【关键词】 大蒜辣素; 生长曲线; 凋亡; DNA周期; HepG2肝癌细胞; Western blot; 免疫印迹
Abstract:ObjectiveTo investigate the effects of apoptosis on heptoma cell lines HepG2 caused by allicin. MethodsCCK-8 was exerted to investigate the proliferation of the HepG2 cell and cytometry machine was used to detect the apoptosis and mortality rate, finally western blot method was used to study the apoptosis protein expression. ResultsExperimental results showed that allicin could inhibit the proliferation of cells and cause the cell apoptosis and death, and the highest ratio of apoptosis and death were 4.13% and 70.38%, respectively. Cell cycle experimental results indicated that the DNA metabolism was disturbance and most of them were blocked in G0/G1 period resulting in cells mitosis were blocked too. Western blot results showed that allicin the actived apoptosis gene of cells.ConclusionAllicin can inhibit the HepG2 cell proliferation and cause cell death and apoptosis, furthermore Allicin can cause cell DNA metabolism disturbance.Western blot experiments results show that the apoptosis gene can increase expression.
2.6 免疫印迹(Western blot)实验肝癌细胞的暂时保存及洗涤都在磷酸盐缓冲液(PBS)中完成。收集细胞,PBS洗涤细胞两次,加细胞裂解液,超声破碎细胞。用抗凋亡抗体在40℃孵育肝细胞溶胞产物1 h,孵育在含牛血清白蛋白(BSA)、吐温(Tween)浓度均为0.1%的PBS中进行。用SDS-PAGE提取凋亡蛋白,并通过电转移使之转录到聚偏二氟乙烯(PVDF)撑体中[8,9]。运用ECL Western blot 检验系统检测免疫复合物。
【参考文献】
[1] Li Y, Xu YS, Sun DW, Preparation of garlic powder with high allicin content by using combined microwave-vacuum and vacuum drying as well as microencapsulation[J].Jour of Food Engin,2007, 83(1): 76.
[2] Seong-Jun Cho, Dong-Kwon Rhee, Suhkneung Pyo.Allicin, a major component of garlic, inhibits apoptosis of macrophage in a depleted nutritional state [J].Nutrition,2006, 22(11-12): 1177.
[3] Curtis H, Noll U, Strmann J, et al, Broad-spectrum activity of the volatile phytoanticipin allicin in extracts of garlic (Allium sativum L.) against plant pathogenic bacteria, fungi and Oomycetes[J].Physi and Mole Plant Path,2004, 65(2): 79.
[4] Oommen S, John A R, Srinivas G, et al, Allicin (from garlic) induces caspase-mediated apoptosis in cancer cells[J].Euro Jour of Pharma,2004, 485(1-3): 97.
[5] Miron T, SivaRaman H, Rabinkov A, A method for continuous production of allicin using immobilized alliinase,[J].Analyti Biochem,2006, 351(1): 152.
[6] European Directorate for the Quality of Medicines & HealthCare, The European Pharmacopoeia 5th Edition(Ep5.0)[M]. :1651
[7] Choi J, Hwang YK, Sung KW, et al, Expression of Livin, an antiapoptotic protein, is an independent favorable prognostic factor in childhood acute lymphoblastic leukemia[J].Blood,2007, 109(2):471.
[8] Son E W, Mo S J, Rhee D K, et al, Inhibition of ICAM-1 expression by garlic component, allicin, in gamma-irradiated human vascular endothelial cells via downregulation of the JNK signaling pathway[J].Inter Immunopharm,2006, 6(12): 1788.
[10] European Directorate for the Quality of Medicines & HealthCare, The European Pharmacopoeia 5th Edition(Ep5.0)[M].1637.
[11] Singh S V, Pan S S, Srivastava S K, et al, Differential induction of NAD(P) H: quinine oxidaoreductase by anti-carcinogenic organosulfides from garlic[J].Biochem Biophys Res Commun, 1998, 244(3): 917.
[12] Li Y, Lu Y Y. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells[J]. Wold J Gastroenterol, 2002, 8(2): 213.
[13] ElkayamA, Mirelman D, Peleg E, et al, The effects of allicin and enalapril in fructose-induced hyperinsulinemic hyperlipidemic hypertensive rats[J].Am J Hypertens 2001;14:377.
[14] Wu C C, Sheen L Y, Chen H W, et al, Differential effects of garlic oil and its three major orgnosulfur components on the hepatic detoxification system in rats[J].J Agric Food Chem, 2002, 50(2): 378.
[15] Josling P.Preventing the common cold with a garlic supplement: a double-binding, placebo-conrolled survey[J].Adv Ther, 2001, 18(4): 189.