亚硒酸化β乳球蛋白诱导K562细胞凋亡及其机制研究

发布时间：2018-06-20 18:50

本文选题：亚硒酸化β-乳球蛋白 + K562细胞　；参考：《天津科技大学》2015年硕士论文

【摘要】：本文以亚硒酸化p-乳球蛋白为研究对象对其体外抗肿瘤活性进行测定,在与K562细胞共培养后,研究其对K562细胞增殖的抑制作用和诱导凋亡作用。通过MTT法、HE染色、流式细胞术、免疫荧光法和荧光定量PCR等方法从细胞形态学的变化、细胞周期的变化和相关周期蛋白及其mRNA的表达等方面考察了亚硒酸化β-乳球蛋白对K562细胞的影响。首先,对亚硒酸化p-乳球蛋白诱导K562细胞凋亡的现象进行研究。MTT实验结果表明,随着浓度和培养时间的升高,亚硒酸化p-乳球蛋白对K562细胞的抑制作用逐渐变强,10μg/mL药物作用K562细胞48 h后,抑制率达到90%,而正常小鼠成纤维细胞C2C12在经过相同处理后仍然正常生长；HE染色和PI/Hoechst双染均观察到亚硒酸化β-乳球蛋白使K562细胞出现体积缩小、核质分离、出现凋亡小体等典型的凋亡现象；Annexin V-FITC/PI双染结果表明,经过48 h与亚硒酸化p-乳球蛋白共培养的K562细胞的凋亡率为18.75%,而共培养72 h后凋亡率上升为40.76%；流式细胞术结果表明亚硒酸化p-乳球蛋白能够使细胞G1期和G2期发生显著变化,细胞分别被阻滞于S→G2期和G2→M期。其次,研究了亚硒酸化p-乳球蛋白诱导K562细胞凋亡的信号途径。免疫荧光实验和ELISA法共同确定了经过亚硒酸化β-乳球蛋白处理的K562细胞,其cyclinB1蛋白含量显著降低,p21蛋白含量显著上升；亚硒酸化p-乳球蛋白还使K562细胞的cyclinB1 mRNA水平下调,极显著上调p21mRNA水平,是对照组的21倍,并且还显著下调蛋白激酶CDK4mRNA的表达水平。综上所述,亚硒酸化p-乳球蛋白能够在体外有效的抑制白血病细胞K562的增殖并且诱导其凋亡,是一种很有潜力的抗癌活性物质。
[Abstract]:In this paper, the antitumor activity of selenated p- lactoglobulin was determined in vitro. After co-culture with K562 cells, the inhibition of proliferation and the induction of apoptosis of K562 cells were studied. The changes of cell morphology were detected by MTT staining, flow cytometry, immunofluorescence and fluorescence quantitative PCR. The effects of 尾 -lactoglobulin selenite on K562 cells were investigated in terms of cell cycle changes and the expression of related cyclins and their mRNA. Firstly, the apoptosis of K562 cells induced by selenite p- lactoglobulin was studied. The results of MTT assay showed that the apoptosis of K562 cells increased with the increase of concentration and culture time. The inhibitory effect of selenated p- lactoglobulin on K562 cells increased gradually after treatment with 10 渭 g / mL of drugs for 48 h, the inhibition rate reached 90%, while the normal mouse fibroblasts C2C12 still grew normally after the same treatment. He staining and Pi / Hoechst double staining showed that 尾 -lactoglobulin selenite reduced K562 cell volume, separated nucleus and cytoplasm, and appeared apoptotic bodies. The results of Annexin V-FITC / Pi double staining showed that: 1. The apoptosis rate of K562 cells co-cultured with selenite p- lactoglobulin for 48 h was 18.75, while that of K562 cells co-cultured for 72 h was increased to 40.76.The results of flow cytometry showed that selenated p-lactoglobulin could significantly change the G1 and G2 phases of K562 cells. The cells were blocked in S G 2 phase and G 2 M phase, respectively. Secondly, the signaling pathway of selenated p- lactoglobulin induced apoptosis in K562 cells was studied. Immunofluorescence assay and Elisa combined to determine that the content of cyclin B1 in K562 cells treated with selenated 尾 -lactoglobulin significantly decreased the content of p21 protein, and the level of cyclin B1 mRNA in K562 cells was down-regulated by selenated p- lactoglobulin. The p21 mRNA level was significantly up-regulated, 21 times higher than that in the control group, and the expression level of protein kinase CDK4 mRNA was significantly down-regulated. In conclusion, selenated p- lactoglobulin can effectively inhibit the proliferation and induce apoptosis of leukemia cell line K562 in vitro, which is a potential anticancer active substance.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R96

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