中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2009年
1期
61-64
,共4页
孙磊%王雯%刘晓梅%金明华%杜海英%彭丹冰%孙志伟
孫磊%王雯%劉曉梅%金明華%杜海英%彭丹冰%孫誌偉
손뢰%왕문%류효매%금명화%두해영%팽단빙%손지위
甲基汞化合物%细胞凋亡%线粒体膜%膜电位
甲基汞化閤物%細胞凋亡%線粒體膜%膜電位
갑기홍화합물%세포조망%선립체막%막전위
Methylmercury compounds%Apoptosis%Mitochondrial membranes%Membrane potential
目的 研究甲基汞对体外培养的人肝细胞株HL-7702的凋亡作用及其机制.方法 实验分为阴性对照组及甲基汞受试物组,甲基汞的浓度分别为10、20、30、40、50 μmol/L.采用丫啶橙(AO)/溴化乙锭(EB)染色法和流式细胞技术检测甲基汞对细胞凋亡的影响;流式细胞技术检测甲基汞对细胞线粒体膜电位的影响;免疫细胞化学方法 检测甲基汞对凋亡相关蛋白表达的影响.结果 不同浓度氯化甲基汞作用24 h均可诱导细胞凋亡,AO/EB染色法检测阴性对照组细胞凋亡率为(2.62±0.19)%,10～50μmol/L 甲基汞受试物组细胞凋亡率分别为(7.97±0.64)%、(12.66±0.76)%、(19.16±0.87)%、(18.42±0.88)%、(11.52±0.63)%,各剂量组细胞凋亡率明显高于对照组(q值分别为17.057、32.009、52.732、50.373、28.375;P＜0.05).随着作用浓度的升高细胞线粒体膜电位明显下降,阴性对照组线粒体膜电位为(10.23±3.43)mV,10～50 μmol/L 甲基汞受试物组线粒体膜电位分别为(3.25±0.66)、(3.03±0.35)、(1.68±1.26)、(1.69±1.13)、(1.77±0.88)mV,各剂量组明显低于对照组(q值分别为9.569、9.871、11.722、11.708、11.598;P＜0.05).随着甲基汞作用浓度的升高 Bax、Bel-2、CytC、Caspase-3 及AIF表达有上升的趋势,且Bax/Bcl-2值升高.阴性对照组Bax表达的积分吸光度值(IOD)为21 295.86±1969.81,10、20、30μmol/L 组Bax表达的IOD分别为42 807.87±4416.64、55 651.65±4662.72、72 708.56±910.10,各剂量组明显高于对照组(g值分别为14.191、14.320、33.917;P＜0.05);阴性对照组Bcl-2表达的IOD为12 588.33±4091.02,10、20、30μmol/L组Bel-2表达的IOD分别为20 539.16±4906.09、23 689.97±2281.42、28692.80±4655.86,各剂量组明显高于对照组(g值分别为4.322、6.035、8.754;P＜0.05);阴性对照组AIF表达的IOD为12 942.72±457.94、10、20、30、40μmol/L组AIF表达的IOD分别为16 973.57±1922.87、29 998.91±6803.58、52 467.16±1916.25、106 342.53±1273.19,20、30及40 μmol/L组明显高于对照组(q值分别为11.449、26.530、62.692;P＜0.05).结论 甲基汞可以通过线粒体通路诱导体外培养的人肝细胞株HL-7702发生凋亡.
