树突状细胞的体外培养及其抗癌作用

Culture of Dendritic Cells in vitro and Its Anti-tumor Immonotherapy

背景与目的恶性肿瘤患者免疫力低下,缺乏强有力的抗肿瘤免疫应答,其原因是肿瘤细胞的抗原性较弱,而且抗原提呈细胞的功能低下,使肿瘤抗原不能有效地提呈给淋巴细胞。因此如何能有效地诱导出抗肿瘤免疫效应是一个非常关键的问题。本研究通过建立体外培养树突状细胞(dendritic cells,DC)的方法并观察其诱导的抗肺癌作用的研究,为以DC为基础的肺癌免疫瘤苗的临床应用提供实验依据。方法3mol/L氯化钾法提取人肺腺癌细胞GLC-82的可溶性抗原多肽;从人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中用GM-CSF、IL-4和TNF-α体外诱导扩增DC,用流式细胞仪FCM及免疫染色法分析鉴定DC;肺癌肿瘤可溶性抗原(tumor soluble antigen,TSA)和超抗原金黄色葡萄球菌肠毒素A(staphylococcal enterotoxin A,SEA)联合修饰致敏DC;以联合抗原修饰后的DC、单纯肺癌抗原TSA致敏的DC、单纯超抗原SEA修饰的DC和未经抗原修饰的DC分别与外周血T淋巴细胞共同孵育,刺激T淋巴细胞活化增殖,诱导产生具有识别肺癌抗原的特异性CTL(作为效应细胞分别称为TSA-SEA-DCL、TSA-DCL、SEA-DCL、DCL);采用MTT法检测各效应细胞对靶细胞GLC-82、肺癌CALU-6和人红白血病K562细胞的抗癌活性;镜下观察DC、效应细胞形态及对靶细胞的杀伤过程。结果诱导出高表达CD1a、HLA-DR、CD80具有典型树突状细胞特性的DC;TSA-SEA-DCL对靶细胞GLC-82的杀伤率明显高于TSA-DCL、SEA-DCL及DCL,亦明显高于对K562细胞的杀伤率;与靶细胞共育时镜下发现效应细胞CTL靠近并聚集在肿瘤细胞周围,致使肿瘤细胞发生变性坏死及凋亡。结论本实验方法能成功诱导出具有典型特征的成熟DC,肺癌TSA联合超抗原SEA诱导的DC疫苗对肺癌细胞有高效特异杀伤作用。

Background and objective Immunocompromised patients with malignant tumor always lack of strong anti-tumor immune response, because the antigenicity of tumor cells is weak, and antigen-presenting cell function is low, so that can not be effectively presenting tumor antigens to the lymphocytes. Therefore, how to effectively induce anti-tumor immune response is the key issue. Through the study on establishing a method to culture dendritic cells （DC） in vitro and to observe the anti-lung cancer immunological effect induced by DC, we provided definite experiment basis for the clinic application of vaccine based on DC. Methods Through the experiment we get the soluble antigen polypeptide from lung cancer cells GLC-82 by 3 mol/L potassium chloride. DCs are cultured and obtained from peripheral blood mononuclear cell by GM-CSF, IL-4 and TNF-a. DCs are identified by flow cytometer （FCM） and immunostaining. DCs modified by lung cancer tumor soluble antigen （TSA） and staphylococcal enterotox in A （SEA）, DCs modified by TSA or DCs modified by SEA or DCs modified by nothing were cultivated together with T lymphocyte, and the obtained cells are named TSA-SEA-DCL or TSA-DCL or SEA-DCL or DCL as effector cells. The anti-tumor activity of every effector cells against target cells was assayed with MTT method. Shape of DCs and effector cells, and the process of killing target cells were observed in microscope. Results Induced DCs expressed more CD1a, CD80 and HLA-DR, which had typical cell traits such as tree branch. The killing ratio of the TSA-SEA-DCL in vitro to GLC-82 is larger than TSA-DCL, SEA-DCL and DCL, also larger than to K562. When the effector cells cultivate with target cells, we can observe the CTL approach and gather to the cancer cell, induce it necrosis and apoptosis. Conclusion Ripe DCs that have typical characteristic and phenotype could be induced successfully. High potency and relatively specific antilung caner effect can be prepared in virtue of DC Bacterin Induced by lung caner TSA and SEA.