中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2014年
1期
48-52
,共5页
蔡德丰%范建高%马东礼%吴跃平%陆元善
蔡德豐%範建高%馬東禮%吳躍平%陸元善
채덕봉%범건고%마동례%오약평%륙원선
细胞凋亡%硬脂酰辅酶A去饱和酶-1%软脂酸
細胞凋亡%硬脂酰輔酶A去飽和酶-1%軟脂痠
세포조망%경지선보매A거포화매-1%연지산
Apoptosis%Stearoyl-CoA desaturase 1%Palmitate
目的 探讨硬脂酰辅酶A去饱和酶-1(SCD1)过表达影响大鼠BRL肝细胞凋亡的机制. 方法 锥虫兰染色检测软脂酸诱导BRL肝细胞的死亡率以确定下游实验软脂酸添加浓度.预实验确定慢病毒pGC-FU-GFP在大鼠BRL肝细胞中复感染指数.细胞培养分为两大类:非软脂酸加入组,包括一般培养细胞组(CON)、一般培养加阴性病毒对照组(NC)及一般培养过表达病毒组(SCD1-LV)；软脂酸加入组,包括一般培养加软脂酸组(CON+)、一般培养加阴性病毒和软脂酸组(NC+)及一般培养加过表达病毒和软脂酸组(SCD1-LV+).实时荧光定量PCR检测SCD1mRNA,碘化丙啶染色检测各组细胞凋亡情况并进行周期分析.对数据进行方差分析或非参数秩和检验的Kruskal-Wallis H检验、Mann-Whitney U检验.结果 400μmol/L的软脂酸诱导72h后BRL细胞的死亡率显著上升(P＜0.01),病毒的复感染指数为20.与CON和NC组相比,CON+和NC+组SCD1 mRNA表达水平明显下降,SCD1-LV和SCD1-LV+表达水平上升,各组SCD1mRNA的相对表达水平分别为2.36±0.59、2.17±0.46、1.20±0.44、1.00±0.36、7.11±0.55、13.64±0.63,F=289,P＜0.01.CON、NC及SCD1-LV组S期细胞比例差异无统计学意义(P=0.137)；CON+和NC+组出现明显的凋亡峰,且细胞周期中S期细胞比例显著下降,细胞增殖能力下降,SCD1-LV+组凋亡细胞比例显著减少. 结论 pGC-FU-SCD1-GFP慢病毒载体感染BRL肝细胞,SCD1表达水平显著升高,增强细胞对饱和脂肪酸的去饱和作用,过表达SCD1降低软脂酸诱导的脂毒性和凋亡作用.
目的 探討硬脂酰輔酶A去飽和酶-1(SCD1)過錶達影響大鼠BRL肝細胞凋亡的機製. 方法 錐蟲蘭染色檢測軟脂痠誘導BRL肝細胞的死亡率以確定下遊實驗軟脂痠添加濃度.預實驗確定慢病毒pGC-FU-GFP在大鼠BRL肝細胞中複感染指數.細胞培養分為兩大類:非軟脂痠加入組,包括一般培養細胞組(CON)、一般培養加陰性病毒對照組(NC)及一般培養過錶達病毒組(SCD1-LV)；軟脂痠加入組,包括一般培養加軟脂痠組(CON+)、一般培養加陰性病毒和軟脂痠組(NC+)及一般培養加過錶達病毒和軟脂痠組(SCD1-LV+).實時熒光定量PCR檢測SCD1mRNA,碘化丙啶染色檢測各組細胞凋亡情況併進行週期分析.對數據進行方差分析或非參數秩和檢驗的Kruskal-Wallis H檢驗、Mann-Whitney U檢驗.結果 400μmol/L的軟脂痠誘導72h後BRL細胞的死亡率顯著上升(P＜0.01),病毒的複感染指數為20.與CON和NC組相比,CON+和NC+組SCD1 mRNA錶達水平明顯下降,SCD1-LV和SCD1-LV+錶達水平上升,各組SCD1mRNA的相對錶達水平分彆為2.36±0.59、2.17±0.46、1.20±0.44、1.00±0.36、7.11±0.55、13.64±0.63,F=289,P＜0.01.CON、NC及SCD1-LV組S期細胞比例差異無統計學意義(P=0.137)；CON+和NC+組齣現明顯的凋亡峰,且細胞週期中S期細胞比例顯著下降,細胞增殖能力下降,SCD1-LV+組凋亡細胞比例顯著減少. 結論 pGC-FU-SCD1-GFP慢病毒載體感染BRL肝細胞,SCD1錶達水平顯著升高,增彊細胞對飽和脂肪痠的去飽和作用,過錶達SCD1降低軟脂痠誘導的脂毒性和凋亡作用.
