中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2014年
11期
1472-1476
,共5页
王艳婕%牛志国%郭继强%王辉%牛新清
王豔婕%牛誌國%郭繼彊%王輝%牛新清
왕염첩%우지국%곽계강%왕휘%우신청
三氧化二砷%自噬%凋亡%急性T细胞白血病
三氧化二砷%自噬%凋亡%急性T細胞白血病
삼양화이신%자서%조망%급성T세포백혈병
Arsenic trioxide%Autophagy%Apoptosis%T-cell acute lymphoblastic leukemia
目的:探讨自噬特异性抑制剂3-甲基腺嘌呤(3-MA)对三氧化二砷(As2O3)诱导急性T细胞白血病系Jurkat细胞系Jurkat细胞凋亡的影响及机制。方法:XTT法检测三氧化二砷( As2 O3)对急性T细胞白血病细胞系Jurkat细胞的增殖抑制作用。电镜下观察不同浓度As2 O3作用Jurkat细胞24 h后细胞形态。免疫印迹和流式细胞术检测微管相关蛋白1轻链3B(LC-3B)蛋白的表达变化。 AnnexinV-FITC/PI双染法流式细胞术检测3-MA对AS2O3诱导急性T细胞白血病系Jurkat细胞凋亡的影响。结果:As2 O3可以抑制急性T淋巴细胞白血病细胞株Jurkat细胞的生长,这种作用呈剂量和时间依赖性。2.5、5、10μmol/L AS2 O3作用Jurkat细胞24 h后在电镜下可观察到自噬、凋亡、坏死的不同形态,并且自噬体数目不断增多。5μmol/L As2 O3处理Jurkat细胞0、24、48 h后,LC-3B的平均荧光强度相对倍数分别(3.1±0.2)倍、(4.6±0.31)倍、(34.2±4.5)倍,组间相比,差异有统计学意义(P<0.05),与生长抑制率一样呈现时间依赖性增强;免疫印迹也显示As2O3处理24 h和48 h后,LC-3B的蛋白表达逐渐增强;相对于As2O3组(33.4±9.1)%的生长抑制率,3-甲基腺嘌呤(3-MA)联合As2O3处理细胞后,48 h的生长抑制率为(60.6±8.3)%,差异明显,而LC-3B的表达明显降低。联合应用自噬抑制剂3-MA后Jurkat细胞的凋亡率(44.96±3.60)%,与单用As2 O3组(2.94±0.26)%相比明显增加,差异有统计学意义。结论:自噬抑制剂3-MA可以增加As2 O3引起的急性T细胞白血病细胞系Jurkat细胞的凋亡细胞百分数,其作用机制与诱导凋亡及抑制自噬密切相关。
目的:探討自噬特異性抑製劑3-甲基腺嘌呤(3-MA)對三氧化二砷(As2O3)誘導急性T細胞白血病繫Jurkat細胞繫Jurkat細胞凋亡的影響及機製。方法:XTT法檢測三氧化二砷( As2 O3)對急性T細胞白血病細胞繫Jurkat細胞的增殖抑製作用。電鏡下觀察不同濃度As2 O3作用Jurkat細胞24 h後細胞形態。免疫印跡和流式細胞術檢測微管相關蛋白1輕鏈3B(LC-3B)蛋白的錶達變化。 AnnexinV-FITC/PI雙染法流式細胞術檢測3-MA對AS2O3誘導急性T細胞白血病繫Jurkat細胞凋亡的影響。結果:As2 O3可以抑製急性T淋巴細胞白血病細胞株Jurkat細胞的生長,這種作用呈劑量和時間依賴性。2.5、5、10μmol/L AS2 O3作用Jurkat細胞24 h後在電鏡下可觀察到自噬、凋亡、壞死的不同形態,併且自噬體數目不斷增多。5μmol/L As2 O3處理Jurkat細胞0、24、48 h後,LC-3B的平均熒光彊度相對倍數分彆(3.1±0.2)倍、(4.6±0.31)倍、(34.2±4.5)倍,組間相比,差異有統計學意義(P<0.05),與生長抑製率一樣呈現時間依賴性增彊;免疫印跡也顯示As2O3處理24 h和48 h後,LC-3B的蛋白錶達逐漸增彊;相對于As2O3組(33.4±9.1)%的生長抑製率,3-甲基腺嘌呤(3-MA)聯閤As2O3處理細胞後,48 h的生長抑製率為(60.6±8.3)%,差異明顯,而LC-3B的錶達明顯降低。聯閤應用自噬抑製劑3-MA後Jurkat細胞的凋亡率(44.96±3.60)%,與單用As2 O3組(2.94±0.26)%相比明顯增加,差異有統計學意義。結論:自噬抑製劑3-MA可以增加As2 O3引起的急性T細胞白血病細胞繫Jurkat細胞的凋亡細胞百分數,其作用機製與誘導凋亡及抑製自噬密切相關。
목적:탐토자서특이성억제제3-갑기선표령(3-MA)대삼양화이신(As2O3)유도급성T세포백혈병계Jurkat세포계Jurkat세포조망적영향급궤제。방법:XTT법검측삼양화이신( As2 O3)대급성T세포백혈병세포계Jurkat세포적증식억제작용。전경하관찰불동농도As2 O3작용Jurkat세포24 h후세포형태。면역인적화류식세포술검측미관상관단백1경련3B(LC-3B)단백적표체변화。 AnnexinV-FITC/PI쌍염법류식세포술검측3-MA대AS2O3유도급성T세포백혈병계Jurkat세포조망적영향。결과:As2 O3가이억제급성T림파세포백혈병세포주Jurkat세포적생장,저충작용정제량화시간의뢰성。2.5、5、10μmol/L AS2 O3작용Jurkat세포24 h후재전경하가관찰도자서、조망、배사적불동형태,병차자서체수목불단증다。5μmol/L As2 O3처리Jurkat세포0、24、48 h후,LC-3B적평균형광강도상대배수분별(3.1±0.2)배、(4.6±0.31)배、(34.2±4.5)배,조간상비,차이유통계학의의(P<0.05),여생장억제솔일양정현시간의뢰성증강;면역인적야현시As2O3처리24 h화48 h후,LC-3B적단백표체축점증강;상대우As2O3조(33.4±9.1)%적생장억제솔,3-갑기선표령(3-MA)연합As2O3처리세포후,48 h적생장억제솔위(60.6±8.3)%,차이명현,이LC-3B적표체명현강저。연합응용자서억제제3-MA후Jurkat세포적조망솔(44.96±3.60)%,여단용As2 O3조(2.94±0.26)%상비명현증가,차이유통계학의의。결론:자서억제제3-MA가이증가As2 O3인기적급성T세포백혈병세포계Jurkat세포적조망세포백분수,기작용궤제여유도조망급억제자서밀절상관。
