【摘要】 背景与目的:Mer是Axl受体酪氨酸激酶家族中的一员,其配体Gas6和Mer结合后可以激活Mer的酪氨酸激酶活性以及下游信号转导途径,在细胞炎症、凋亡等方面发挥作用。近年来对于Mer及其家族成员功能的研究不断完善,但关于其在恶性疾病方面的研究报道甚少。本实验探讨Mer在恶性T淋巴细胞白血病细胞株Jurkat中的表达及其对Jurkat细胞凋亡过程的影响,并对其抗凋亡机制进行初步探究。方法:利用流式细胞仪分别检测正常外周血、骨髓中T淋巴细胞以及Jurkat细胞中Mer的表达。利用RNAi技术敲除Jurkat细胞中Mer的表达后采用噻唑蓝(MTT)法检测Mer对于Jurkat细胞增殖的影响,AnnexinV/PI双染流式细胞术检测Mer对于Jurkat细胞血清撤离诱导凋亡过程的影响。最后利用荧光定量PCR检测RNA干扰后凋亡相关基因Bcl-2和Caspase-3以探讨Mer在Jurkat细胞中抗凋亡机制。结果:Mer在正常外周血和骨髓的T细胞中均未见表达,而在Jurkat细胞中高表达(51.1%)。特异性的siRNA(第1403位点)可以抑制86.0%Mer在Jurkat细胞中的表达。在血清撤离48h导致的凋亡实验中正常Jurkat细胞仅有1.5%的凋亡率,而敲除Mer后的Jurkat细胞凋亡率达到15.3%,而MTT增殖实验中阻断Mer前后差异无统计学意义(P>0.05)。定量PCR结果显示敲除Mer基因后Jurkat细胞中Bcl-2明显下调,为对照组的42.7%,而Caspase-3变化不显著。结论:Mer在Jurkat细胞中异常高表达,并可能通过Bcl-2途径影响细胞凋亡过程。更多还原

【Abstract】 BACKGROUND & OBJECTIVE: Mer is a member of Axl receptor tyrosine kinase family; its ligand Gas6 can bind Mer,then stimulate the tyrosine kinase activity and downstream cell signal pathway of Mer,and take part in cell inflammation and apoptosis. There are more and more reports on Mer function,while few on its association with malignant diseases. This study was to detect the expression of Mer on T-cell leukemia cell line Jurkat,and investigate its anti-apoptosis function and the mechanism. METHODS: Flow cytometry (FCM) was used to detect Mer expression on normal T cells and Jurkat cells. RNA interference (RNAi) was used to block the expression of Mer on Jurkat cells. The effect of Mer on the proliferation of Jurkat cells was assessed by MTT assay,and its effect on serum starvation-induced apoptosis was evaluated by FCM with Annexin V/PI double staining. The expression of apoptosis-associated genes Bcl-2 and Caspase-3 in Jurkat cells was detected by real-time polymerase chain reaction (PCR) after Mer blocking. RESULTS: Mer was not expressed on normal T cells both from peripheral blood and bone marrow,but highly expressed on Jurkat cells with a positive rate of 51.1%. The inhibition rate of Mer expression on Jurkat cells by RNAi was 86.0%. After 48-hour serum starvation,the apoptosis rate was 15.3% in Mer-blocking Jurkat cells,and only 1.5% in control Jurkat cells. There was no significant difference in the proliferation rate of Jurkat cells between these 2 groups. After Mer blocking,Bcl-2 expression was decreased by 42.7% of control,Caspase-3 expression showed no significant change. CONCLUSION: Mer is highly expressed on Jurkat cells,and could inhibit cell apoptosis via Bcl-2 signaling pathway.更多还原