【作者】 易岚； 曾希； 苏琦； 葛玲； 解娜；

【Author】 Yi Lan Zeng Xi Su Qi Ge Ling Xie Na

【机构】 南华大学肿瘤研究所；

【摘要】 目的:寻找二烯丙基二硫(diallyl disulfide,DADS) 诱导人白血病HL-60细胞凋亡的启动点,建立DADS启动 HL-60细胞凋亡模型。在此基础上,应用蛋白质组学技术研究DADS启动人白血病细胞凋亡过程中差异表达的蛋白质, 鉴定启动凋亡相关关蛋白,在蛋白质水平上探讨DADS诱导肿瘤细胞凋亡的分子机理。方法:实验设未处理组、处理组和撤药组,分别采用倒置显微镜和普通光学显微镜观察、细胞计数、流式细胞术、DNA凝胶电泳、western blot等方法, 进行细胞形态学观察,绘制生长曲线,检测凋亡率、活化的 caspase-3以及DNA Ladder、凋亡相关蛋白的检测,建立 DADS启动HL-60细胞凋亡模型。然后提取对照组和凋亡启动点的HL-60细胞的总蛋白,双向电泳分离细胞总蛋白质; 考马斯亮蓝染色对凝胶上的蛋白质进行可视化检测,运用计算机辅助图像分析软件(PDQUEST)分析考染的蛋白质图谱, 切割差异表达的蛋白质点,胶上原位酶解,通过基质辅助激光解吸附电离飞行时间质谱(MALDI-TOF-MS)分析, 并采用生物信息学方法搜索NCBInr、EMBL、SWISS—PROT 等数据库,进行蛋白质鉴定。结果:3．6mg L—1DADS作用 HL-60细胞2d-6d,细胞数分别比对照组减少24．1％、36．5％、 44．2％、52％、53．6％。处理后,细胞核浓缩,染色质边聚,可见凋亡小体。DADS作用1d、2d,凋亡率分别为3．1％、4．3％, 与对照组(3．0％)无显著性差异,而作用3d、4d、5d的凋亡率分别达到8．5％,15．2％,27．4％,均高于对照组(P<0．05)。 DADS作用2d-5d撤药后再培养至第6d,凋亡率分别为7．9%, 12．4％,16．5％,18．8％,高于对照组的3．3％(P<0．05)。处理2d- 5d后,活化的caspase-3的表达率为10．0％,10．4％,14．9％, 17．3％,均高于对照组5．4％(P<0．05)。DNA凝胶电泳显示, 处理组4d后出现明显梯状条带,而撤药组从2d-5d撤药均出现明显梯状条带。western blot结果表明,caspase-3从作用 2d起开始表达上调,而Bcl-2从2d起表达开始下调。分别提取3．6mg L-1DADS作用2d和对照组HL-60细胞总蛋白进行双向电泳,获得重复性较好的双向电泳考染图谱。扫描成像及PDQuest软件分析,识别的蛋白质斑点数分别为对照组 467±24个,处理组385±18个,其平均匹配率分别为82％和74％。两组图谱中有387个蛋白质斑点匹配,93个点未被匹配,蛋白质表达量相差2倍以上的29个,其中22个上调, 7个下调。质谱分析和数据库搜索鉴定其中的9个蛋白质点。网上数据库搜索后,得分值>64分(P<0．05),获得7个有意义蛋白。其中,Ras相关C3肉毒毒素酶解物2(Ras-related C3 botulinum toxin substrate2,Rac2),肌动蛋白(A CTB protein,Actin),核结合因子β亚基(Core binding factor, beta subunit isoform 1),富小脯氨酸蛋白3(Small pro— line-rich protein 3,SPRR3 protein),酯酶D／谷胱甘肽甲酰基水解酶(Esterase D／formylglutathione hydrolase), 蛋白激酶C相互作用蛋白底物类似物(PKCI-substrate analog)蛋白表达明显上调,而延长因子(eukaryotic elon- gation factor,EF)则表达下调。这些蛋白功能分别与RNA 转录、转录调控、细胞骨架、细胞代谢及氧化还原作用调节等有关。结论:3．6mg·L—1DADS可诱导人白血病HL-60细胞凋亡,且诱导凋亡的启动点为处理2d。DADS可能通过上调Rac2、Actin、核结合因子β亚基、富小脯氯酸蛋白3、酯酶D／谷胱甘肽甲酰基水解酶、蛋白激酶C相互作用蛋白底物类似物和下调真核细胞延长因子启动HL-60细胞的凋亡。更多还原

【Abstract】 Objective: Aim to search the apoptosis initiation spot induced by DADS, establish the apoptosis initiation model. After this, to study the related proteins of apoptosis initiation in human leukemia HL-60 cells induced by diallyl disulfide and from the protein level research its related molecular mechanisms. Methods: this study designed control group, treated group and DADS-withdraw group. After incubation of HL-60 cells with 3. 6mg · L-1 DADS, the effects of DADS on HL-60 cells growth inhibition were estimated by growth curve, and inverted microscope, light microscope, flow cytometry method, the expression of actived caspase-3, DNA agar-ose electrophoresis were used to determine the induction of apoptosis. and protein expression of caspase-3, Bcl-2 were tested by western-blotting. After this, Cells were processed for two-dimensional (2-D) electrophoresis. proteins were visualized by Coomassie brilliant blue staining. PDQuest 2-DE software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) and public domain NCBInr, EMBL and SWISS-PROT databases were used to separate and identify the differential proteomic expressions in apoptosis initiation phase in human leukemia HL-60 cells induced by diallyl disulfide Results: Growth curve show the growth of HL-60 cells could not be inhibited at day l,but remarkably reduced the growth rate at day2 to day6 (24.1%, 36.5%, 442%, 52%,53.6%,P<0.05), After HL-60 cells were exposed to DADS, the typical apoptotic morphological changes were observed under the light microscope and inverted microscope. Flow cytometry analysis showed that the apoptotic rate of DADS treated cells at day 1 and day 2 was 3.1%, 4.3%, with no difference from control cells(3.0%), while from day 3 to day 5, the apoptotic rate was 8.5%, 15.2%, 27.4% respectively. Have great difference from control cells, the DADS-withdrawal group from day 2 to day 5 the apoptotic rate was 7.9%, 12.4%,16.5%,18.8% respectively, significantly increased than the control group and the 1 day with DADS withdrawl(P<0.05).the expression of actived caspase-3 from day 2 to day 5 was 6.3%,10.0%,10.4%,14. 9%,17.3% respectively, was higher than control group and DADS treated 1 day(P<0.05), DNA agarose electrophoresis was showed the DADS treated group from day 4 and the DADS-withdrawal group from day 2 had DNA ladder, and western-blotting showed caspase-3 was upregulated and Bcl-2 was downregulated from day 2. the good 2-DE pattern including high resolution and reproducibility was obtained. After Coomassie brilliant blue staining, the 2-DE image analysis by PDQuest 2-DE software detected average (467 ± 24) spots in HL-60 cell, and (385 ± 18) spots in DADS treated HL-60 cell. And the average matching rate was 82% and 74% respectively. The differential proteomic expression analysis found that there were 387 spots matched and 93 spots unmatched between HL-60 and DADS treated HL-60更多还原