hAFP(137-145)肽段修饰树突细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应

目的:通过体外杀伤试验,研究人肝癌特异性甲胎蛋白hAFP(137-145)短肽段修饰树突细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异杀伤效应,并与完整hAFP蛋白的杀伤效应相比较。方法:选取健康人外周血单个核细胞,体外诱导成熟DC;构建hAFP表达载体,在BL21细菌中异丙基硫代半乳糖苷(IPTG)诱导原核表达后纯化,与合成的hAFP(137-145)短肽段分别修饰诱导DC细胞,体外刺激细胞毒性T细胞(CTL),通过流式细胞仪法和细胞毒性检测试剂盒法检测修饰后的DC所诱导的CTL在体外对肝癌细胞株的杀伤作用;构建荷瘤裸鼠人HepG2肝癌模型,检测该肽段修饰DC后诱导的CTL细胞在裸鼠过继体内实验中对人肝癌转移瘤的杀伤作用。结果:成功构建了pET-hAFP表达载体,原核表达并纯化后用Western blot检测到hAFP蛋白的表达;hAFP蛋白和hAFP(137-145)短肽段均能在体外修饰和诱导DC细胞的增殖和成熟;hAFP蛋白和hAFP(137-145)短肽段修饰DC体外均能诱导特异性CTL,并通过体外实验验证了其对于肝癌细胞的杀伤效应,其中hAFP(137-145)短肽段诱导CTL的能力和所诱导CTL对于肝癌细胞的杀伤能力相对全长hAFP蛋白较强;动物实验证实,该hAFP(137-145)肽段修饰DC后诱导的CTL细胞在荷瘤裸鼠过继体内实验中对人肝癌转移瘤有明显的杀伤作用,杀伤效率优于完整hAFP蛋白。结论:hAFP蛋白和hAFP(137-145)短肽段修饰DC均具有单独体外诱导特异性CTL的作用,体外实验和动物实验均证实此CTL有特异地杀伤HegG2肝癌细胞的作用;hAFP(137-145)短肽段诱导CTL效率和杀伤肿瘤细胞效率均优于完整hAFP蛋白。

Induction of Specific Anti-Hepatocelluar Cancer Cell Line Immunity of CTLs Activated by hAFP(137-145) Modified Dendritic Cells

Objective:To investigate the strategies and methods of hepatocelluar cancer immunotherapy with cytotoxic T lymphocytes(CTLs) activated by dendritic cells(DCs),which were modified by hAFP(137-145) peptide.Methods:Prokaryotic expression vector of hAFP gene was constructed and expressed to obtain 6 His-Tag fusion protein.And the pET-hAFP plasmid was transformed into BL21(DE3) plysS for expression.Then the hAFP protein was purified by the His-Bind Purification Kit.Dendritic cells derived from normal human peripheral monocyte were induced by GM-CSF and IL-4.MTT method was applied to analyze the proliferation activities of DCs.To achieve the specific T lymphocyte cells,DCs modified with hAFP(137-145) peptide or hAFP protein were cocultured with monocyte-derived CD8+ lymphocytes for 48 hours.Then these activated lymphocytes were cocultured with HepG2 cells at ratio of effect target 80:1,40:1,and 20:1 in vitro and the killing activities were detected by flow-cytometry and Non-Radiactive Cytotoxity assay.Mouse model of migrated hepatocellular cancer was established,and the acquired CTLs were engrafted to testify the killing activities in vivo.Results:The pET-hAFP vector was successfully constructed and sequenced,and high purity hAFP protein was obtained.T cells stimulated by hAFP(137-145) peptide modified DCs showed more specific cytotoxic activity to HepG2 than lymphocytes activated by DCs with integrate hAFP protein in vitro.In the mouse model of migrated human hepaocellular cancer,the adoptive transfer of acquired CTLs activated by hAFP(137-145) peptide also had a marked killing activity on the migrated cancer cell in vivo,which showed a higher efficiency than the integrate hAFP protein.Conclusion:High purity hAFP were extracted in this study for clinical individual immunotherapy from hepatocellular cancer cells by using His-Bind Purification Kit.DCs vaccine modified with hAFP protein and hAFP(137-145) peptide has significant killing effect against hepatocellular cancer cell in vitro and in vivo,and the hAFP(137-145) peptide has a relatively high efficiency.