大蒜素对LM-8细胞Bcl-2及Bax表达的影响

Effect of allicin on the expression of Bcl-2 and Bax protein in LM-8 cells

背景:有研究证实,大蒜素对LM-8细胞增殖及凋亡有影响。目的:观察大蒜素干预C3H小鼠骨肉瘤细胞株LM-8细胞Bcl-2、Bax凋亡蛋白的表达,以及LM-8细胞的形态学变化与Bcl-2及Bax变化的关系。设计:随机分组,非盲法,细胞水平体外实验。单位:实验于2006-09/2007-05在华中科技大学同济医学院附属协和医院中心实验室完成。材料:实验所用C3H小鼠LM-8骨肉瘤细胞株购自解放军第四军医大学实验动物中心。大蒜素为武汉汇海公司产品,是从大蒜球茎中分离出的硫化物。CellCountingKit-8试剂购于上海同仁化学研究所,Bcl-2和Bax抗体及SP试剂盒购于福州迈新生物技术开发公司。方法:以含体积分数为0.1新生牛血清的1640完全培养基,培养LM-8细胞,制作细胞爬片。SP免疫细胞组织化学技术检测细胞爬片中Bcl-2和Bax蛋白表达;倒置显微镜下观察5.0,10.0和15.0mg/L质量浓度大蒜素作用前后LM-8细胞形态变化;将LM-8细胞调整至7.5×107L-1接种于96孔板,用1.0,5.0,10.0和15.0mg/L质量浓度的大蒜素处理细胞,设立不加任何处理因素的对照组及不含细胞和药物的培养基空白组,孵育24,48,72h后取出培养板,加入CCK-8测定吸光度值,计算LM-8细胞生长抑制率;以5.0,10.0和15.0mg/L质量浓度大蒜素干预LM-8细胞24,48,72h,消化离心收集细胞,以流式细胞仪分析细胞凋亡率的变化。主要观察指标:LM-8细胞中Bcl-2和Bax蛋白表达;LM-8细胞形态及增殖、凋亡情况。结果:①Bcl-2和Bax蛋白表达:大蒜素可以降低Bcl-2表达,增强Bax表达,经15.0mg/L大蒜素作用72h,Bcl-2和Bax蛋白表达阳性率及Bcl-2/Bax表达与对照组比较,差异有显著性意义(P<0.01)。②LM-8细胞的形态:经不同质量浓度大蒜素干预后均出现明显凋亡表现。③LM-8细胞的增殖:大蒜素能明显抑制LM-8细胞的增殖,当大蒜素浓度从1.0mg/L增加到15.0mg/L,LM-8细胞72h时生长抑制率由(23.87±3.26)%增至(58.32±5.38)%,在72h其药物半数抑制率浓度是11.09mg/L。④LM-8细胞的凋亡:5.0,10.0和15.0mg/L质量浓度的大蒜素作用72h后可诱导LM-8细胞凋亡,且呈浓度依赖性(P<0.05)。结论:①大蒜素可抑制LM-8细胞增殖,该效应与大蒜素的浓度和时间有关。②大蒜素可诱导LM-8细胞凋亡,作用呈浓度依赖性。③大蒜素抑制LM-8细胞增殖和诱导LM-8细胞凋亡的机制可能与下调Bcl-2的表达和上调Bax的表达有关。

BACKGROUND： Allicin has been proved to influence the proliferation and apoptosis of LM-8 cells. OBJECTIVE： To study the effects of allicin on the expression of apoptosis protein Bcl-2 and Bax in C3H mouse osteosarcoma cell line LM-8, and the correlation of the morphological changes of LM-8 cells with the changes of Bcl-2 and Bax. DESIGN： Randomized grouping, non-blind, cell level, in vitro study. SETTING： The experiment was carried out from September 2006 to May 2007 at the Center Laboratory of Union Hospital, Tongji Medical College, Huazhong University of Science ＆ Technology. MATERIALS： Osteosarcoma cell line LM-8 was purchased from the Animal Center of the Fourth Military Medical University of Chinese PLA, Allicin presented by Wuhan Huihai Co., was a sulphide isolated from garlic corn. Cell Counting Kit-8 （CCK-8） from Dojindo Laboratories; Bcl-2 antibody, Bax antibody and SP immunohistochemistry kit from Fuzhou Maixin Biotech Co, METHODS： LM-8 cells were cultured in RPMI 1640 culture medium, then cell climbing films were made, and SP immunocyte-histochemistry was used to detect the expression of Bcl-2 and Bax protein. Inverted microscope was performed to observe the morphologic changes of LM-8 cells before and after adding with 5.0, 10.0 and 15.0 mg/L allicin. Serial subcultivated LM-8 cells were adjusted to 7.5×10^7 L^-1 in concentration, added to 96-pore culture plate, and treated with allicin at a serial concentration of 1.0, 5.0, 10.0 and 15.0 mg/L. Additionally blank culture medium pores without cells and allicin and pores without allicin were taken as control groups. The 96-pore culture plate was maintained for 24, 48 and 72 hours, and then fetched. CCK-8 was added to determine the optical density value. The inhibition rate of LM-8 cell growth was also calculated. LM-8 cells were treated with different concentrations of allicin （5.0, 10.0 and 15.0 mg/L） for 24, 48 and 72 hours, and then we carried out an apoptosis analysis by flow cytometry. MAIN OUTCOME MEASURES： Expression of Bcl-2 and Bax protein in LM-8 cells; cell morphous, cell proliferation, and cell apoptosis. RESULTS： （1）Expression of Bcl-2 and Bax protein： Allicin could lower the expression of Bcl-2 protein and enhance the expression of Bax. The positive expression of Bcl-2, Bax and Bcl-2/Bax expression all showed significant differences compared with control group （P 〈 0.01）.（2）LM-8 cell morphous： LM-8 Cells treated with allicin presented typical apoptosis changes under microscope.（3）LM-8 cell proliferation： Allicin could inhibit the growth of osteosarcoma cell line LM-8, when concentration of allicin added from 1.0 mg/L to 15.0 mg/L, the inhibition rate of LM-8 cells at 72 hours increased from （23.87±3.26）% to （58.32±5.38）%, and 50% inhibiting concentration was 11.09 mg/L.（4）LM-8 cell apoptosis： After 72 hours with treatment of allicin of 5.0, 10.0 and 15.0 mg/L in concentration, apoptosis rate of LM-8 cells revealed a dose-dependent relationship （P 〈 0.05）. CONCLUSION： （1）Allicin can inhibit the proliferation of LM-8 cells in a dose-time-dependent manner.（2）Allicin can induce the apoptosis of LM-8 cells in a dose-dependent manner.（3）Allicin can inhibit LM-8 cell proliferation and induce LM-8 cell apoptosis, which is related to down-regulate Bcl-2 protein expression and up-regulate Bax protein expression.