D-柠檬烯对K562细胞增殖及凋亡影响.docVIP

D-柠檬烯对K562细胞增殖及凋亡影响 　【摘要】 　　本研究探讨D-柠檬烯对K562白血病细胞增殖的影响及机制。应用MTT试验观察不同浓度D-柠檬烯作用后K562细胞增殖的改变；应用细胞形态学检查、流式细胞术、DNA琼脂糖凝胶电泳观察和检测细胞凋亡。结果表明： 在0.125-1.0 mmol/L浓度范围内，D-柠檬烯对K562细胞增殖的抑制作用呈现剂量依赖的关系。 典型的细胞形态学改变、DNA琼脂糖凝胶电泳梯形条带及流式细胞仪检测出亚G1峰共同证实了D-柠檬烯能诱导K562白血病细胞凋亡。结论： D-柠檬烯抑制K562细胞的增殖并呈剂量依赖的方式，使细胞滞留于G1期，诱导其凋亡。 【关键词】 D-柠檬烯； K562细胞； 细胞凋亡； 细胞增殖 　　Proliferation Inhibition and Apoptosis Induction of K562 Cells by D-limonene 　　AbstractThis study was aimed to investigate the effect of D-limonene on K562 leukemia cells and its mechanism. Inhibitory effect of D-limonene on proliferation of K562 leukemia cells was assayed by MTT method and cell apoptosis was detected by flow cytometry and DNA agarose gel electrophoresis, the morphologic change of K562 cells was observed by microscopy. The results showed that when K562 cells were treated with 0.125-1.0 mmol/L of D-limonene for 48 hours, the proliferation of K562 cells was obviously inhibited in dose-dependent manner. Typical morphological changes and the typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis were significantly appeared in D-limonene treated K562 cells. Simultaneously, the sub-G1 peak was found in FCM analysis. It is concluded that the D-limonene can inhibit proliferation of K562 cells in dose-dependent manner, cause cell detained at G1 phase and induce apoptosis of K562 cells. 　　Key wordsD-limonene; K562 cell; cell apoptosis; cell proliferation 　　D-柠檬烯（D-limonene）是一种单萜烯类化合物，广泛存在于芸香科柠檬、柑桔精油及其他植物中，具有抗氧化、抗炎、利胆溶石等作用。近年来，人们发现D-柠檬烯具有抗癌活性，用于预防及治疗化学致癌物诱导的结肠癌、乳腺癌、胃癌、肺癌、皮肤癌、肝癌等［1-6］ ，取得了一定的效果。目前对其抗癌作用的研究主要集中在实体瘤方面，对非实体瘤作用的研究报道尚少。我们以K562白血病细胞作为靶细胞，探讨D-柠檬烯对其抑制增殖、诱导凋亡的作用。 　　材料和方法 　　主要试剂 　　RPMI 1640培养液（Gibco公司产品）；小牛血清（杭州四季青公司产品）；淋巴细胞分离液（密度1.077，上海试剂二厂生产）；溴化噻唑蓝四唑（thiazolyl blue tetrazolium bromide， MTT）和D-柠檬烯（Sigma公司产品）。 　　细胞系和细胞培养 　　K562细胞株由大连医科大学组胚教研室提供，K562细胞悬浮于含15%小牛血清的RPMI 1640培养液中，置于37℃、 5% CO2、饱和湿度的培养箱内培养，每2-3天传代1次，取对数生长期细胞调成适当浓度进行实验，台盼蓝拒染率＞95%。 　　细胞增殖抑制的MTT测定 　　2×104/ml K562细胞悬液接种于96孔培养板，每孔 0.2 ml。实验组：加