Effect of dendritic cell modified by gp96-peptide complex on antitumor effect in H22 cell

Objective： To investigate the antitumor effect of dendritic cell （DC） modified by gp96-peptide complexes both in vitro and in vivo. Methods：Gp96-peptide complexes were acquired from H22 liver cancer cells in mice. DC were cultured from bone marrow cells and modified by gp96-peptide complexes. Spleen lymphocytes of mice were activated by modified DC and the cytotoxicity were detected by ^51Cr release method. Modified DC, gp96-peptide complexes and inactivated H22 cells were injected into mice bearing H22 liver cancer cells to observe the levels of IL-10, IFN-y in serum and the alteration of proportions of CD8^＋-IFNy^＋ and CD8^＋-IL-10^＋ cells, CD4^＋-IFNy^＋ and CD4^＋-IL-10^＋ cells. Results： DC modified by gp96-peptide complexes can activate spleen lymphocyte and the latter can specifically kill H22 cells but not Ehrilich ascites carcinoma cells. Modified DC can improve the host＇s antitumor immune response and the proportions of Thl cells, inhibiting tumor growth. Conclusion： Gp96-peptide complexes can activate DC effectively, making DC a good vaccine.

Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic cell on mice of H22 liver cancer

Objective： To improve DC-based tumor vaccination, we studied whether dendritic ceils （DCs） which cocultured with H22 liver cancer cells-derived heat shock protein （HSP） glycoprotein 96 （gp96） affect the T cell-activating potential in vitro and the induction of tumor immunity in vivo. Methods： Maturation of murine bone marrow-derived DC was induced by GM-CSF plus IL-4, which mimiced the immunostimulatory effect of DC. Cocultured DC and gp96-peptide complexes were used to vaccine H22 liver cancer cells of mice. Using murine models we compared the immunogenecity of DC modified by gp96-peptides complexes derived from murine liver cancer cells alone or inactive tumor cells. To verify the specificity of the vaccine, in vitro assays were executed. Serum cytokine levels were quantified to explore the supposed pathway of DC modified by gp96 peptide complexes and its effect on antitumor immune response. Results： DC modified by gp96-peptide complexes can activate spleen lymphocyte and the latter can specifically kill H22 cells but not Ehrilich ascites carcinoma cells. Modified DC can induce potent tumor-antigenspecific immune response, augment the proliferation of Thl cells, and inhibit tumor growth. Conclusion： In this study, we have developed a novel DC-mediated tumor vaccine by combing the gp96 antigenic peptides complexes and inducing immune response against specific tumor cells, gp96 can be identified as a potent DC activator.

gp96-肽复合物体外诱导CTL反应及其抗肿瘤效应研究

目的研究小鼠肝癌腹水瘤H22细胞来源的gp96-肽复合物体外诱导小鼠脾淋巴细胞特异性细胞毒T细胞(CTL)的产生及其抗肿瘤效应。方法利用蛋白提取纯化技术、Western blot法、细胞培养技术、流式细胞术、CCK-8法、RT-PCR法等研究gp96-肽复合物体外诱导扩增CTL及其抗肿瘤效应。结果流式细胞仪检测表明,经gp96-肽复合物诱导后的CD8^+T细胞比例达到近70％,远远高于对照组的35％、26％。该活化的CTL细胞在效靶比为50：1时的肿瘤细胞杀伤率达72％与对照组相比具有统计学意义；将活化的CTL细胞回输小鼠体内能产生对该肿瘤细胞良好的过继性免疫保护,延长荷瘤小鼠的生存时间；RT-PCR法检测该活化的脾细胞较正常脾淋巴细胞高表达IFN-γ mRNA。结论小鼠肝癌腹水瘤H22细胞来源的热休克蛋白gp96-肽复合物能诱导小鼠脾淋巴细胞的CTL反应,并增强脾细胞IFN-γ mRNA的表达,该CTL具有特异性抗H22肿瘤细胞的免疫作用。

人腺样囊性癌细胞热休克蛋白gp96肽复合物的纯化和鉴定

目的:建立从人腺样囊性癌细胞系中提取gp96-肽复合物的方法。方法:培养人腺样囊性癌(ACC-M和ACC-2)细胞系,经裂解、盐析、过Con A亲和柱、透析、DEAE-Sepharose离子交换层析后得到目的蛋白,对进行蛋白质浓度、分子量及性质进行鉴定。结果:取湿重同为1g的两株细胞,纯化后得到总量分别为77μg和85μg的产物,电泳结果显示纯化蛋白分子量约为96KD,western blot显示产物是gp96-肽复合物。结论:采取上述方法从腺样囊性癌细胞中得到了较高纯度的gp96-肽复合物。