Objective: To analyze the effect of down-regulating the CD59 gene expression by RNAi lentivirus as vector on Jurkat cell line of acute T-lineage leukemia.
Methods: The expression of CD59 in Jurkat cell line of acute T-line leukemia was induced to decrease by RNAi lentivirus as vector. The transfection of RNA lentivirus and the localization of CD59 molecule were analyzed by laser confocal technique. The relative expression of CD59 gene in blank control, negative control and RNAi lentivirus transfected group was detected by real-time fluorescence quantitative PCR, and the enzyme-linked immunosorbent assay was used to detect the expression of TNF-β and IL-3 in supernatants of cultured cells in 3 groups. The expression levels of apoptosis-related molecules including Caspase-3, Survivin, BCL-2 and BCL-2-associated X protein (BAX) were measured by Western blot.
Results: The transfection efficiency for Jurkat cells was higher than 90%. CD59 was mainly located on the cell membrane. Compared with the blank control group and the negative control group, the expression level of CD59 mRNA and protein in the RNAi lentivirus transfected group significantly decreased (P<0.05). Compared with the blank control group and the negative control group, the expression of TNF-β and IL-3 in the RNAi lentivirus transfected group were significantly higher and lower (P<0.05) respectively. The expression levels of Survivin and BCL-2 in the RNAi lentivirus transfected group were significantly lower than those in the blank control group and the negative control group, while the expression levels of Caspase-3 and BAX in the RNAi lentivirus transfected group were significantly higher than those in the blank control group and the negative control group (P< 0.05).
Conclusion: The down-regulation of CD59 gene expression induced by RNAi lenti-virus can decrease the expression of proliferation and differentiation-promoting molecule such as IL-3 and increase the expression of TNF-related factor in Jurkat cell line of acute T-lineage leukemia, which also can increase the expression of apoptosis-related proteins such as Caspase-3 and BAX, and decrease the expression of anti-apoptosis-related proteins such as Survivin and BCL-2.
题目: 以RNAi慢病毒为载体下调CD59对急性T系白血病Jurkat细胞株的表达影响.
目的: 分析以RNAi慢病毒为载体下调CD59基因表达对急性T系白血病Jurkat细胞株的表达影响.
方法: 通过RNAi慢病毒作为载体，诱导急性T系白血病Jurkat细胞株中的CD59表达降低；采用激光共聚焦技术分析RNAi慢病毒转染情况和CD59分子的定位情况；应用实时荧光定量PCR法检测空白对照组、阴性对照组和RNAi慢病毒转染组中CD59 mRNA的相对表达量；用酶联免疫吸附试验法测定3组细胞培养上清中肿瘤坏死因子-β （TNF-β）和白介素-3（IL-3）的表达，用免疫印迹法（Western blot）测定3组细胞中凋亡相关分子半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、生存素（Survivin）、B淋巴细胞瘤-2（BCL-2）及BCL-2相关X蛋白（BAX）蛋白表达水平.
结果: Jurkat细胞转染效率高于90%，CD59主要定位于细胞膜中。相比空白对照组和阴性对照组，RNAi慢病毒转染组中CD59 mRNA表达明显下调（P<0.05）。RNAi慢病毒转染组中CD59蛋白表达水平较空白对照组和阴性对照组明显降低（P<0.05）。相比空白对照组和阴性对照组，RNAi慢病毒转染组中TNF-β表达水平明显升高，IL-3表达水平明显降低（P<0.05）。RNAi慢病毒转染组Survivin和BCL-2表达水平较空白对照组和阴性对照组明显降低，Caspase-3和BAX蛋白表达较空白对照组和阴性对照组明显升高（P<0.05）.
结论: 通过转染RNAi慢病毒载体促使CD59基因表达下调可降低急性T系白血病Jurkat细胞株中促增殖分化相关分子IL-3的表达，提高肿瘤坏死相关因子TNF-β的表达，同时可提高细胞中促凋亡相关蛋白Caspase-3和BAX的表达，降低抗凋亡蛋白Survivin和BCL-2的表达.