合成多肽体外诱导乙肝病毒抗原特异性T杀伤细胞动态观察

Study on dynamic induction of hepatitis B virus antigen specific T killer by synthetic peptides derived from HBcAg in vitro

根据乙肝病毒核心抗原（ＨＢｃＡｇ）的ＭＨＣ－Ⅰ类分子结合关键序列合成抗原性多肽２段（ＰｅｐⅠａａ：１８～２７及ＰｅｐⅡａａ：１４１～１５１），分别与转铁蛋白（Ｔｆ）、抗ＣＤ３的单克隆抗体（ＣＤ３ＭｃＡｂ）和ＩＬ－２联合体外诱导乙肝病毒抗原特异性细胞毒Ｔ细胞（ＨＢＶ－ＣＴＬｓ），动态观察其增殖状态和细胞毒活性。结果显示，ＨＢＶ－ＣＴＬｓ对转染ＨＢＶＤＮＡ的肝癌细胞（２．２．１５细胞）在第７天出现特异性细胞毒活性，第１４天达高峰，分别为６７％（ＰｅｐⅠ＋Ｔｆ＋ＣＤ３ＭｃＡｂ＋ＩＬ－２）、５５％（ＰｅｐⅡ＋Ｔｆ＋ＣＤ３ＭｃＡｂ＋ＩＬ－２），明显高于ＬＡＫ细胞组（２０％）（ＩＬ－２）及ＣＤ３－ＡＫ细胞组（３４％）（ＩＬ－２＋ＣＤ３ＭｃＡｂ＋Ｔｆ），Ｐ＜０．０５。而ＨＢＶ－ＣＴＬｓ对无ＨＢＶＤＮＡ转染的肝癌细胞（ＨｅｐＧ２细胞）非特异性杀伤率，与ＬＡＫ细胞及ＣＤ３－ＡＫ细胞相比，无显著性差异（Ｐ＞０．０５）。ＨＢＶ－ＣＴＬｓ体外培养３天后开始增殖，２１天达高峰，扩增倍数比ＬＡＫ细胞高１０倍。细胞培养上清一氧化氮（ＮＯ）含量动态分析显示，ＨＢＶ－ＣＴＬｓ细胞培养上清ＮＯ含量与细胞毒活性存在正相关关系（Ｒ＝０．８，Ｐ＜０?

To study the optimal induction of HBV antigen specific T cells (HBV CTLs),the PBMC isolated from healthy donors blood were costimulated with the synthetic peptides derived from HBcAg (PepⅠ:amino acid 18￣27 and PepⅡ:aa 141￣151),which were equal to the published HLA A2 binding motif (CTL epitope).The specific lysis of HBV CTLs against HBV DNA transfected HepG2 cells(2.2.15) could be detected after stimulation for 7 days and the peak specific lysis could be obtained on the 14th day,which were 67%(PepⅠ) and 52% (PepⅡ),respectively,significantly high than that of LAK cells (25%) and CD3 AK cells(30%)( P <0 05).But no significant difference was found between the nonspecific cytotoxicity of HBV CTLs and LAK/CD3 AK cells against HepG2 cells.The number of HBV CTLs expanded by 8 fold and 20 fold after 14 and 20 days cultivation,respectively,5￣10 times higher than that of LAK cells.The population of HBV specific CTLs were mainly CD8 positive.The positive relationship between the level of nitrogen oxide in supernatant and the specific cytotoxicity of HBV CTLs could also be found.Our study of dynamic induction suggests that induction of HBV CTLs stimulated with synthetic Peptide Ⅰ(aa 18￣27) derived from HBcAg in vitro for 14 days might be optimal.