大蒜素对LM-8细胞Bcl-2及Bax表达的影响

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.Cell Counting Kit-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15.0 mg/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15.0 mg/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24,48,72 h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15.0 mg/L质量浓度大蒜素干预LM-8细胞24,48,72 h,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1.0 mg/L增加到15.0 mg/L,LM-8细胞72 h时生长抑制率由(23.87±3.26)%增至(58.32±5.38)%,在72 h其药物半数抑制率浓度是11.09 mg/L.④LM-8细胞的凋亡:5.0,10.0和15.0 mg/L质量浓度的大蒜素作用72 h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.

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

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 carded 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× 107 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 rag/L) for 24, 48 and72 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: ①Expression 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).②LM-8 cell morphous: LM-8 Cells treated with allicin presented typical apoptosis changes under microscope.③LM-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.④LM-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: ①Allicin can inhibit the proliferation of LM-8 cells in a dose-time-dependent manner.②Allicin can induce the apoptosis of LM-8 cells in a dose-dependent manner.③Allicin 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.