目的：应用晚期乳腺癌患者外周血来源的单个核细胞体外培养诱导产生新型（ Cytokine in-duced killer cells ，CIK）细胞的可能性，为乳腺癌患者应用自体免疫细胞治疗提供理论基础。方法取8例晚期乳腺癌患者外周血提取单个核细胞，经体外培养诱导增殖，并应用细胞计数法和流式细胞仪检测增殖细胞表面特异性标志CD3、CD16和CD56，应用51Cr release assay 及MTT方法测定其对MCF7及BT20乳腺癌细胞株的杀伤能力。应用ELISA试验方法对诱导得到的新型CIK细胞的培养上清液进行解析。结果经过体外18天培养，平均得到8．2×108个以上纯度为95．2％～98．1％的CD16＋、CD56＋和CD16＋CD56＋阳性高纯度NK细胞的新型CIK细胞，且对乳腺癌细胞株MCF7及BT20具有明显的抑制作用。结论成功应用晚期乳腺癌外周血单个核细胞选择性扩增诱导高纯度NK细胞的新型CIK细胞，且证明其对MCF7及BT20乳腺癌细胞具有明显抑制作用，为应用自体高纯度NK细胞的新型CIK细胞为基础的过继性免疫细胞治疗乳腺癌提供理论研究基础。%Objective To investigate new type cytokine induced killer cells expansion using advanced breast cancer′s peripheral blood .Methods peripheral blood mononuclear cells were isolated from 8 advanced breast cancer volunteers and co -cultured with Cytokine induced killer cells .These cells were placed in plastic flasks containing CIK-MediumTM supplemented with 10% auto-plasma in the presence of IL -2 ( 1 000 IU/mL) .The cultures were fed with CIK-MediumTM supplemented with IL -2 following the proliferation capacity . Cell proliferation was measured by cell counting during the cultivation .Fourteen days after cultivation ,cell mark-ers CD3/CD16/CD56 were examined by flow cytometry .51Cr and MTT assays were employed in cytotoxicity as-says.Cytokines were assayed by ELISA method .Results CD16+,CD16+CD56+,CD56+CIK cells were 5.8~11.6%in 2 ×107 fresh PBMCs and 95.2~97.6%in co-cultured cells after 18 days cultivation .The in vitro ex-pansion rate of new type cytokine induced killer cells was up to more than 8.2 ×108 in total,the cytotoxicity are ef-fective killing cells against MCF 7 and BT20 breast cancer cell lines .New type cytokine induced killer cells expand-ed from all PBMCs and secreted cytokines IFN -and TNF-.Conclusion The present culture could be useful to clarify the mechanisms of CIK cells expansion in vitro and feasible for breast cancer immmuno cell therapy .
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