【作者】 曾勇智；

【导师】 唐圣松；

【作者基本信息】 南华大学 ， 药理学， 2005， 硕士

【摘要】 研究背景 DADS是从大蒜中分离出的脂溶性单体有机硫化合物,可通过抑制细胞增殖、诱导细胞凋亡和细胞周期停滞等方式抑制多种肿瘤细胞的生长。我们前期的研究显示:DADS能诱导HL-60细胞生长阻滞在G₂/M期,但机制尚不完全清楚。cyclinB1是细胞周期G₂/M期的特异性周期素,是唯一随细胞周期变化而发生亚细胞分布改变的细胞周期素,cyclinB1的核转位是启动细胞有丝分裂的必要条件。p21是细胞周期蛋白依赖性激酶（cycle dependent kinase,CDK）抑制剂,可与CDK、cyclinB1和增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）形成四元复合物,在细胞周期推进过程中发挥重要作用。研究表明:p21与cyclinB1诱导细胞G₂/M期生长阻滞可能涉及如下两种机制:形成p21-PCNA-cdc2/cyclinB1四元复合物竞争性抑制cdc25与PCAN结合,阻断cdc25催化cdc2去磷酸化;这种四元复合物阻断cdc2活化激酶（cdc2 activating kinase,CAK）磷酸化cdc2,以上两种机制协同抑制cdc2激酶的活化,诱导细胞G₂/M期生长阻滞。但是迄今为止,cyclinB1亚细胞分布和p21在DADS诱导肿瘤细胞G₂/M期生长阻滞中的作用尚未得到证实。 方法 用20μM DADS处理HL-60细胞,MTT法测定DADS对HL-60细胞增殖活性的影响,流式细胞术测定DADS对细胞增殖周期的影响,Western blot测定DADS对cyclinB1亚细胞分布、p21与c-myc的表达水平的影响,免疫共沉淀分析cyclinB1与p21两者相互作用,重组质粒pcDNA3-c-myc和对照空质粒pcDNA3分别转染HL-60细胞,用流式细胞术、Western blot以及丫啶橙染色法检测p21受到抑制后,DADS对细胞增殖周期和细胞命运的影响。 结果 为了探讨DADS对HL-60细胞增殖的影响,经20μM DADS、DMSO、及10nM全反式维甲酸（ATRA）孵育HL-60细胞24h,结果显示:DADS能抑制HL-60细胞增殖,20μM DADS对细胞增殖的抑制率与ATRA（10nM）对细胞的抑制率接近。为更多还原

【Abstract】 Background Diallyl disulfide （DADS）, an oil-soluble allyl sulfur compound found inprocessed garlic, was found to inhibit the growth of various tumors by decreasing the cell growth rate, inducing apoptosis and cell cycle arrest. The previous studies in our laboratory showed that DADS induced the growth arrest of HL-60 cells in the G2/M phase, but the detail mechanism by which DADS induced the cell cycle arrest remains obscure. CyclinBl, as a specific G2/M phase cyclin, translocates to the nucleus during prophase. it is necessary for nuclear targeting of cyclinBl to initiate the mitosis. p21, which is a cyclin-dependent kinase inhibitor, could inhibit the cdc2 activation by preventing cdc2 dephosphorylation by cdc25 and/or interfering with CAK-mediated cdc2 phosphorylation by forming p21-PCNA-cdc2-cyclinBl. But the role of the subcelullar distribution of cyclinBl and p21 expression during the initiation of G₂/M checkpoint by DADS in HL-60 cells remains to be clarified.Methods HL-60 cells were treated with 20μM DADS for the indicated time, and then cell proliferation was determined by MTT assay. Cell cycle was assayed by flowcytometry. The subcelullar distribution of cyclinBl, the expression of p21 and c-myc were measured by Western blot. The status and interaction between cyclinBl and p21 in DADS-treated HL-60 cells was measured by immunoprecipitation. The effects of c-myc on the p21 expression and the determination of HL-60 cell fate were analysed by transfecting recombinant plasmid pcDNA3-c- myc into the HL-60 cells.Results To explore the effect of DADS on cellular proliferation in HL-60 cell growth,the cells were treated with 20μM DADS, DMSO, 10nM ATRA for 24h. The results showed that DADS could inhibit HL-60 cell proliferation. The inhibitory efficiency of 20 μM DADS was similar to that of 10 nM ATRA. To determine the phase at which the cell growth arrest, the cell cycle was measured by flow cytometry. The results indicated that the percentage of G₂/M phase cells reached maxium at 12h after treated with 20μM DADS,These results suggested that DADS could inhibit the proliferation of HL-60 cells and induce the cell gowth arrest in G2/M phase.It is reported that the initiation of mitosis is regulated by the activation of cdc2-cyclinBl, and that the nuclear targeting of cyclinBl is necessary for the initiation of the mitosis. To explore the relationship between the subcelullar distribution of cyclinBl and the initiation of G2/M checkpoint by DADS, the cells were treated with 20/uM DADS. The results showed that the expression of cyclinBl increased steadily following 20/uM DADS incubation, and that the expression of cyctoplasmic cyclinBl reached the peak at 12h after treated with DADS, While the expression of nuclear cyclinBl was obviously inhibited. These results suggested that the initiation of G2/M checkpoint by DADS was associated with the cytoplasmic targeting of cyclinBl.To explore the effect of p21 on the initiation of G2/M checkpoint by DADS, HL-60 cells were treated with DADS for 0h²4h. The results indicated that DADS could induce steadily the expression of p21, and that p21 level achieved the peak at 12h after treated with DADS. To further ananlyze the status and interaction between p21 and cyclinBl, the cyclinBl from DADS-treated HL-60 cells precipitated with anti-cyclinBl antibody and followed by ProteinA/G PLUS-Agarose. The precipitation was blotted with anti-p21 monoclonal antibody. The result showed that p21 was precipitated with cyclinBl. So p21 existed in cyclinBl-bound state, and p21 conferred its biogical effects by binding to cyclinBl. These results suggested that the initiation of G2/M checkpoint by DADS was possiblly involved in p21 expression.To further confirm the possible roles of p21 in the initiation of G2/M checkpoint by DADS, recombinant pcDNA3-c-myc plasmid and pcDNA3 plasmid were transfected into HL-60 cells, respectively. The results showed that c-myc could express in c-myc -transfected-HL-60 cells, and that DADS could inhibit the c-Myc expression. Moreover, the level of p21 expression in HL-60 cells transfected c-myc gene was down-regulated by c-Myc, suggesting that the expreassion of c-myc in HL-60 cells could inhibit p21 expression induced by DADS. To explore the effects of p21 down-regulation by c-Myc on the initiation of G2/M checkpoint by DADS, the cell cycle was analyzed by flow cytometry. The result indicated that the percentage of the G2/M phase cells in c-myc-tansfected-HL-60更多还原