CD40配体对转CD40 cDNA的人肺癌细胞功能调节的研究

Functional modulating effect of CD40 ligand on CD40-transfected human lung carcinomas

目的 研究CD4 0配体 (CD4 0L ,CD15 4 )对CD4 0阴性肺癌细胞系转染CD4 0cDNA后 (即CD4 0高表达细胞系 )的调节作用 ,评价CD4 0作为治疗靶抗原的潜能。方法 通过流式细胞术筛选CD4 0阴性细胞系 ,即GLC 82细胞。以 3D5细胞为模板 ,以pCDNA3为载体 ,通过基因克隆技术制备CD4 0cDNA并转染到GLC 82细胞中 ,建立CD4 0高表达细胞系 ,即GLC 82 /CD4 0。在细胞培养液中加入 0 .1μg/mlCD4 0L ,通过流式细胞术和MTT试验检测CD4 0L对细胞表型、细胞生长、细胞周期及凋亡的影响。结果 Western蛋白印迹检测、限制性酶切电泳、DNA测序和流式细胞仪测定均证明了CD4 0cDNA转染成功 ,转染后细胞的CD4 0表达率高达 95 .9%。CD4 0L与CD4 0作用后 ,使GLC 82 /CD4 0细胞系上MHC I、ICAM 1和Fas分子的表达增强 ,EGFR表达减弱。细胞生长受到抑制 ,第 5天受抑最明显 ,受抑率为 30 % ,但无周期特异性变化。CD4 0L停用 4 8h后 ,各种指标的变化有所恢复。CD4 0L对原有高表达CD4 0的Calu 3肺癌细胞上述指标的影响较GLC 82 /CD4 0更明显 ,而对不表达CD4 0的GLC 82细胞无明显影响。所有肺癌细胞系均未出现细胞凋亡。结论 CD4 0L对转染CD4 0cDNA后CD4 0高表达肺癌细胞系具有免疫导向治疗的潜能。

ObjectiveTo determine the modulating effect of CD40 ligand (CD40L, CD154) on CD40-transfected human lung carcinomas and to assess the potential of CD40 as a therapeutic target. MethodsTumor cells of a CD40-negative lung cancer cell lines (GLC-82) were transfected with a vector expressing CD40 cDNA. The transfected cell line, GLC-82/CD40, was shown to express hign levels of CD40. GLC-82/ CD40 cells after being exposed to 0.1 μg/ml CD40L were examined for their surface expression, cell cycle, apoptosis and cell growth by flow cytometry and MTT assay. ResultsThe expression of MHC-I, ICAM-1 and Fas in GLC-82/CD40 cells was significantly increased, whereas that of EGFR was decreased. Cell proliferation was significantly inhibited by CD40L with an inhibition rate of 30% on day 5, but no change in cell cycle. All of the changes disappeared after 48 h incubation with CD40L. More significant changes were observed in Calu-3 cell lines which expressed high levels of CD40, but the CD40-negative GLC-82 cells were unresponsive to CD40L. None of the 3 cell lines showed significant changes in apoptosis upon CD40L treatment. ConclusionCD40, if over-expressed in tumor cells, could be considered as a potential therapeutic target.