CAJ | 학술논문

目的：：探讨饥饿条件下自噬与凋亡在小鼠成骨细胞中的发生情况与相互关系。方法：培养初代MC3T3-E1小鼠成骨细胞系,将其分为饥饿诱导组与3-甲基腺素(3-MA)预处理饥饿诱导组。其中,饥饿诱导组采用单纯饥饿诱导法,用厄尔氏平衡盐溶液( earle’ s balanced salt solution,EBSS)培养1 h、2 h、3 h、4 h、5 h、6 h。3-MA是一种特异性自噬抑制剂,在3-MA预处理饥饿诱导组中,正常培养的小鼠成骨细胞加入3-MA预处理1 h后,再用EBSS平衡盐溶液培养1 h、2 h、3 h、4 h、5 h、6 h。应用透射电子显微镜观察饥饿诱导组小鼠成骨细胞自噬与凋亡的形态学变化,应用Western blot免疫印迹法和Annex-in V-FITC/PI双标记流式细胞术检测小鼠成骨细胞中自噬相关蛋白LC3,凋亡相关蛋白caspase 3表达水平的变化。结果：透射电镜结果显示单纯饥饿诱导条件下,细胞在2 h前以自噬为主,2 h后以凋亡为主,并随着时间的延长,凋亡水平逐渐增高。Western blot免疫印迹法结果显示与饥饿诱导组相比,3-MA预处理饥饿诱导组的LC3转化水平在1 h、2 h、3 h、4 h、5 h、6 h都出产生了明显的下降趋势(P<0．05)；caspase 3蛋白表达水平在1 h、2 h、3 h和4 h增高(P<0．05),但0 h、5 h和6 h无差异(P>0．05)。 Annexin V-FITC/PI双标记流式细胞术检测结果和单纯饥饿诱导组相比,3-MA预处理组仅在1 h、2 h、3 h和4 h促进了细胞凋亡(P<0．05),而并未影响到0 h、5 h和6 h的细胞凋亡(P>0．05)。结论：饥饿发生后,小鼠成骨细胞的自噬作用通过降低caspase 3的活化水平,在细胞饥饿2 h前拮抗凋亡作用,随着饥饿时间的不断延长,细胞自噬的表达水平在3 h和4 h时不断下降,自噬对细胞的保护不断降低,最终走向凋亡,此时自噬对凋亡的发生将不再有任何作用。
목적：：탐토기아조건하자서여조망재소서성골세포중적발생정황여상호관계。방법：배양초대MC3T3-E1소서성골세포계,장기분위기아유도조여3-갑기선소(3-MA)예처리기아유도조。기중,기아유도조채용단순기아유도법,용액이씨평형염용액( earle’ s balanced salt solution,EBSS)배양1 h、2 h、3 h、4 h、5 h、6 h。3-MA시일충특이성자서억제제,재3-MA예처리기아유도조중,정상배양적소서성골세포가입3-MA예처리1 h후,재용EBSS평형염용액배양1 h、2 h、3 h、4 h、5 h、6 h。응용투사전자현미경관찰기아유도조소서성골세포자서여조망적형태학변화,응용Western blot면역인적법화Annex-in V-FITC/PI쌍표기류식세포술검측소서성골세포중자서상관단백LC3,조망상관단백caspase 3표체수평적변화。결과：투사전경결과현시단순기아유도조건하,세포재2 h전이자서위주,2 h후이조망위주,병수착시간적연장,조망수평축점증고。Western blot면역인적법결과현시여기아유도조상비,3-MA예처리기아유도조적LC3전화수평재1 h、2 h、3 h、4 h、5 h、6 h도출산생료명현적하강추세(P<0．05)；caspase 3단백표체수평재1 h、2 h、3 h화4 h증고(P<0．05),단0 h、5 h화6 h무차이(P>0．05)。 Annexin V-FITC/PI쌍표기류식세포술검측결과화단순기아유도조상비,3-MA예처리조부재1 h、2 h、3 h화4 h촉진료세포조망(P<0．05),이병미영향도0 h、5 h화6 h적세포조망(P>0．05)。결론：기아발생후,소서성골세포적자서작용통과강저caspase 3적활화수평,재세포기아2 h전길항조망작용,수착기아시간적불단연장,세포자서적표체수평재3 h화4 h시불단하강,자서대세포적보호불단강저,최종주향조망,차시자서대조망적발생장불재유임하작용。
Objective:To explore the interaction between autophagy and apoptosis of MC3T3-E1 at starvation state. Methods:MC3T3-E1 of 3-MA group was pretreated with 3-MA for 1 hour before being cultured in earle’s balanced salt solution(EBSS)for anoth-er 6 hours,and the samples without 3-MA pretreatment were taken as starvation group. MC3T3-E1 of different groups were sampled hourly. The morphological change of MC3T3-E1 form starvation group was observed by TEM. To determine the expression of LC3 and caspase 3,western blot and flow cytometry were performed. Results:The observation of TEM showed that MC3T3-E1 from starvation group was dominated by autophagy in the first two hours of the 6-hours period cultured in EBSS,whereas apoptosis was the main process after the first two hours and the level increased with time. The observation of western blot showed that,compared with those from starva-tion group,MC3T3-E1 from 3-MA group presented a reduced LC3 expression level (P<0. 05) throughout the 6-hours period,and an en-hanced caspase 3 expression level in the first 4 hours ( P<0. 05 ) , while there was no difference observed between the two groups (P>0. 05) in the last 2 hours(P>0. 05). The observation of flow cytometry showed that,compared with those from starvation group, MC3T3-E1 from 3-MA group presented an enhanced apoptosis level in the first 4 hours (P<0. 05),while there was no difference ob-served between the two groups (P>0. 05) in the last 2 hours(P>0. 05). Conclusion:Under starvation condition,autophagy of MC3T3-E1 may antagonize apoptosis in the first 2 hours by inhibiting the activity of caspase 3. While the autophagy level reduces in the middle 2 hours,apoptosis may not be affected by autophagy.