HTLV-1 Tax蛋白阳性T细胞中EGR-1表达调控机制的研究

Study of the regulatory mechanism of EGR-1 expression in HTLV-1 virus Tax protein positive-T cells

目的：探讨成人T淋巴细胞白血病病毒1型（ HTLV-1病毒） Tax蛋白阳性T细胞TaxP中早期生长反应基因-1（EGR-1）表达调控的机制。方法构建不同长度的EGR-1调控序列报告基因，将其转染TaxP细胞，加入NF-κB抑制剂BAY 11-7082或同等体积的二甲基亚砜（DMSO），24 h后收集细胞检测荧光素酶活性；TaxP细胞上清中加入BAY 11-7082或同等体积的DMSO，24 h后免疫印迹检测EGR-1蛋白表达；将Tax及其突变体M22和M47分别转染293 T细胞，24 h后免疫印迹检测EGR-1蛋白表达。结果成功构建了不同长度及突变体的EGR-1调控序列荧光素酶报告基因；荧光素酶报告基因检测显示：相对于E1、E2而言， E3、DelE、MutE的荧光素酶活性明显降低，加入BAY 11-7082后，E1、E2的荧光素酶活性明显降低（P＜0．01），而E3、DelE、MutE无明显变化；同样地，免疫印迹检测显示加入BAY 10-7082后，EGR-1蛋白表达明显降低；相对于野生型和M47，转染M22突变体后，293 T细胞EGR-1蛋白表达明显降低。结论 NF-κB是Tax蛋白阳性T细胞中EGR-1表达调控的关键核因子。

Objective To explore the expression of early growth response gene-1 （EGR-1） in T cells that were positive for Tax protein of human T-cell leukemia virus type 1 （HTLV-1） and its possible reg-ulatory mechanism .Methods A series of expression structures carrying the regulatory elements of EGR-1 in different length and luciferase reporter genes were constructed .TaxP cells were transfected with the con-structs containing reporter genes and cultured with 5μmol/L of NF-κB inhibitor BAY 11-7082 or equal vol-ume of DMSO.After cultured for 24 hours the cells were collected to test the luciferase activity .BAY 11-7082 or equal volume of DMSO was added into the supernatant of TaxP cell culture to test the expression of EGR-1 protein by Western blot after 24 hours of culture .Tax and its mutants M22 and M47 were transfected into 293 T cells respectively to test the expression of EGR-1 protein by Western blot after 24 hours of culture . Results The expression structures carrying the regulatory elements of EGR-1 in different length and their mutants followed by luciferase reporter genes were successfully constructed .The luciferase activity in the cells transfected with the constructs containing the elements E 1 and E2 were higher than that transfected with E3, DelE and MutE, but the reporter gene expressions were decreased with the interference of BAY 11-7082 （P〈0.01）.However, there were no significant changes with the luciferase activity in the cells transfected by elements E3, DelE and MutE.Western blot analysis indicated that the expression of EGR-1 protein was significantly decreased with the interference of BAY 10-7082 .The expression of EGR-1 protein in M22 mu-tants-transfected 293 T cells were decreased significantly in comparison with those by wild type tax-and M47-transfected cells .Conclusion NF-κB was the key nuclear factor in regulating the expression of EGR-1 pro-tein in Tax-positive T cells .