突变的肝细胞生长因子受体被肝癌细胞HLA-A2分子递呈的实验研究

Epitope of somatic mutations in the hepatocyte growth factor receptor naturally processed and presented by HLA A2 on human hepatocellular carcinoma

目的 寻找肝癌细胞抗原肽。方法 应用细胞膜酸洗技术使抗原肽从人肝癌细胞膜表面脱落 ,经过凝胶层析 ,应用反相高效液相色谱层析得到不同组份多肽 ,细胞表位重建及生物学鉴定确定其活性 ,高效液相色谱 质谱仪联用技术获得抗原肽的一级结构 ,在互联网上进行氨基酸同源性分析确定其同源性序列。结果 经过反相高效液相色谱层析后可以获得 2 0多个多肽组份 ,生物活性鉴定有 2个活性组份 ,其中 1个组份经过液质联用鉴定和氨基酸同源性分析 ,确定其序列为SLIVHLNEV ,是肝细胞生长因子受体前体第 1175～ 1183肽段发生突变 ,第 1180位点的苯丙氨酸变为亮氨酸。结论 液质联用技术为寻找抗原肽的有效方法 。

Objective To isolate and identify peptides bound to HLA I on human hepatocellular carcinoma. Methods MHC associated peptides were extracted by mild acid wash of viable hepatocellular carcinomas cells, collected by gel filtration, and fractioned by reversed phase high pressured liquid chromatography (RP HPLC). Peptides of individual fractions were reconstitution of T cell epitopes and were identified by cytotoxicity T lymophacyte assay. Actively extracted sample analyses were performed by HPLC MS MS (tandom mass spectrometry). Protein database in the internet was used as an additional tool for structure analysis and for determination of protein source of the eluted peptides. Result RP HPLC showed that there were over 20 different fractions of peptides derived from human hepatocellular carcinoma, and only two active peaks were identified by cytotoxicity T lymophocyte assay. The most promising candidate for T cell epitope was nonamers peptide (SLIVHLNEV), derived from met/hepatocyte growth factor receptor, point mutation was 1180F to L. Conclusion Sensitive sequencing by HPLC MS may provide a powerful method of identifying tumor specific antigenic peptides, and nonamers peptide (SLIVHLNEV) is the hepatocellular carcinoma antigenic peptide.