EpCAM特异性免疫效应细胞对EpCAMhigh腺癌细胞的体外杀伤实验研究
| 作者: |
1,2周晔,
1,2张树交,
1,2高飞,
1,2蔡建辉
1 河北医科大学，石家庄 050017 2 河北省人民医院， 石家庄 050051 |
| 通讯: | |
| DOI: | 10.3978/j.issn.2095-6959.2015.06.S134 |
摘要
背景：上皮细胞粘附分子(EpCAM CD326)高表达于多种实体腺癌细胞表面，包括结肠癌、胃癌、乳腺癌等。EpCAM在胃肠道肿瘤细胞>95%，与肿瘤细胞的增殖及肿瘤预后有密切关系。近年来，EpCAM已经成为肿瘤免疫治疗的一个新靶点，培养EpCAM特异性免疫效应细胞(Ep-effector)制备新高效特异性杀伤细胞。目的：探讨体外制备的EpCAM抗原特异性免疫效应细胞的特异性杀伤功能。并比较其与腺癌细胞裂解物特异性免疫效应细胞、CIK细胞杀伤功能的差异。方法：采集健康成人外周血，分离淋巴细胞及imDCs。体外诱导淋巴细胞中T细胞转化为CIK细胞并扩增。本实验首次以纯化EpCAM多肽抗原负载imDCs生成EpCAM-mDCs(Ep-mDCs)，Ep-mDCs与自体CIK细胞体外共培养，制备成纯化EpCAM抗原特异性DC-CIK-CTL效应细胞(Ep-effector)。相似的方法，制备LS174-T腺癌细胞全抗原特异性DCCIK-CTL效应细胞(LS-effector)。设单纯CIK组、LSeffector组及Ep-effector组，选择EpCAMhighLS174-T结肠腺癌细胞及EpCAMlowPaCa-2胰腺癌细胞作为靶细胞。细胞毒试验(CCK-8法)检测不同效靶比时各组效应细胞对EpCAMhigh的LS174-T及EpCAMlow的PaCa-2的杀伤效率。结果：体外制备LS-mDCs及Ep-mDCs表面分子表达量无差异(P>0.05)。随效靶比(E∶T)增高，各组效应细胞对LS174-T及PaCa-2的杀伤效率均升高。Ep-effector组对LS174-T的杀伤效率高于LSeffector组及单纯CIK组，LS-effector组与单纯CIK组无统计学差异；对PaCa-2的杀伤效率，两两组间比较无统计学差异(P>0.05)。结论：本实验首次应用EpCAM负载人外周血来源DC细胞制备Ep-mDCs，并证实其拥有激活T细胞生成特异性CTL的功能，由其制备的效应细胞(Ep-effector)对EpCAMhigh腺癌细胞具有更高的特异性杀伤活性。
关键词：
上皮细胞粘附分子
腺癌细胞
肿瘤免疫治疗
Ep-mDCs
The research of cytotoxicity of EpCAM antigen-specific immune effectors to EpCAMhigh adenocarcinoma cells in vitro
DOI: 10.3978/j.issn.2095-6959.2015.06.S134
Abstract
Background: Epithelial cell adhesion molecule (EpCAM) is overexpressed abnormally in nearly all adenocarcinomas including colon cancer, gastric cancer, breast cancer and so on. The highest expression levels of EpCAM on gastrointestinal adenocarcinomas are more than 95%. EpCAM is closely related to the proliferation of cancer cells and the prognosis of the tumor. In recent years, EpCAM has been regarded as the new target of immunitherapy of tumor. Culturing EpCAM specific effectors is used to generate new effectors that with better specific killing effective. Objective: To explore the killing efficiency of EpCAM antigen-specific immune effector cells cultured in vitro. Compare the killing rates between EpCAM antigen-specific immune effectors and full antigens of EpCAMhigh adenocarcinoma cells -specific immune effectors. Methods: Col lected mononuclear cel ls (PBMCs) from peripheral blood of healthy adults. Induction of T lymphocytes into CIK cells and amplification of CIK cells in vitro. imDCs pushed with purified EpCAM polypeptide antigen to generated EpCAM antigen mature DCs that named Ep-mDCs. Ep-mDCs and autologous CIK cells were co-cultured in vitro, part of CIK cells turn to EpCAM antigen-specific cytotoxic CTL after EpCAM antigens information were presented by Ep-mDCs, and then EpCAM antigen-specific DC-CIK-CTL effector cells (Ep-effector) were generated. EpCAMhigh LS174-T colon adenocarcinoma cells and EpCAMlow PaCa-2 pancreatic cells were selected to be target cells. Detected the killing rates of each group of effector cells to EpCAMhigh LS174-T and EpCAMlow PaCa-2 at different E:T by Cytotoxicity assay (CCK-8 method), and analyzed the statistical differences. Results: The expressions of surface molecules in LS-mDCs and Ep-mDCs have no significant difference. With the target ratio (E:T) increased, the killing rates of effector cells in each group were all raised. The killing rates to LS174-T cells in Ep-effector group were higher than the others, but have no difference between CIK group and Ls-effector group. The killing rates to PaCa-2 have no difference between each other group. Conclusion: This is the first time that we use EpCAM antigen to push imDCs to generate Ep-mDCs. And the function that Ep-mDCs can active T cells to generate CTLs had been provided.Epeffector has a higher specific cytotoxicity for EpCAMhigh adenocacinoma cells.