目的 研究甲基汞對體外培養的人肝細胞株HL-7702的凋亡作用及其機製.方法 實驗分為陰性對照組及甲基汞受試物組,甲基汞的濃度分彆為10、20、30、40、50 μmol/L.採用丫啶橙(AO)/溴化乙錠(EB)染色法和流式細胞技術檢測甲基汞對細胞凋亡的影響;流式細胞技術檢測甲基汞對細胞線粒體膜電位的影響;免疫細胞化學方法 檢測甲基汞對凋亡相關蛋白錶達的影響.結果 不同濃度氯化甲基汞作用24 h均可誘導細胞凋亡,AO/EB染色法檢測陰性對照組細胞凋亡率為(2.62±0.19)%,10～50μmol/L 甲基汞受試物組細胞凋亡率分彆為(7.97±0.64)%、(12.66±0.76)%、(19.16±0.87)%、(18.42±0.88)%、(11.52±0.63)%,各劑量組細胞凋亡率明顯高于對照組(q值分彆為17.057、32.009、52.732、50.373、28.375;P＜0.05).隨著作用濃度的升高細胞線粒體膜電位明顯下降,陰性對照組線粒體膜電位為(10.23±3.43)mV,10～50 μmol/L 甲基汞受試物組線粒體膜電位分彆為(3.25±0.66)、(3.03±0.35)、(1.68±1.26)、(1.69±1.13)、(1.77±0.88)mV,各劑量組明顯低于對照組(q值分彆為9.569、9.871、11.722、11.708、11.598;P＜0.05).隨著甲基汞作用濃度的升高 Bax、Bel-2、CytC、Caspase-3 及AIF錶達有上升的趨勢,且Bax/Bcl-2值升高.陰性對照組Bax錶達的積分吸光度值(IOD)為21 295.86±1969.81,10、20、30μmol/L 組Bax錶達的IOD分彆為42 807.87±4416.64、55 651.65±4662.72、72 708.56±910.10,各劑量組明顯高于對照組(g值分彆為14.191、14.320、33.917;P＜0.05);陰性對照組Bcl-2錶達的IOD為12 588.33±4091.02,10、20、30μmol/L組Bel-2錶達的IOD分彆為20 539.16±4906.09、23 689.97±2281.42、28692.80±4655.86,各劑量組明顯高于對照組(g值分彆為4.322、6.035、8.754;P＜0.05);陰性對照組AIF錶達的IOD為12 942.72±457.94、10、20、30、40μmol/L組AIF錶達的IOD分彆為16 973.57±1922.87、29 998.91±6803.58、52 467.16±1916.25、106 342.53±1273.19,20、30及40 μmol/L組明顯高于對照組(q值分彆為11.449、26.530、62.692;P＜0.05).結論 甲基汞可以通過線粒體通路誘導體外培養的人肝細胞株HL-7702髮生凋亡.
목적 연구갑기홍대체외배양적인간세포주HL-7702적조망작용급기궤제.방법 실험분위음성대조조급갑기홍수시물조,갑기홍적농도분별위10、20、30、40、50 μmol/L.채용아정등(AO)/추화을정(EB)염색법화류식세포기술검측갑기홍대세포조망적영향;류식세포기술검측갑기홍대세포선립체막전위적영향;면역세포화학방법 검측갑기홍대조망상관단백표체적영향.결과 불동농도록화갑기홍작용24 h균가유도세포조망,AO/EB염색법검측음성대조조세포조망솔위(2.62±0.19)%,10～50μmol/L 갑기홍수시물조세포조망솔분별위(7.97±0.64)%、(12.66±0.76)%、(19.16±0.87)%、(18.42±0.88)%、(11.52±0.63)%,각제량조세포조망솔명현고우대조조(q치분별위17.057、32.009、52.732、50.373、28.375;P＜0.05).수착작용농도적승고세포선립체막전위명현하강,음성대조조선립체막전위위(10.23±3.43)mV,10～50 μmol/L 갑기홍수시물조선립체막전위분별위(3.25±0.66)、(3.03±0.35)、(1.68±1.26)、(1.69±1.13)、(1.77±0.88)mV,각제량조명현저우대조조(q치분별위9.569、9.871、11.722、11.708、11.598;P＜0.05).수착갑기홍작용농도적승고 Bax、Bel-2、CytC、Caspase-3 급AIF표체유상승적추세,차Bax/Bcl-2치승고.음성대조조Bax표체적적분흡광도치(IOD)위21 295.86±1969.81,10、20、30μmol/L 조Bax표체적IOD분별위42 807.87±4416.64、55 651.65±4662.72、72 708.56±910.10,각제량조명현고우대조조(g치분별위14.191、14.320、33.917;P＜0.05);음성대조조Bcl-2표체적IOD위12 588.33±4091.02,10、20、30μmol/L조Bel-2표체적IOD분별위20 539.16±4906.09、23 689.97±2281.42、28692.80±4655.86,각제량조명현고우대조조(g치분별위4.322、6.035、8.754;P＜0.05);음성대조조AIF표체적IOD위12 942.72±457.94、10、20、30、40μmol/L조AIF표체적IOD분별위16 973.57±1922.87、29 998.91±6803.58、52 467.16±1916.25、106 342.53±1273.19,20、30급40 μmol/L조명현고우대조조(q치분별위11.449、26.530、62.692;P＜0.05).결론 갑기홍가이통과선립체통로유도체외배양적인간세포주HL-7702발생조망.
