【摘要】 目的研究猪苓多糖联合卡介苗刺激T24膀胱癌细胞株后,细胞外HSP的表达及其对J774A.1巨噬细胞TLR2、TLR4,胞内钙离子(Ca2+)、一氧化氮(NO)和活性氧(ROS)表达的影响,探讨膀胱内灌注卡介苗治疗浅表性膀胱癌的免疫启动和免疫调节机制。方法用ELISA的方法检测猪苓多糖和卡介苗联合刺激T24膀胱癌细胞后胞外HSP90、HSP70、HSP60、HSP27的表达,胞外HSP作用于J774A.1巨噬细胞后,用流式细胞术检测J774A.1巨噬细胞表面TLR4和TLR2的表达,应用激光共聚焦扫描技术,检测J774A.1细胞内Ca2+、NO和ROS的动态变化。结果猪苓多糖和卡介苗联合作用组胞外HSP的表达明显高于猪苓多糖和BCG单独使用组(P<0.05),48h时胞外HSP的表达至峰值,且胞外HSP70呈优势表达;胞外HSP可上调J774A.1巨噬细胞TLR4的表达(P<0.05),对J774A.1细胞内Ca2+、NO和ROS有明显的调节作用(P<0.05)。结论卡介苗体外直接刺激人T24膀胱癌细胞后,可表达细胞外HSP90、HSP70、HSP60、HSP27,猪苓多糖和卡介苗联合使用可增加其表达。细胞外HSP对J774巨噬细胞有一定的免疫调节作用。更多还原

【Abstract】 Objective To detect the expressions of extracellular HSPs (eHSPs) in T24 human bladder cancer cell line stimulated by polyporus polysaccharide (PPS) combined with BCG,and to evaluate the effects of eHSPs on TLR2,TLR4,intracellular free calcium (Ca2+),nitric oxide(NO),and reactive oxygen species (ROS) of J774A.1 macrophages and for studying the immunoregulatory and immunological priming mechanism of intravesical injection treatment of BCG on superficial bladder tumors. Methods ELISA was used to detect the expressions of eHSPs after the T24 human bladder cancer cells were stimulated with PPS combined with BCG. After J774A.1 macrophages were stimulated by eHSPs,the expressions of TLR2 and TLR4 were detected with flow cytometry (FCM). Dynamic changes of Ca2+,NO,and ROS were determined with laser scanning confocal microscopy (LSCM). Results The concentrations of eHSPs were the highest in T24 cells stimulated with combination of BCG and PPS (P<0.05,vs single use of BCG or PPS respectively). eHSPs upgraded the expression of TLR4 but regulated the concentrations of Ca2+,NO,and ROS (P<0.05,vs control). Conclusion After in vitro stimulation of BCG and/or PPS,T24 human bladder cancer cell line may express eHSPs,which can be enhanced by the combination of BCG and PPS. Furthermore,eHSPs show immunoregulatory effects on the J774 macrophages.更多还原