목적 탐토경지선보매A거포화매-1(SCD1)과표체영향대서BRL간세포조망적궤제. 방법 추충란염색검측연지산유도BRL간세포적사망솔이학정하유실험연지산첨가농도.예실험학정만병독pGC-FU-GFP재대서BRL간세포중복감염지수.세포배양분위량대류:비연지산가입조,포괄일반배양세포조(CON)、일반배양가음성병독대조조(NC)급일반배양과표체병독조(SCD1-LV)；연지산가입조,포괄일반배양가연지산조(CON+)、일반배양가음성병독화연지산조(NC+)급일반배양가과표체병독화연지산조(SCD1-LV+).실시형광정량PCR검측SCD1mRNA,전화병정염색검측각조세포조망정황병진행주기분석.대수거진행방차분석혹비삼수질화검험적Kruskal-Wallis H검험、Mann-Whitney U검험.결과 400μmol/L적연지산유도72h후BRL세포적사망솔현저상승(P＜0.01),병독적복감염지수위20.여CON화NC조상비,CON+화NC+조SCD1 mRNA표체수평명현하강,SCD1-LV화SCD1-LV+표체수평상승,각조SCD1mRNA적상대표체수평분별위2.36±0.59、2.17±0.46、1.20±0.44、1.00±0.36、7.11±0.55、13.64±0.63,F=289,P＜0.01.CON、NC급SCD1-LV조S기세포비례차이무통계학의의(P=0.137)；CON+화NC+조출현명현적조망봉,차세포주기중S기세포비례현저하강,세포증식능력하강,SCD1-LV+조조망세포비례현저감소. 결론 pGC-FU-SCD1-GFP만병독재체감염BRL간세포,SCD1표체수평현저승고,증강세포대포화지방산적거포화작용,과표체SCD1강저연지산유도적지독성화조망작용.
Objective To investigate the protective mechanism of stearoyl-CoA desaturase 1 (SCD1) over-expression against the pro-apoptotic affects of palmitic acid on hepatocytes using the rat BRL cell line.Methods Concentration effect curves were generated using the trypan blue exclusion test to assess the death rate of BRL ceils upon exposure to a dilution series of palmitic acid.The multiplicity of infection (MOI) of a lentiviral expression vector,pGC-FU-GFP,was determined for the BRL cells.Unmanipulated BRL cells were divided into two groups:the non-palmitate groups were composed of ordinary cultured cells (CON) alone,infected with lentivirus empty expression vector (negative control,NC),and infected with lentivirus overexpressing SCD1 (SCD1-LV); the palmitate groups were composed of ordinary cultured cells plus palmitate (CON+) alone,infected with lentivirus empty expression vector plus palmitate (NC+),and infected with lentivirus overexpressing SCD1 plus palmitate (SCD1-LV+).SCD1 mRNA expression was detected by real-time PCR.Propidium iodide (PI) single-staining was used to detect apoptosis and assess the cell cycle.Inter-group differences were analyzed statistically.Results The death rate of BRL cells increased significantly after 72 h of exposure to 400 μmol/L palmitate (P ＜ 0.01).The MOI of pGC-FU-GFP in BRL cells was 20.The expression of SCD1 was significantly higher in the SCD1-LV and SCD1-LV+ groups than in the respective controls (vs.CON:F =289,P ＜ 0.01; vs.CON+:F =1522,P ＜ 0.01).Palmitate exposure led to decreased expression of SCD1 (CON+ vs.CON,F =22,P ＜ 0.05 and NC+ vs.NC:F =34,P ＜ 0.05).The ratio of S stage cells was similar in all non-palmitate groups (CON,NC and SCD1-LV,P =0.137).However,there was a significant apoptotic peak and lower ratio of S stage cells in the control palmitate groups (CON+ and NC+) and the activity of cell proliferation was decreased as well.The ratio of apoptotic cells was decreased significantly in the SCD1-LV+ group compared to the CON+ group (P ＜ 0.01).Conclusion The expression of SCD 1 and its desaturation activity increased in BRL cells upon infection with the pGC-FU-SCD1-GFP lentiviral vector,suggesting that SCD1 over-expression can decrease palmitic acid-induced toxicity and apoptosis in hepatocytes.
