CAJ | 학술논문

目的探讨p53凋亡刺激蛋白2(ASPP2)对饥饿诱导的大肠癌HCT116 p53+/+(p53野生型)细胞凋亡、周期和自噬的影响.方法实验分6组:①对照组；②绿色荧光蛋白腺病毒(rAd-GFP)感染组；③ASPP2腺病毒(rAd-ASPP2)感染组；④饥饿处理组；⑤rAd-GFP+饥饿组；⑥rAd-ASPP2+饥饿组.利用rAd-ASPP2感染使细胞过表达ASPP2基因.无血清培养基培养24h诱导凋亡、自噬和细胞周期改变.钙黄绿素(Calcein)/碘化丙啶(PI)吸收试验观察各组细胞调亡水平.细胞转染红色荧光蛋白标记的CFP-Lc3自噬质粒,荧光显微镜下观察各组细胞自噬水平.流式细胞术观察细胞周期改变.组间比较采用单因素方差分析进行统计学分析.结果 ASPP2过表达显著促进了饥饿诱导的细胞凋亡、自噬及G2-M期阻滞,各组细胞的凋亡率为:rAd-GFP+饥饿组10.00％±1.42％,rAd-ASPP2+饥饿组18.44％ ±2.06％(q=9.548,P=0.000)；各组细胞的自噬发生率为:rAd-GFP+饥饿组35.00％±5.34％,rAd-ASPP2+饥饿组57.61％ ±6.06％(q=7.657,P=0.000).但无饥饿诱导时ASPP2过表达使G0-G1、G2-M期都发生阻滞.结论 ASPP2过表达促进饥饿诱导的大肠癌HCT116 p53+/+细胞凋亡和自噬,显著改变细胞周期进程.
목적 탐토p53조망자격단백2(ASPP2)대기아유도적대장암HCT116 p53+/+(p53야생형)세포조망、주기화자서적영향.방법 실험분6조:①대조조；②록색형광단백선병독(rAd-GFP)감염조；③ASPP2선병독(rAd-ASPP2)감염조；④기아처리조；⑤rAd-GFP+기아조；⑥rAd-ASPP2+기아조.이용rAd-ASPP2감염사세포과표체ASPP2기인.무혈청배양기배양24h유도조망、자서화세포주기개변.개황록소(Calcein)/전화병정(PI)흡수시험관찰각조세포조망수평.세포전염홍색형광단백표기적CFP-Lc3자서질립,형광현미경하관찰각조세포자서수평.류식세포술관찰세포주기개변.조간비교채용단인소방차분석진행통계학분석.결과 ASPP2과표체현저촉진료기아유도적세포조망、자서급G2-M기조체,각조세포적조망솔위:rAd-GFP+기아조10.00％±1.42％,rAd-ASPP2+기아조18.44％ ±2.06％(q=9.548,P=0.000)；각조세포적자서발생솔위:rAd-GFP+기아조35.00％±5.34％,rAd-ASPP2+기아조57.61％ ±6.06％(q=7.657,P=0.000).단무기아유도시ASPP2과표체사G0-G1、G2-M기도발생조체.결론 ASPP2과표체촉진기아유도적대장암HCT116 p53+/+세포조망화자서,현저개변세포주기진정.
Objective To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2)in the apoptosis,cell cycle and autophagy of starvation-induced colorectal cancer HCT116 p53 +/+ (p53 wild-type) cell line.Methods Six groups were included:(1) control group; (2) green fluorescent protein adenovirus (rAd-GFP) infection group; (3)ASPP2 adenovirus (rAd-ASPP2) infection group; (4)starvation group; (5)rAd-GFP + starvation group; (6) rAd-ASPP2 + starvation group.HCT116 cells were infected with ASPP2 adenovirus (rAd-ASPP2),resulting ASPP2 gene over-expression.The apoptosis,autophagy and cell cycle changes were induced by culturing with serum-free medium for 24 h.Apoptosis was evaluated by Calcein/PI uptaking test,and autophagy was observed by counting the red fluorescent protein autophagy plasmid CFP-Lc3 which was transfected into cytoplasm.Cell cycle was detected by flow cytometry.Statistical analysis was performed by one-way analysis of variance (ANOVA).Results Over-expressed ASPP2 was found to significantly promote starvation-induced HCT116 apoptosis and autophagy.The cell apoptosis rate in rAd-GFP + starvation group was 10.00％ ± 1.42％,and 18.44％ ±2.06％ in rAd-ASPP2 + starvation group(q =9.548,P =0.000).The cell autophagy rate in rAd-GFP+ starvation group and rAd-ASPP2 + starvation group was 35.00％ ± 5.34％ and 57.61％ ± 6.06％ respectively(q =7.657,P =0.000).Over-expressed ASPP2 accelerated HCT116 G2/M arrest under starvation,but resulted in both G0/G1 and G2/M arrest without starvation.Conclusion These results suggest that ASPP2 can promote starvation-induced HCT116 p53 +/+ cells apoptosis and autophagy,and affect the cell cycle.