摘要
从黑色素瘤病人的瘤块和淋巴结中制备黑色素瘤细胞和肿瘤浸润性淋巴细胞（ＴＩＬ）系，通过流式细胞仪对黑色素瘤细胞表型分析。经去垢剂处理、免疫沉淀和亲和层析法从ＨＬＡＡ２阳性患者黑色素瘤细胞中分离纯化ＨＬＡＡ２蛋白，其纯度通过４％～２０％梯度ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｍｂｌｏｔ鉴定。１００℃沸水浴和弱酸处理ＨＬＡＡ２蛋白经反相高压液相色谱层析（ＲＰＨＰＬＣ）得到不同组份多肽，将其与抗原加工缺陷的ＨＬＡＡ２阳性Ｔ２细胞反应，进行重建瘤细胞特异性表位测定。ＴＩＬ杀伤活性结果表明，来源于６２４黑色素瘤细胞的不同组份多肽中有三个活性峰值，而来源于Ｃｈａｐ黑色素瘤细胞的不同组份多肽中仅一个与上述细胞组份位置相同的单一活性峰值。提示具有活性组份的多肽与ＨＬＡＡ２限制性ＴＩＬ识别的人黑色素瘤细胞抗原肽有关。
The identification of antigenic
peptides presented on the melanoma cell surface by HLAA2 molecules and recognized by
HLAA2 restricted tumor infilting lymphocytes (TIL) could lead to develop a peptide vaccine
capable of inducing protective cellular immunity TIL and melanoma cell lines were derived from
lymph nodes and tumor masses taken from the human melanoma patients,and the phenotypes
of melanoma cells in the expression of HLAA2 molecules have been identified by using flow
cytometry, and the HLAA2 molecules were immunochemically purified from the melanoma cells
by immunoprecipitation and immunoaffinity column chromatography of detergentsolubilized cell
pellets Purification of HLAA2 molecules was determined by 4%20% gradient SDSPAGE and
Western blot Treatment of peptides bound to the HLAA2 molecules with 100 boiled water bath
and weak acid was fractionated by reversed phase high pressure liquid chromatography
(RPHPLC) Peptides of individual fractions were assessed for their ability to reconstitute tumor
cellspecific epitopes by adding to the HLAA2 positive Agprocessing mutant cell (T2) The results
of TIL killing activities showed that there were three active peaks in the different fractions of
peptides derived from 624melanoma cells,whereas only one peak from those derived from
Chapmelanoma cells with the same location of maximal peak from 624 melanoma cells. These
results suggested that the active peptide fractions were related to the antigenic peptides of
HLA2restricted and TILrecognized human melanoma cells,and the identification of peptides
could result in the development of broadly applicable vaccine therapy for melanoma
出处
《上海免疫学杂志》
CSCD
北大核心
1999年第3期142-146,共5页
Shanghai Journal of Immunology
关键词
黑色素瘤
抗原肽
HLA-A2
亲和层析
melanoma
antigenic peptides
HLAA2
immunoaffinity column chromatography
reversed
phase high pressure liquid chromatography
