中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2010年
1期
10-13
,共4页
关杨%姜袆群%高莹%陈浩%曾学思%孙建方
關楊%薑袆群%高瑩%陳浩%曾學思%孫建方
관양%강위군%고형%진호%증학사%손건방
肿瘤细胞%培养的%黑色素瘤%细胞增殖%细胞凋亡%组氨酸三聚体核苷结合蛋白1
腫瘤細胞%培養的%黑色素瘤%細胞增殖%細胞凋亡%組氨痠三聚體覈苷結閤蛋白1
종류세포%배양적%흑색소류%세포증식%세포조망%조안산삼취체핵감결합단백1
Tumor cells,cultured%Melanoma%Cell proliferation%Apoptosis%HINT1
目的 探讨HINTl基因对人黑素瘤细胞A375增殖、凋亡的影响及其作用机制.方法 应用构建的peDNA.3.1/mye-His(-)A-HINTl真核表达载体,建立稳定转染且能高表达HINTI的A375细胞模型.用M1rr法检测细胞增殖活性变化,流式细胞仪检测细胞周期及细胞凋亡率,TUNEL法检测细胞凋亡.分光光度法检测Caspase 3,Caspase 8及Caspase 9的相对活性.Western印迹法检测bcl-2、bax、细胞色素C及p53的表达.结果 与空载体-A375及未转染A375细胞相比,HINT1-A375细胞生长速度明显减慢(P<0.05);Gl期细胞增多(73.17%±3.99%,F=25.65,P<0.05),S期细胞减少(16.75%±1.62%.F=75.48,P<0.01),细胞出现明显的晚期凋亡(23.57%±9.58%,F=11.71,P<0.01). TUNEL法显示凋亡阳性细胞百分比(12%±1%)显著增高(F=358.02,P<0.01);Caspase 9(0.45±0.03,F=135.62,P<0.01)和Caspase 3表达(0.46±0.04,F=90.28,P<0.01)升高.凋亡相关蛋白bax、细胞色素C及p53表达增加,bel-2表达降低.结论 高表达H1NT1可抑制A375细胞的增殖,促进其凋亡,细胞周期出现G_1期阻滞,提示HINT1可能是人黑素瘤的抑癌基因.
目的 探討HINTl基因對人黑素瘤細胞A375增殖、凋亡的影響及其作用機製.方法 應用構建的peDNA.3.1/mye-His(-)A-HINTl真覈錶達載體,建立穩定轉染且能高錶達HINTI的A375細胞模型.用M1rr法檢測細胞增殖活性變化,流式細胞儀檢測細胞週期及細胞凋亡率,TUNEL法檢測細胞凋亡.分光光度法檢測Caspase 3,Caspase 8及Caspase 9的相對活性.Western印跡法檢測bcl-2、bax、細胞色素C及p53的錶達.結果 與空載體-A375及未轉染A375細胞相比,HINT1-A375細胞生長速度明顯減慢(P<0.05);Gl期細胞增多(73.17%±3.99%,F=25.65,P<0.05),S期細胞減少(16.75%±1.62%.F=75.48,P<0.01),細胞齣現明顯的晚期凋亡(23.57%±9.58%,F=11.71,P<0.01). TUNEL法顯示凋亡暘性細胞百分比(12%±1%)顯著增高(F=358.02,P<0.01);Caspase 9(0.45±0.03,F=135.62,P<0.01)和Caspase 3錶達(0.46±0.04,F=90.28,P<0.01)升高.凋亡相關蛋白bax、細胞色素C及p53錶達增加,bel-2錶達降低.結論 高錶達H1NT1可抑製A375細胞的增殖,促進其凋亡,細胞週期齣現G_1期阻滯,提示HINT1可能是人黑素瘤的抑癌基因.
목적 탐토HINTl기인대인흑소류세포A375증식、조망적영향급기작용궤제.방법 응용구건적peDNA.3.1/mye-His(-)A-HINTl진핵표체재체,건립은정전염차능고표체HINTI적A375세포모형.용M1rr법검측세포증식활성변화,류식세포의검측세포주기급세포조망솔,TUNEL법검측세포조망.분광광도법검측Caspase 3,Caspase 8급Caspase 9적상대활성.Western인적법검측bcl-2、bax、세포색소C급p53적표체.결과 여공재체-A375급미전염A375세포상비,HINT1-A375세포생장속도명현감만(P<0.05);Gl기세포증다(73.17%±3.99%,F=25.65,P<0.05),S기세포감소(16.75%±1.62%.F=75.48,P<0.01),세포출현명현적만기조망(23.57%±9.58%,F=11.71,P<0.01). TUNEL법현시조망양성세포백분비(12%±1%)현저증고(F=358.02,P<0.01);Caspase 9(0.45±0.03,F=135.62,P<0.01)화Caspase 3표체(0.46±0.04,F=90.28,P<0.01)승고.조망상관단백bax、세포색소C급p53표체증가,bel-2표체강저.결론 고표체H1NT1가억제A375세포적증식,촉진기조망,세포주기출현G_1기조체,제시HINT1가능시인흑소류적억암기인.
Objective To explore the effects of HINT1 on the proliferation and apoptosis of human melanoma cell line A375.Methods Three cell clones were used in the experiment.including A375 cells that were previously transfected with eukaryotic expressing vector pcDNA.3.1/myc-His(-)A-HINT1 and highly expressed HINTI(HINT1-A375),A375 cells transfected with pcDNA.3.1/myc-His(-)A empty vector(neo-A375)and untransfected A375 cells.M3T assay,flow cytometry and terminal deoxynucleotjdyl transferase (TdT)-mediated dUTP nick end labeling(TUNEL)were performed to detect the proliferation,cell cycle and apoptosis of the three kinds of cells,respectively.The relative activity of Caspase 3,8 and 9 was measured by spectrophotometry,and the protein expression of Bcl-2,Bax,Cytoehrome C and p53 by Western blot. Results Compared with neo-A375 and untransfected A375 cells,HINT1-A375 cells grew more slowly(P<0.05)with an increase in G1-phase population(73.17%±3.99%,F=25.65,P<0.05).a decrease in S-phase population(16.75%±1.62%,F=75.48,P<0.01),and a pronounced late apoptosis(23.57%±9.58%,F=11.71,P<0.01).A significant increase was also observed in the percentage of apoptotic cells(12%±1%,F=358.02,P<0.01)as well as in the relative activity of Caspase 9(0.45±0.03,F=135.62,P<0.01)and Caspase 3(0.46±0.04,F=90.28,P<0.01)in HINT1-A375 cells compared with the other two kinds of cells.Western blot showed upregulated expressions of Bax,Cytochrome C and p53 but downregulated expres-sion of Bel-2 in HINT1-A375 cells.Conclusions The overexpression of HINT1 could inhibit the proliferation and promote the apoptosis of A375 cells with cell cycle arrest in G1 phase,hinting that HINT1 may be a tumor suppressor gene in human melanoma.