Objective To study the apoptotie effect and mechanisms of methylmercury (MeHg) on HL-7702 cell line in vitro.Methods In this study,the cell apoptosis was observed by AO/EB method and FCM method:the mitochondrial membrane potential was detected by FCM;and the expression of proteins related to apoptosis was measured by immunocytochemical method.Results After exposure to MeHg for 24 h in ditierent doses,apoptotic rate ascended with tlle increasing of MeHg concentration.By AO/EB method,cell apoptotie ratio of negative control group was (2.62±0.19)%,cell apoptotie ratio of 10-50 μmol/L exposure groups were (7.97±0.64)%,(12.66±0.76)%,(19.16±0.87)%,(18.42±0.88)%,and (11.52±0.63)%,there were significant differences between the exposure and negative control groups (q values were 17.057,32.009,52.732.50.373,28.375;P＜0.05).Mitochondrial membrane potential descended with the increase of MeHg,mitoehondrial membrane potential of negative control group was (10.23±3.43)mV,mitochondrial membrane potential of 10-50 μmol/L exposure groups were (3.25±0.66),(3.03±0.35),(1.68±1.26),(1.69±1.13)and(1.77±0.88)mV,and there was significant differences between exposure and negative control groups (q values were 9.569,9.871,11.722,11.708,11.598:P＜0.05).The expression of Bax,Bcl-2,CytC,Caspase-3 and AIF enhanced with the increase of MeHg,Bax/Bcl-2 ratio also appeared a trend of increase.Bax expression integral optical density(IOD)of negative control group was (21 295.86±1969.81),Bax expression IOD of 10,20,30 μmol/L groups were 42 807.87±4416.64,55 651.65±4662.72,and 72 708.56±910.10,there were significant differences in Bax expression between 10,20,30 μmol/L groups and negative control group (q values were 14.191,14.320,33.917;P＜0.05);Bcl-2 expression IOD of negative control group was (12 588.33±4091.02),Bcl-2 expression IOD of 10,20,30 μmol/L groups were 20 539.16±4906.09,23 689.97±2281.42,and 28 692.80±4655.86,there were significant differences in Bcl-2 expression between 10,20.30 μmol/L groups and negative control group (q values were 4.322,6.035,8.754;P＜0.05);and AIF expression IOD of negative control group was (12 942.72±457.94),AIF expression IOD of 10,20,30,40 μmol/L groups were 16 973.57±1922.87.29 998.91±6803.58.52 467.16±1916.25 and 106 342.53±1273.19,there were significant differences in AIF expression between 20.30 and 40 μmol/L groups and negative control group (q values were 11.449,26.530,62.692;P＜0.05).Conclusion MeHg could induce apoptosis on HL-7702 cell line in vitro.The mechanisms could be related to mitoehondfial pathway in apoptosis.
