【摘要】 目的运用RNAi慢病毒沉默CD59的表达,观察其对急性T淋巴细胞白血病Jurkat细胞株增殖、凋亡的影响。方法构建CD59 RNAi-EGFP融合蛋白慢病毒载体,转染急性T系白血病Jurkat细胞株,筛选出稳定转染的细胞系,空病毒组为阴性对照,未处理的Jurkat细胞系作为空白对照;荧光显微镜和流式细胞仪观察各组的细胞转染效率;ELISA检测各组细胞CD59的表达情况;RT-PCR检测各组CD59基因以及凋亡相关基因Bcl-2/Bax m RNA的表达;CCK8表达检测细胞增殖效率的改变;流式细胞术检测各组细胞凋亡情况。结果荧光显微镜和FCM观察转染效率在90%以上;ELISA结果显示实验组细胞CD59蛋白表达降低;RT-PCR结果显示实验组CD59、Bcl-2 m RNA表达水平降低(P<0.05),Bax m RNA表达水平升高(P<0.05);CCK8结果显示实验组细胞增殖效率明显降低(P<0.05);流式细胞仪结果显示沉默CD59的表达能够促进细胞凋亡(P<0.05)。结论沉默D59基因表达可抑制急性T系白血病Jurkat细胞株的增殖能力并诱导细胞凋亡,为临床急性T系白血病的治疗提供了新靶标、新思路。更多还原

【Abstract】 To observe the function of CD59 in proliferation and apoptosis of acute T lymphocyte Jurkat cell lines,RNA interference(RNAi) was used to silence the expression of CD59 gene by lentivirus.CD59 RNAi-EGFP lentivirus vector(RNAi-CD59) was built and transfect to acute T lymphocyte Jurkat cell lines,then stable transfection cell line was selected.Empty lentivirus with EGFP was used as negative control group(RNAi-NC) and the Jurkat as the blank control group.Fluorescence microscope and flow cytometer(FCM) was applied to test the transfection efficiency,while ELISA was employed to detect the expression of CD59 in each group;the expression of CD59 Bcl-2/Bax mRNA was detected by Real-time PCR(RT-PCR);CCK8 was used to analyze the change of cell proliferation,while the cell apoptosis was tested by FCM.Data showed that the transfection efficiency was over 90%,the expression of CD59 protein was reduced and the expression of CD59 and Bcl-2 mRNA were decreased in experimental group while Bax was increased;the cell proliferation of experimental group was significantly lower than that of the control group;the apoptosis rate of experimental group was significantly higher than that of the control group.In conclusion,silencing the expression of CD59 gene can inhibit the proliferation of acute T lymphocyte Jurkat cell lines and induce the apoptosis,which provides a new target and a new idea for clinical therapy of acute T leukemia.更多还原