医学毕业酪氨酸激酶抑制剂对转染p15基因的k562细胞的作用.docxVIP

XX大学 毕业论文 酪氨酸激酶抑制剂对转染卩15基因的K562细胞的作用 姓 名： 2014年6月25日 【摘要】 本研究探讨酪氨酸激酶抑制剂伊马替尼(imatinib)和pl5基因克 隆联合作用对K562细胞的增殖、细胞周期及凋亡等的调控作用。用RT PCR 扩增pl5基因，胶纯化回收后连接到T载体测序，确认序列正确后构建 pl5 pcDNA3」载体，将pl5 pcDNA3.1与空载体分别以脂质体转染入pl5基 因突变的K562细胞，经筛选得到G418抗性的K562细胞株；用Western blot检 测转染后P15蛋白的表达；用MTT法测定细胞存活率；流式细胞术检测细胞周 期和凋亡。结果表明：从对照K562细胞中扩增的DNA片段，在第174-180位 点有7个碱基缺失，这证实对照K562细胞屮pl5基因部位基因缺失。表达外源 性P15蛋白的pl5 pcDNA3.1 K562细胞株生长速度明显慢于对照K562细胞 株；流式细胞术显示G0/G1期细胞增多，S期细胞明显减少； pl5 pcDNA3.1 K562细胞与伊马替尼联合应用后凋亡细胞比例明显上升， MTT法显示细胞存活率较对照细胞明显下降。结论：表达外源P15蛋白联合 应用伊马替尼对抑制K562细胞增殖及促凋亡方面具有协同作用。 【关键词】pl5基因氨酸激酶抑制剂伊马替尼K562细胞 Effect of Tyrosine kinase Inhibitor on pl5 Gene Transfected K562 Cells Abstract The objective of study was to investigate the combined effect of tyrosine kinase inhibitor(imatinib) and pl5 gene on the proliferation, cell cycle and apoptosis of chronic myeloid leukemia cell line K562. pl5 gene was amplified from peripheral blood mononuclear cells by RT PCR, and confirmed by DNA sequencing, then the recombinant pl5 pcDNA3.1 vector was constructed and transfected into K562 cell line by Lipofectine. After screening with G41 pl5 pcDNA3.1 K562 cell clone stably expressing P15 was isolated. P15 protein was identified by Western blot. The cell survival rate was determined by MTT, cell cycle and apoptosis were detected by flow cytometry? The results showed that partial deletion of pl5 gene in K562 cells was verified by DNA sequencing, leading to the function of P15 protein to be lost. The expression of P15 protein can be detected by Western blot in pl5 pcDNa3A K562 cells. A strong inhibition of cell proliferation was observed in pl5 pcDNA3.1 K562 cells as compared with that of the control K562 cell. The cells of G0/G1 phase in pl5 pcDNA3.1 K562 cells increased apparently, and S phase cells declined signifcantly. Cell cycle was arrested in G0/G1 phase. The percentage of apoptotic cells greatly increased after transfection with pl5 pcDNA3.1 K562 cells combined with imatinib, and cell survival rate notably