GP73+AFP+HCC/DC杂交细胞诱生特异性抗人肝癌CTL疫苗的研究

Our previous study found that the Dendrite Cell vaccine transfected AFP gene can induce CTL against AFP positive hepatoma carcinoma cell（AFP+HCC）, but not the AFP negative HCC. Therefore, how to induce anti-AFP-HCC CTL is the top difficulty concerned by many researchers. GP73 is a novel tumor marker, which is highly expressed in hepatoma carcinoma tissues, with better sensitivity and specificity than AFP in diagnosis of HCC. Especially GP73 is positive even in most AFP negative HCC patients. Accordingly, we speculated that the GP73 combined with AFP as the target antigen for immunotherapy in liver cancer, can induce CTL specifically targeting GP73+AFP+, GP73+AFP-, or GP73-AFP+HCC cells. It will be important in improving the AFP negative targeted immunotherapy. In this study, we first isolate subsets of GP73+AFP+, GP73+ AFP-, GP73-AFP+ and GP73- AFP-HCC cells by FACS and later fuse with autologous DC. The hybrid cells are co-cultured with autologous T cells to induce specific targeted CTL for cytotoxicity experiments in vitro. To study the anti-tumor effect and mechanism of specific CTLs in vivo, we first establish a mouse model by implanting the primary cultured human hepatoma cells into SCID mice which have reconstructed immune system of HCC patients. And then the induced specific CTLs aboved were infused to this animal for Anti-tumor efficacy and mechanism study in vivo.

我们前期研究发现转染AFP基因的DC疫苗可诱导抗AFP+肝癌CTL ，而对AFP-肝癌无效。因此，诱导出抗AFP-肝癌CTL成为研究者迫切解决的难题。GP73是新一代肝癌标志物，在肝癌组织中高表达，尤其在大部分AFP-表达的肝癌组织中呈阳性表达。我们推测，若将GP73和AFP联合作为靶抗原，可诱导出靶向杀伤GP73+AFP+、GP73+AFP-、GP73-AFP+肝癌细胞的CTL，对解决靶向治疗AFP-肝癌的难题有重要意义。本课题拟从患者肝癌组织中流式分选出GP73+AFP+、GP73+AFP-、GP73-AFP+、GP73-AFP-肝癌细胞，与体外诱导的患者DC融合成杂交细胞，进一步在体外诱生抗肝癌CTL，完成体外细胞毒实验，确定其对表达不同抗原的肝癌细胞的特异性杀伤作用；将CTL回输至荷人肝癌SCID鼠体内，观察其治疗作用及机制。本课题的完成，可为肝癌的免疫治聊提供新的治疗手段。

人肝癌组织表达AFP和GP73达94%，设想利用高表达AFP和GP73的肝癌细胞与树突状细胞（DC）融合制备疫苗，可激发强烈的抗肝癌免疫。肝癌干细胞与肝癌的发生密切相关，设想靶向肝癌干细胞的免疫治疗可从源头上防治肝癌的复发转移。通过这两方面研究，为肝癌治疗和防治复发转移提供新的策略。主要研究成果有：.（1）AFP+GP73+HepG2/DC疫苗的抗肝癌作用。采用流式细胞仪筛选获得AFP和GP73表达均很低（仅为0.1%和0.6%）的HepG2作为AFP-GP73-肝癌细胞。分别构建携带AFP、GP73基因的慢病毒转染AFP-GP73-HepG2，获得AFP+GP73-、 AFP-GP73+、AFP+GP73+的HepG2细胞。采用PEG诱导DC与AFP+GP73+HepG2融合（融合率达60%）制备AFP+GP73+HepG2/DC疫苗，该疫苗诱导的CTL对4组细胞均有杀伤作用，对AFP+GP73+HepG2的杀伤作用更强。该疫苗对AFP+GP73+HepG2组裸鼠的肿瘤生长抑制效应最显著。.（2）肝癌干细胞DC疫苗的抗肝癌作用。以CD90作为干细胞标记分子对HepG2细胞进行流式分选及富集培养得到99.5±1.3% CD90+HepG2细胞。将CD90+HepG2与DC融合（融合率为57.2%）制备CD90+HepG2/DC疫苗。该疫苗分泌IL-12p70、刺激T细胞增殖、活化T细胞分泌IFN-γ水平，与混合细胞DC组和单纯DC组比较，差异有统计学意义（P＜0.05），与HepG2/DC组比较差异无统计学意义。而CD90+HepG2/DC诱导的CTL较其他3组细胞诱导的CTL具有更显著的杀伤CD90+HepG2的效应；该疫苗能够显著抑制裸鼠CD90+HepG2移植瘤生长。.结论：AFP+GP73+HepG2/DC疫苗、CD90+HepG2/DC疫苗都具有良好的抗肝癌免疫效应。