奥沙利铂联合Exosome诱导的效应性T细胞抑制HepG2细胞增殖的研究

目的观察奥沙利铂(OXA)联合负载HepG2细胞裂解物的Exosome诱导的效应性T细胞对肝癌HepG2细胞系增殖的抑制作用。方法自健康人外周血中提取单个核细胞和T淋巴细胞,并以GM-CSF+IL-4的培养方案获得树突状细胞(DC)。将反复冻融后产生的HepG2细胞裂解物体外致敏DC,收集培养上清后以超高速离心法分离得到Exosome,电镜下观察其形态特征。用致敏DC及其分泌的Exosome刺激T淋巴细胞的增殖和活化。以ELISA试验检测不同刺激环境下T细胞分泌IL-1β、IL-2、IL-4、IL-6、IL-10、IFN-γ和TNF-α的能力。以细胞杀伤实验(MTT法)检测不同因素刺激后的T细胞、OXA(2.5、5.0、10.0mg.L-1)以及两者联合应用对HepG2细胞生长的抑制效应。结果与未致敏DC及其分泌的Exosome相比,HepG2细胞冻融裂解物致敏的DC及其分泌的Exosome能显著促进T细胞的增殖,并使其活化和分泌大量的IL-1β、IL-2、IL-6、IFN-γ和TFN-α等Th1型细胞因子。活化后的T细胞、OXA以及两者联合应用对HepG2细胞体外增殖均有抑制作用,而活化T细胞联合10mg.L-1OXA对HepG2细胞的杀伤率最高,但与联合5 mg.L-1OXA的效应相比,差异无统计学意义(P0.05)。结论负载肿瘤细胞抗原的DC所分泌的Exosome可在体外刺激T细胞,使其活化为具有抗肿瘤效应的细胞毒性T细胞,该细胞联合低浓度OXA能显著抑制肝癌细胞的体外增殖,并降低OXA的用量。

Effect of oxaliplatin combined with effector T lymphocyte induced by exosome on inhibiting proliferation of HepG2 cell

Objective To study the effect of oxaliplatin(OXA) combined with the effector T lymphocytes activated by exosome pulsed HepG2 cells’ whole lysates on inhibiting proliferation of HepG2 cells.Methods Mononuclear cells and T lymphocytes were isolated from peripheral blood of healthy people.Dendritic cells(DCs) were derived from mononuclear cells by the culture method of GM-CSF + IL-4.Whole cell lysates of HepG2 cells were extracted by freeze thawing method,then the monocyte-derived DCs were sensitized with this cellular antigen.Subsequently,exosomes were prepared from the supernatants of the DCs by ultracentrifugation and observed by transmission electron microscopy.After T lymphocytes were incubated with sensitized DCs or sensitized exosomes,the effects on promoting T lymphocyte proliferation were detected by MTT method and the concentrations of IL-1β,IL-2,IL-4,IL-6,IL-10,IFN-γ,and TNF-α were measured by enzyme linked immunosorbent assay(ELISA) in different culture enviroments.At last,the ability of different concentration OXA(2.5,5.0,10.0 mg·L-1) combined with sensitized T cells to kill HepG2 cells were checked by MTT methods,respectively.Results Compared with unsensitized DC and its exosomes,the DCs and its exosomes pulsed whole cell lysates of HepG2 cells could significantly stimulate T cells proliferation and secrete Th1 type cytokines including IL-1β,IL-2,IL-4,IL-6,IL-10,IFN-γ and TNF-α.All of senstitized T cells group,OXA groups and the combined groups obviously inhibited the proliferation of HepG2 cells,and the killing rate of activitaed T cells + 10 mg·L-1 OXA group was highest.However,there was no statistical difference for the killing rate between activitaed T cells + 10 mg·L-1 OXA group and activitaed T cells + 5 mg·L-1OXA group.Conclusion The exosomes that are secreted by DCs sensitized by HepG2 cells whole lysates can stimulate T cells and make it turn to be anti-cancer cytotoxcity T cells(CTLs).And then,the combination of CTLs and OXA have the negative effect on proliferation of HepG2 cells and reduce the dosage of OVA.