CD40不同形式的活化对CD40突变的RPMI48226细胞增殖和表型的影响  

Effect of different patterns activation forms of CD40 on cloning of CD40 mutant and its proliferation and phenotype in RPMI8226 cell line

作　　者：郑璐[1] 马泓冰[1] 戚春建[1] 董秋明[1] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,215007

出　　处：《中华血液学杂志》2008年第2期101-104,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金（30571690）;苏州大学医学发展基金（EE120507）

摘　　要：目的分析CIM0抗体或配体不同形式刺激对RPMI8226细胞增殖及表面共刺激分子和黏附分子表达的影响，探讨RPMI8226细胞CIM0基因突变在其生物学行为中的作用。方法用RT—PCR方法和DNA序列测定检测RPMI8226细胞CIM0基因突变体，分析RPMI8226细胞经CIM0抗体或配体四种不同方式（CD40单抗、CD40单抗包板、rhsCD40L和CIMOL转基因细胞）刺激后，其体外生长曲线、细胞表型及细胞周期的变化，并利用激光共聚焦显微镜对RPMI8226细胞表面CIM0信号转导体的形成进行初步分析。结果RPMI8226细胞表达CIM0突变体（TCA→TTA，Ser→ku），但是该点突变并未影响hmuCD40的抗原表位。三种激发CD40活化的分子（CD40单抗、rhsCD40L和CD40L转基因细胞）并不影响RPMI8226细胞的体外增殖，但是CIM0单抗包板能明显抑制RPMI8226细胞的增殖[（2．5±0．6）×10^5vs（7．8±1．2）×10^5，P〈0．05]，并产生明显的G．期阻滞[（58．0±3．6）％vs（42．0±2．3）％，P〈0．05]。RPMI8226细胞上黏附分子和共刺激分子的表达无明显变化。激光共聚焦显微镜分析结果显示，在CD40被激活后能形成CD40信号传导的复合体。结论RPMI8226细胞高表达一种CIM0基因突变体，该突变体能在被激活后形成相应的信号传导复合体。Objective To analyze the cloning result of CD40 mutant from RPMI8226 cells, a multiple myeloma（MM） cell line, and study the change of the expressions of costimulatory molecules and the apoptosis of RPMI8226 cells after activated with CD40. Methods CD40 gene mutant in RPMI8226 cell was detected by RT-PCR and DNA sequencing. The cell lines were cultured with sCD40L, L929/CD40L, soluble 5C11 （an anti-CD40 mAb） plate-bound 5C11 and their respective controls. Their growth curves, change of phenotypes and cell cycles were detected. The signalosome of CD40 on RPMI8226 cells were analyzed with laser scanning confocal microscope. Results There was a single base substitution （TCA→TTA） in the open reading frame of CD40 from RPMI8226 cells, resulting in the conversion of a amino acid （Ser124Leu）. Only plate-bound antibody could inhibit RPMI8226 cell proliferation [ （2.5 ± 0.6）×10^5 vs （7.8 ± 1.2） ×10^5 , P〈0.05] and cause G1 arrested [（58.0 ±3.6）% vs （42.0 ±2.3）% , P〈0.05]. muCDD40 was translocated to CD40 signalosome while CD40 activated. Conclusion The mutated CD40 in RPMI8226 cell might decrease its affinity to CD40L, leading to the disorder of CD40 signal.

关 键 词：RPMI8226细胞 抗体 CIM0 CD40配体 DNA突变分析 

分 类 号：R686[医药卫生—骨科学]