EpCAM蛋白激活DC诱导抗原特异性CTL治疗卵巢癌的实验研究

摘要

目的:探讨EpCAM蛋白激活树突细胞(DC)诱导产生CD8+细胞毒T淋巴细胞(CD8+ CTL)进行卵巢癌免疫治疗的效果,为卵巢癌的临床治疗提供帮助.方法:利用EpCAM蛋白诱导成熟DC同时检测DC表面分子和白介素(IL)-10与IL-12表达量的变化,随后通过EpCAM-DC诱导EpCAM抗原特异性CD8+ CTL,继而检测EpCAM-DC-CD8+ CTL对正常卵巢上皮细胞IOSE80和卵巢癌细胞SKVO3的杀伤效果,同时检测干扰素(IFN)-γ释放量.随后进一步检测EpCAM-DC-CD8+ CTL对卵巢癌移植裸鼠的肿瘤抑制程度,并通过病理学染色检测治疗后肿瘤组织变化情况.结果:与PBS刺激相比,EpCAM蛋白能够显著上调DC表面分子DC80、DC83、DC86和HLA-DR水平,依次达到4.79、4.85、4.60和10.91倍;同时EpCAM蛋白显著提高IL-12释放和显著抑制IL-10分泌(P<0.05).DC-CD8+ CTL与EpCAM-DC-CD8+ CTL均引起少量IOSE80细胞凋亡(P>0.05),但EpCAM-DC-CD8+ CTL对SKVO3细胞杀伤率是DC-CD8+ CTL的6.82倍(P<0.05).动物实验表明,经EpCAM-DC-CD8+ CTL治疗后,BALB/c-nu/nu卵巢癌移植肿瘤体积比明显低于PBS组以及DC-CD8+ CTL组,分别达到0.27和0.28倍(P<0.05).HE染色显示 EpCAM-DC-CD8+CTL治疗导致肿瘤组织出现明显的病理学改变.结论:EpCAM蛋白刺激促进了DC成熟继而诱导产生EpCAM特异性CD8+ CTL,EpCAM-DC-CD8+ CTL能够高效的杀伤肿瘤细胞并延迟肿瘤生长,对卵巢癌临床免疫治疗具有重要意义.%Objective:To observe the effect on ovarian cancer immunotherapy by dendritic cells (DC) which activated by Epithelial cell adhesion molecule (EpCAM) induce antigen-specific CD8+ cytotoxic T lymphocytes (CTL) and to provide some help to ovarian cancer immunotherapy.Methods: Interleukin (IL)-12,and IL-10 of DC were tested after inducing by EpCAM.Subsequently,EpCAM specific CTL CD8+ was induced by EpCAM-DC.The therapeutic effect and interferon (IFN)-γ of EpCAM-DC-CD8+ CTL on normal ovarian epithelial cells IOSE80 and ovarian cancer cell SKVO3 was detected.After treatment of EpCAM-DC-CD8+ CTL,the volume of ovarian tumor of bearing BALB/c-nu/nu mice was detected.Meanwhile,the morphology changes of tumor tissue were observed by HE staining.Results: Compared with PBS,EpCAM stimulation significantly inceased surface markers DC80,DC83,DC86 and HLA-DR levels,and added up to 4.79,4.85,4.60 and 10.91 times (P<0.05).EpCAM stimulation significantly increased the expression of IL-12 and reduced the secretion of IL-10 (P<0.05).Both of DC-CD8+ CTL and EpCAM-DC-CD8+ CTL resulted in minute amount of IOSE80 cell killing (P>0.05).However,the killing rate of EpCAM-DC-CD8+ CTL on SKVO3 cells was 6.82-folds as much as that of DC-CD8+ CTL.Animal experiments showed that ovarian cancer transplantation tumor volume ratio after EpCAM-DC-CD8+ CTL treatment,was significantly lower than PBS group and DC-CD8+ CTL group,which reached 0.27 and 0.28 times,respectively (P<0.05).HE staining showed that EpCAM-DC-CD8+ CTL treatment resulted in significant changes of tumor tissues in pathology.Conclusion: EpCAM protein stimulated the maturation of DC that induced the production of EpCAM specific CD8+ CTL.EpCAM-DC-CD8+ CTL can effectively kill ovarian tumor cells and delay the growth of tumor,which is of great significance for the immunotherapy of ovarian cancer.