树突状细胞吞噬PLA-AFP 218-226微球后诱导细胞毒性T淋巴细胞反应

目的研究树突状细胞（DC）吞噬包被有甲胎蛋白HLA—A2限制性表位肽（AFP218-226，LLNQHACAV）的聚乳酸微球（PLA—AFP218-226）后诱导的特异性细胞毒性T淋巴细胞（CTL）对肝癌细胞株HepG2和负载有AFP218-226的T2细胞株的毒性作用。方法用GM—CSF和IL-4诱导HLA—A2^＋的健康志愿者外周血单核细胞，LPS诱导成熟，使其分化为高纯度DC，在诱导过程中加入PLA—AFP218-226微球，用吞噬了PLA—AFP218-226微球的DC诱导自身T淋巴细胞，使其成为肝细胞癌（HCC）特异性CTL。用流氏细胞仪检测DC膜分子标志，T2细胞与AFP218-226结合实验检测HLA—A2分子与AFP218-226之间的亲合力，MTT法检测特异性CTL杀伤HepG2和T2细胞株的能力。结果AFP218-226与HLA—A2分子具有较高的亲合力，吞噬微球后的成熟DC高表达CD83、CD86、CD40等膜分子，其诱导的特异性CTL对HepG2和负载有AFP218-226的T2细胞株具有强的细胞毒作用。结论PLA—AFP218-226被DC细胞吞噬后，能够诱导HCC特异性CTL的产生，可能成为一种新型的抗肿瘤表位肽疫苗，在肝癌的防治中得到应用。

Cytotoxicity of Cytotoxic T Lymphocytes Induced by the Dendritic Cells Phagocytosing PLA-AFP 218-226 Micospheres Against Hepatocellular Carcinoma Cell Lines

Objective To assess the cytotoxicity of cytotoxic T lymphocytes (CTLs) induced by the dendritic cells phagocytosing HLA-A2~+ restricted epitope peptides encapsulated in polylactic acid(PLA) microspheres (PLA-AFP_ 218-226 ) against cell lines HepG2 and T2-loaded with HLA-A2~+ restricted epitope peptides derived from alpha fetoprotein (AFP_ 218-226 ,LLNQHACAV). Methods Mature dendritic cells (DCs) were obtained by inducing the monocytes isolated from peripheral blood cells of HLA-A2~+ healthy donors with GM-CSF and IL-4. On day 3 from onset of the culture, PLA- AFP_ 218-226 was added to the culture medium, and on day 6, lipoplysaccharide (LPS) was added to it for inducing the immature DCs to mature. The surface phenotype of the mature DCs was determined with fluorescence activated cell sorting (FACS) assay; the cytotoxicity of CTLs induced by the DCs for 7 days against cell lines HepG2 and T2-loaded with AFP_ 218-226 was determined with MTT method; and the avidity between HLA-A2 and AFP_ 218-226 was determined with T2-peptide binding experiment. Results There was high avidity between AFP_ 218-266 and HLA-A2. The DCs phagocytosing PLA-AFP_ 218-226 highly expressed CD83, CD86, CD40, etc., and the CTLs induced by the DCs strongly decomposed the HepG2 and T2-loaded with AFP_ 218-226 . Conclusions The strong cytotoxicity against HepG2 cell lines can be induced in vitro by DCs phagocytosing PLA-AFP_ 218-226 micospheres, suggesting that PLA-AFP_ 218-226 microspheres can serve as a new type of CTL epitope vaccine for the prophylaxis and treatment of hepatocellular carcinoma.