榄香烯复合抗肿瘤疫苗免疫的效应机制

Immunity Effects Mechanism Treated with Elemene- Combo Tumor Vaccine

以小鼠H22腹水型肝癌和L615白血病为实验模型,采用榄香烯或/和热休克等为应激原(stressor),对小鼠H22腹水型肝癌细胞和L615白血病细胞进行不同浓度榄香烯、不同时间热休克处理,用透射电镜观察肿瘤细胞的形态变化;采用直接免疫荧光法流式细胞术(FCM)检测肿瘤细胞凋亡百分率及对肿瘤细胞膜HSP70、HSP90表达的影响;进而分离纯化不同应激原(榄香烯、丝裂霉素-C)诱导的H22和L615肿瘤细胞的HSP70-肽复合物,并用之免疫Balb/c和615小鼠,进行体内抗瘤免疫效应的研究。透射电镜显示,榄香烯或热休克处理都能引起2种肿瘤细胞的凋亡和坏死,L615白血病细胞较H22肝癌细胞更敏感,形态学变化更剧烈;流式细胞术检测进一步证明,榄香烯与热休克都能诱导2种肿瘤细胞发生凋亡,两者复合作用更能促进肿瘤细胞的凋亡,在一定范围内L615细胞凋亡与榄香烯的浓度和热休克的时间呈明显的依赖关系;用榄香烯或MMC诱导的2种肿瘤细胞分离纯化的HSP70-肽复合物给Balb/c和615小鼠进行主动免疫,免疫组小鼠在1个月内全部存活。而buffer D免疫对照组小鼠无一存活。

The possible relationship between heat shock protein (HSP) and the molecular mechanisms of active immunization of elemene-combo tumor cell vaccine (TCV) was tudied by using murine H22 ascitic hepatic carcinoma and L615 leukemia as the experimental models. Stressors, such as elemene or/and heat shock, were applied to study the following items: treated with different concentrations of elemene and different time course of heat shock, transmission electron microscopy were used to observe the morphological influence on tumor cells. Direct immunofluorescent flow cytometry (FCM) was used to detect the apoptotic rate of H22 and L615 tumor cells and influence on the expression of membrane HSP70 and HSP90. The membrane HSP70-peptide complex was separated and purified from H22 and L615 tumor cells induced by different stressors (elemene, MMC) and then a preliminary in vivo active immunoprophylaxis experiment was carried out using those isolated HSP70-peptide complex against H22 on Balb/c mice and L615 on 615 mice. Under transmission electron microscope, the influence of elemene or heat shock on the two kinds of tumor cells is, to some extent, the same or almost the same, which means the apoptosis and necrosis of the cells. The extent of the influence on the two kinds of tumor cells is different, and L615 leukemia was more sensitive and undergoing bigger morphological changes than H22 hepatic carcinoma. It's further proved by FCM that both elemene and heat shock alone can induce the apoptosis of mice H22 hepatic carcinoma or L615 leukemia cells, with the intensifying of this effect by combining use of the two, the apoptosis of cells from mice L615 leukemia relates closely to the concentration of elemene and the time course of heat shock at 42℃. By separating and purifying Balb/c and 615 mice immunized by HSP70-peptide compound of H22 and L615 tumor cell induced by elemene or MMC, the results showed that all mice survived within one month, while none of the mice in control immunized by buffer D survived.