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分别以水、盐、乙醇及NaOH溶液为提取剂,从山杏仁中提取蛋白,通过细胞增殖分析实验,研究山杏仁蛋白对人结肠癌细胞Caco-2生长的影响。结果表明,用有机溶剂乙醇提取的山杏仁蛋白显著抑制Caco-2细胞生长(P<0.01,其IC50值为1.018 mg/mL),而用其他提取剂提取的山杏仁蛋白并没有显示同样活性。以乙二醇、异丙醇及叔丁醇等其他有机溶剂代替乙醇提取的山杏仁蛋白,也没有显示抑制Caco-2细胞生长作用。因用乙醇提取的山杏仁蛋白具有显著抑制Caco-2细胞生长作用,称为活性山杏仁蛋白。SDS-聚丙烯凝胶电泳分析结果表明,21 ku和41 ku 2个亚基存在于活性山杏仁蛋白中。细胞活力检测结果表明,用5 mg/mL浓度活性山杏仁蛋白处理Caco-2细胞48 h后,死细胞占细胞总数的89.5%。TUNEL和Annexin V-FITC染色等细胞凋亡检测结果分别表明,活性山杏仁蛋白诱导了Caco-2细胞凋亡。
The proteins were extracted from the almonds by using water, salt, ethanol and NaOH solution respectively. The proliferation of Corynebacterium salivarius on Caco-2 cells was studied by cell proliferation assay. The results showed that the apricot protein extracted with organic solvent ethanol significantly inhibited the growth of Caco-2 cells (IC50 value was 1.018 mg / mL), whereas the apricot protein extracted with other extractants did not show the same activity. Substitution of ethanol with other organic solvents, such as ethylene glycol, isopropanol and t-butanol, did not show any inhibitory effect on the growth of Caco-2 cells. The almond protein extracted by ethanol has significant inhibitory effect on the growth of Caco-2 cells, which is called active almond protein. SDS-PAGE analysis showed that 21 ku and 41 ku 2 subunits were present in the active almond protein. The results of cell viability assay showed that dead cells accounted for 89.5% of the total cells after Caco-2 cells were treated with 5 mg / mL of active almond protein for 48 h. TUNEL and Annexin V-FITC staining and other apoptosis test results showed that active almond protein induced Caco-2 cell apoptosis.