Objective:To discuss the effect and mechanism of autophagy inhibitor 3-MA on arsenic trioxide inducing apoptosis of acute T-cell leukemia cell line Jurkat cells.Methods:Proliferation inhibition of Jurkat cells treated with arsenic trioxide was detected by XTT.Morphological characteristics of Jurkat cells treated with different concentrations arsenic trioxide were observed by electron mi-croscope.Microtubule-associated protein 1 light chain 3B (LC-3B) protein expression was detected by Western blot and flow cytome-try.Apoptosis rates of Jurkat cells treated with 3-MA combining arsenic trioxide were detected by flow cytometry using AnnexinV-FITC/PI double staining.Results:Arsenic trioxide inhibited the growth of Jurkat cells in a dose and time dependence.We observed different morphological characteristics of autophagy , apoptosis and necrosis accompanying more autophagosomes in Jurkat cells which were treated with arsenic trioxide 2.5,5,10 μmol/L after 24 h.LC3B mean fluorescence intensity (MFI)relative multiples were(3.1±0.2) fold,(4.6±0.31)fold,(34.2±4.5)fold with 5 μmol/L arsenic trioxide treated Jurkat cells 0,24,48 h,and the P values between each of the two groups were less than 0.05,which increased depending time consistently with the growth inhibition rates.LC-3B protein expression gradually increased treated Jurkat cells with arsenic trioxide after 24 h,48 h.The growth inhibition rate (60.6±8.3)%was significantly different treated with arsenic trioxide combining 3-methyl adenine ( 3-MA ) while it was ( 33.4 ±9.1 )% treated with arsenic trioxide alone, however, LC-3B protein expression gradually decreased.Jurkat cell apoptosis rate ( 44.96 ±3.60 )% was significantly increased treated with arsenic trioxide combining autophagy inhibitor(3-MA) while it was (2.94±0.26)% treated with arsenic trioxide alone, and this difference was statistically significant.Conclusion: 3-MA increased apoptosis rates of Jurkat cells inducing by Arsenic trioxide and it may be related with inhibition of autophagy and induction of apoptosis.