CAJ | 학술논문

目的本研究探讨热休克E.G7-OVA肿瘤细胞来源的exosomes的体内抗肿瘤效应.方法通过分级离心和蔗糖密度梯度离心法分离和纯化E.G7-OVA肿瘤细胞来源的exosomes.热休克E.G7-OVA肿瘤细胞来源的exosomes和未热休克E.G7-OVA肿瘤细胞来源的exosomes分别命名为Exo/HS和Exo.通过电镜观察exosomes的形态,Western blot检测exosomes的蛋白成分.以Exo、Exo/HS、PBS免疫小鼠,用E.G7-OVA肿瘤细胞进行攻击,观察各组免疫保护效应;建立E.G7-OVA荷瘤小鼠模型,观察各组免疫治疗效应.通过LDH法检测各组免疫小鼠脾细胞CTL活性.结果电镜下exosomes为双层膜的囊泡样结构,直径为40～100 nm.Western blot结果表明:Exo和Exo/HS都含有HSC70、HSP70、HSP60、HSP90、MHC Ⅰ和OVA分子,而Exo/HS中HSP70和MHC Ⅰ分子的含量更高.免疫保护试验发现,Exo/HS组免疫小鼠90 d的无瘤率显著高于Exo组和PBS组(50%、20%、0%,P<0.01);对荷瘤小鼠的免疫治疗显示,Exo/HS对荷瘤小鼠的肿瘤抑制效应显著高于Exo组和PBS组(P<0.01).CTL结果表明,Exo/HS免疫小鼠诱导的OVA抗原特异性的CTL活性显著高于Exo组和PBS组(P<0.01).结论热休克E.G7-OVA肿瘤细胞来源的exosomes可作为有效的肿瘤疫苗.
목적 본연구탐토열휴극E.G7-OVA종류세포래원적exosomes적체내항종류효응.방법 통과분급리심화자당밀도제도리심법분리화순화E.G7-OVA종류세포래원적exosomes.열휴극E.G7-OVA종류세포래원적exosomes화미열휴극E.G7-OVA종류세포래원적exosomes분별명명위Exo/HS화Exo.통과전경관찰exosomes적형태,Western blot검측exosomes적단백성분.이Exo、Exo/HS、PBS면역소서,용E.G7-OVA종류세포진행공격,관찰각조면역보호효응;건립E.G7-OVA하류소서모형,관찰각조면역치료효응.통과LDH법검측각조면역소서비세포CTL활성.결과 전경하exosomes위쌍층막적낭포양결구,직경위40～100 nm.Western blot결과 표명:Exo화Exo/HS도함유HSC70、HSP70、HSP60、HSP90、MHC Ⅰ화OVA분자,이Exo/HS중HSP70화MHC Ⅰ분자적함량경고.면역보호시험발현,Exo/HS조면역소서90 d적무류솔현저고우Exo조화PBS조(50%、20%、0%,P<0.01);대하류소서적면역치료현시,Exo/HS대하류소서적종류억제효응현저고우Exo조화PBS조(P<0.01).CTL결과 표명,Exo/HS면역소서유도적OVA항원특이성적CTL활성현저고우Exo조화PBS조(P<0.01).결론 열휴극E.G7-OVA종류세포래원적exosomes가작위유효적종류역묘.
Objective To study the antitumor effects of exosomes derived from heat-shocked E.G7-OVA tumor cells in vivo. Methods Exosomes derived from E.G7-OVA tumor cells were isolated and purified by serial centrifugation and sucrose gradients ultracentrifugation. Exosomes from heat-shocked or non-heat-shocked E.G7-OVA tumor cells were named as Exo/HS and Exo correspondingly. Exosomes were viewed by electron microscopy. Protein components of exosomes were detected by Western blot. Exo, Exo/ HS or PBS were injected into mice before injection of E.G7-OVA tumor cells, and antitumor effects were ob-served in each group. Mouse model bearing E.G7-OVA tumor cells were established to examine immunother-apy effects of Exo or Exo/HS. Cytotoxity of spleen CTL were measured by LDH. Results Exosomes con-tained bi-layer membrane and their diameters are between 40 nm and 100 nm under electron microscopy. The Western blot results showed that HSC70, HSP70, HSP60, HSP90, MHC Ⅰ and OVA were present in both Exo and Exo/HS. However, Exo/HS contained more HSP70 and MHC Ⅰ than Exo. Protective antitu-mor immunity suggested that tumor-free survival (90 days) rate in Exo/HS vaccinated mice was significantly higher than those in Exo or PBS vaccinated mice (50%, 20%, 0%, P<0.01). Therapeutic antitumor effects showed that immunization by Exo/HS resulted in dramatically enhanced antitumor effects when com-pared to the Exo- or PBS-treated groups (P<0.01). CTL results showed that immunization with Exo/HS in-duced higher level of OVA-specific CTL responses as compared with those from Exo or PBS (P<0.01). Conclusion Exosomes derived heat-shocked E.G7-OVA tumor cells may be used as potent cancer vaccine.