CAJ | 학술논문

目的：观察骨肉瘤细胞分泌的外泌体体外刺激细胞毒性T细胞对骨肉瘤细胞的杀伤作用。方法采用超滤离心联合蔗糖密度梯度超速离心的方法从骨肉瘤细胞培养上清液中分离外泌体( Texo)。透射电子显微镜鉴定Texo形态,western blot分析Texo表面主要组织相容性复合体-Ⅰ( MHC-Ⅰ)类分子表达。分离培养小鼠骨髓来源树突状细胞( DC),流式细胞仪鉴定细胞表型。噻唑蓝( MTT)法检测负载Texo的DC刺激T淋巴细胞增殖情况及效应淋巴细胞对骨肉瘤细胞的杀伤作用。结果透射电镜下见Texo为圆形或类圆形小体,大小不等,平均直径50~100 nm；Texo表面有MHC-Ⅰ类分子表达。骨髓来源DC边缘有绒毛样突起,呈典型的树突状细胞形态,经脂多糖诱导后DC的CD80、MHC-Ⅰ、MHC-Ⅱ类分子表达明显高于未经诱导的DC,阳性率分别为77.16%,83.21%,91.26%。负载Texo的DC刺激T细胞增殖能力及T细胞对靶细胞的杀伤效应明显强于未负载Texo的DC与Texo(P<0.05)。结论负载Texo的DC能促进T细胞增殖,可体外诱导细胞毒性T细胞应答抑制骨肉瘤细胞生长。
목적：관찰골육류세포분비적외비체체외자격세포독성T세포대골육류세포적살상작용。방법채용초려리심연합자당밀도제도초속리심적방법종골육류세포배양상청액중분리외비체( Texo)。투사전자현미경감정Texo형태,western blot분석Texo표면주요조직상용성복합체-Ⅰ( MHC-Ⅰ)류분자표체。분리배양소서골수래원수돌상세포( DC),류식세포의감정세포표형。새서람( MTT)법검측부재Texo적DC자격T림파세포증식정황급효응림파세포대골육류세포적살상작용。결과투사전경하견Texo위원형혹류원형소체,대소불등,평균직경50~100 nm；Texo표면유MHC-Ⅰ류분자표체。골수래원DC변연유융모양돌기,정전형적수돌상세포형태,경지다당유도후DC적CD80、MHC-Ⅰ、MHC-Ⅱ류분자표체명현고우미경유도적DC,양성솔분별위77.16%,83.21%,91.26%。부재Texo적DC자격T세포증식능력급T세포대파세포적살상효응명현강우미부재Texo적DC여Texo(P<0.05)。결론부재Texo적DC능촉진T세포증식,가체외유도세포독성T세포응답억제골육류세포생장。
Objective To investigate the stimulation of exosome derived from osteosarcoma cells suppressing cytotoxic T cells and the inhibitory effect of active T cells for surpressing osteosarcoma cells. Methods Exosome derived from tumor cells was isolated and purified by ultracentrifugation. Its morphology was observed with transmission electron microscope, and the major histocompatibility complex-I ( MHC-I) molecules were analyzed by western blot. Mice bone marrow-derived dendritic cells were pulsed with exosome. Surface membrane MHC-I molecules were analyzed with flow cytometry. The effect of active T cells on the growth of osteosarcoma cells were detected by MTT assay after the T cells being stimulated by exosome-pulsed dendritic cells. Results The exosome was round or near round corpuscle, and the diameter was about 50-100 nm by transmission electron microscope. The size was relatively homogeneous. Western blot showed that the exosome expressed MHC-Ⅰmolecules. Surface membrane CD80,MHC-I and II molecules were expressed on 77. 16%, 83. 21%, and 91. 26% of LPS-treated dendritic cells, respectively, which were up-regulated compared to untreated cells. Dendritic cells pulsed with exosome derived from osteosarcoma cells caused significantly higher T cells stimulation and osteosarcoma cells inhibition as compared to un-pulsed dendritic cells. (P<0.05). Conclusion T cells can inhibit the growth of osteosarcoma cells after being stimulated by exosome-pulsed dendritic cells in vitro.