目的探讨热休克蛋白70(HSP70)在膀胱癌细胞株EJ中的热诱导表达及其意义。方法 106/ml EJ置于1.5 ml Eppendorf管中,将它们在不同温度水浴分别加热诱导2 h、4 h、6 h、8 h。5%CO2孵箱中(37℃)恢复2 h。流式细胞术(FCM)和蛋白印迹法检测HSP70,反转录-聚合酶链反应(RT-PCR)检测HSP70 m RNA,SPSS16.0统计软件处理数据。结果人膀胱癌细胞株EJ中HSP70随着温度的增高表达增多,差异有统计学意义(P<0.05),但温度超过43℃时,细胞死亡率增高;随着热诱导时间的延长而增多,差异有统计学意义(P<0.05),超过8 h EJ中HSP70不再增高。结论人膀胱癌细胞株EJ中HSP70热诱导表达的最适条件是43℃、6 h,这对基于HSP70膀胱肿瘤的瘤苗制作提供一定的实验基础。
Objective： To investigate whether human dendritic cells （DC） derived from peripheral blood mononuclear cells （PBMC）, which were pulsed by heat shock protein 70 （HSP70） isolated from human bladder tumor cell lines of E J, were able to induce peptide specific cytotoxic T-lymphocytes （CTL） response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods： The E J-derived HSP70 co-cultured with DC from the healthy volunteers＇ PBMC, along with the crude lysate （the supematant before HSP70 purification） from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes （SI） and interferon-y （IFN-γ） were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51^Cr releasing test. Results： Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate （the supernatant before HSP70 purification） （P 〈 0.05）. DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells （P 〈 0.05）. Conclusion： The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.
