LDL). Methods Third-sixth passages of HUVECs were used. Two experiments were conducted. In the first experiment, there was a blank control group, ox-LDL control group (100 mg/L, incubated for 48 hours), and low, medium, and high recombinant human erythropoietin (rhEPO) groups (6.25, 25.00, 100.00 kU/L rhEPO incubated for 24 hours+100 mg/L ox-LDL incubated for 48 hours). Another experimental protocol consisted of groups of the cells pretreated with either caspase-3 inhibitor DEVD-CHO, or caspase-8 inhibitor z-IETD-fmk, or caspase-9 inhibitor z-LEHD-fmk of 25 μmol/L for 24 hours, then HUVECs were exposed ox-LDL (100 mg/L) incubated for 48 hours. The activity of caspase-3, caspase-8, or caspase 9 was determined by caspase colorimetric assay. The cell survival rate was assessed with methyl thiazolyl tetrazolium (MTT) method. The positive expression rate of caspase-3 and apoptotic rate were measured by flow cytometer. Results The activity of caspase-3 was significantly decreased and cell survival rate was increased in the caspase-3 inhibitor group (both P<0.05). The activity of caspase-8 was decreased in the caspase-8 inhibitor group (P<0.05), but the cell survival rate was not significantly different from that of ox-LDL group (P>0.05). The activity of caspase-3 or caspase-9 was lower and cell survival rate was higher in the caspase-9 inhibitior group than that of ox-LDL group (all P<0.05). The pretreatment with rhEPO led to decreased activity of caspase-3, caspase-9, positive expression rate of caspase-3 and apoptotic rate in a dose-dependent manner compared with ox-LDL group (all P<0.05), but the activity of caspase-8 showed no significant difference from rhEPO pretreatment groups (all P>0.05). Conclusion These results demonstrate that rhEPO can significantly inhibit the activity of caspase-3 or caspase-9 in endothelial cell apoptosis in a dose-dependent manner. Activation of caspase-3 or caspase-9 is involved in ox-LDL-induced HUVECs apoptotic signaling pathway, but caspase-8 is not involved.