实验研究
中文摘要
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）进行卵巢癌免疫的效果，为卵巢癌的临床提供帮助。
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SKOV3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效果，并通过病理学染比较前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CD80、CD83、CD86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SKOV3细胞杀伤率是DC-CD8+ CTL的6.82倍(P＜0.05)。动物实验表明，经EpCAM-D
C-CD8+ CTL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染显示 EpCAM-DC-CD8+CTL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
免疫具有重要意义。
关键词：卵巢癌; 免疫; EpCAM; DC; CTL
Experimental study on ovarian cancer immunotherapy by EpCAM activated dendritic cells induce antigen-specific CD8+
cytotoxic T lymphocytes
ABSTRACT
Objective: To observe the effect on ovarian cancer immunotherapy by Epithelial cell adhesion molecule (EpCAM) activated dendritic cells (DC) induce antigen-specific CD8+ cytotoxic T lymphocytes (CTL) and offer help to ovarian cancer immunotherapy.
Methods: After induce by EpCAM, surface molecules, interleukin (IL) -12 and IL-10 of DC were tested. 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 SKOV3 was detected. After treatment of EpCAM-DC-CD8+ CTL, tumor volume of ovarian tumor of bearing BALB/C-nu/nu mice was detected. Meanwhile, the changes of tumor tissue were observed by HE staining.
Results: Compared with PBS, EpCAM stimulation can significantly improve surface markers DC80, DC83, DC86 and HLA-DR levels, and 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 significantly reduced the secretion of IL-10 (P＜0.05). DC-CD8+ CTL and EpCAM-DC-CD8+ CTL both resulted in little amount of IOSE80 cell killing (P＞0.05), but the killing rate of EpCAM-DC-CD8+ CTL on SKOV3 cells was 6.82-folds as much as that of DC-CD8+ CTL. Animal experiments showed that after EpCAM-DC-CD8+ CTL treatment, ovarian cancer transplantation tumor volume ratio 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 pathological changes in tumor tissues.
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.
Key words: Immunotherapy/ovarian cancer/ EpCAM/DC/CTL
EpCAM蛋白激活DC诱导抗原特异性CTL卵巢癌的
实验研究
线形诱导标志1. 前言
卵巢恶性肿瘤是女性生殖器官常见的恶性肿瘤之一，发病率仅次于子宫颈癌和子宫体癌而列居第三位[1, 2]。卵巢癌的目前缺少早期的临床标志物，发病隐匿，2/3女性诊断时便为晚期，5年生存率仅为30%，死亡率居各类妇科肿瘤的首位，对妇女生命造成严重威胁[3-5]。
卵巢癌的流行病学研究发现卵巢癌发病具有明显的地区差异，欧美国家发病率较高，白人发病率约为14/10万[6, 7]；其次是印度，中国和日本等东亚国家发病率最低。近30年来恶性卵巢癌发病率逐年上升，中国卵巢癌发病率近10年来呈逐渐上升趋势，其中北京，上海等城市同济发现其发病率在5-7/10万人，因此推测卵巢癌发病与遗传和环境因素密切相关[8]。此外，晚婚、不育、绝经、抽烟及高脂肪
饮食都会对卵巢癌的发生有影响[9, 10]。有研究发现，初潮早和晚绝经都是卵巢癌的高危因素，自然绝经可减少卵巢癌的发病[11-13]。
尽管卵巢癌死亡率高，但早期诊断可以使其存活率可能达90%左右，因此早期诊断和干预能够提高治愈率、延长生存期和改善生存质量。目前卵巢癌的诊断可以分为三种：（1）定性检查；（2）定位检查；（3）生化检测；（4）细胞学检测[14, 15]。由于卵巢癌发病隐匿，早期无明显的体征，可以通过详细的询问病史和妇科体检定性的进行判别[16]。妇科检查时，肿瘤增大时下腹部能够摸到肿块，早期肿瘤一般单侧生长，表明光滑，而晚期常位于双侧生长，表面较为粗糙。卵巢功能障碍或者泌尿道症状都需要通过进一步的影像学检查[17, 18]，定位检查是通过影像学手段对于卵巢外转移而形成盆腔内散在小结节或者包块进行检查，确定包块的位置。临床上常采用超声波明确肿瘤的大小、位置、形态、内部结构和来源。此外，可以通过腹腔镜检查，窥视盆腹腔脏器，明确有无肿瘤及有
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