抑制核因子-κB的活性减少内皮细胞与T淋巴细胞粘附

Decreasing cell adhesion between endothelial cells to T cells through inhibition of NF-κB activity

目的探讨抑制核因子κB(NFκB)对内皮细胞活化及内皮细胞与T淋巴细胞粘附的影响。方法应用pCMVIκBαM质粒建立稳定表达突变型IκBα蛋白的内皮细胞系,同时以表达野生型IκBα蛋白(pCMVIκBα)的内皮细胞作为对照。通过逆转录聚合酶链反应及流式细胞仪检测细胞表面细胞间粘附分子1(ICAM1)、血管细胞粘附分子1(VCAM1)和P选择素的变化,并将两种转染细胞分别与人T淋巴细胞株Jurkat细胞混合培养,通过相差显微镜下细胞计数来观察内皮细胞与T淋巴细胞的粘附情况。结果高表达突变型IκBα蛋白的内皮细胞的NFκB活性被显著抑制,其ICAM1mRNA和VCAM1mRNA水平明显低于对照细胞(P<0.05),细胞表面的ICAM1、VCAM1和P选择素的表达较对照细胞显著减少(P<0.05),与T淋巴细胞的粘附较对照细胞显著减少(P<0.05)。结论抑制核因子κB的活性能有效减少内皮细胞与T淋巴细胞的粘附。

Objective To investigate the role of inhibition of nuclear factor kappa B (NF-κB) activation in cell adhesion between T cells to endothelial cells. Methods The pCMV-IκBαM was ~stably transfected into human endothelial cell line ECV304 using geneticin (G418). The pCMV-IκBα was also stably transfected as controls. The NF-κB activity was detected with reporter vector pNF-κB Luciferase and electrophoretic mobility shift assay. The cells with over-expression of non-degraded IκBα protein were selected by Western blot using specific antibody. mRNA levels of cell surface molecules (ICAM-1, VCAM-1) were detected by reverse transcription polymerase chain reaction (RT-PCR). The protein levels of surface adhesion molecules including ICAM-1, VCAM-1 and P-selectin were measured by flow cytometery. The adhesion of Jurkat T cells to ECV304 cells was observed through cell number count using phase-contrast microscopy. Results NF-κB activity was inhibited by over-expression of non-degraded IκBα protein. mRNA levels of adhesion molecules ICAM-1, VCAM-1 were significantly decreased in the cells transfected with pCMV-IκBαM compared to the cells ~transfected with pCMV-IκBα. Flow cytometery revealed that the expression of ICAM-1, VCAM-1 and P-selectin was inhibited significantly. Cell adhesion assay showed pCMV-IκBαM could decrease the ~adhesion of Jurkat T cells to ECV cells from (~71.4 ±~5.2 ) % to (~42.2 ±~3.7 ) % (P<~0.05 ) compared to those transfected with pCMV-IκBα. Conclusion The pCMV-IκBαM, which inhibits the activity of NF-κB through over-expression of non-degraded IκBα protein, can inhibit the activation of NF-κB. It may be used for gene therapy in diseases involving NF-κB activation abnormally like organ ~transplantation via decreasing cell adhesion.