EpCAM蛋白激活DC诱导抗原特异性CTL治疗卵巢癌的实验研究

Experimental study on ovarian cancer immunotherapy by EpCAM activated dendritic cells induce antigen-specific CD8^+ cytotoxic T lymphocytes

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EpCAMDC-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 ） -1 2 , 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）-7 of EpCAM-DC-CD8＋ CTL on nor^nal 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. 0 5 ） . EpCAM stimulation significantly increased the ex-pression 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.