CAJ | 학술논문

目的制备白细胞介素2(IL-2)锚定的肿瘤细胞来源的exosomes疫苗,探讨其体外诱导细胞毒性T淋巴细胞(CTL)的抗肿瘤效应.方法构建含糖基化磷脂酰肌醇(GPI)信号肽序列与人IL-2的融合基因的pEGFP-N1-IL2gpi质粒,建立稳定表达GPI-IL-2的T24细胞系(T24/GPI-IL-2).采用超速和蔗糖梯度密度离心法提取Ex/GPI-IL-2(即T24/GPI-IL-2细胞培养液中提取的exosomes),并对其进行形态学观察和分子标志检测.通过混合淋巴细胞实验和细胞毒实验,观察Ex/GPI-IL-2对T细胞的增殖和诱导杀伤效应.结果成功构建重组质粒pEGFP-N1-IL2gpi,建立了稳定表达GPI-IL-2蛋白的细胞系.Ex/GPI-IL-2为直径约30～90nm的类圆碟形小囊泡,表达细胞间黏附分子1(ICAM-1)、热休克蛋白70(HSP70)、MAGE-1和GPI-IL-2等免疫相关蛋白.Ex/GPI-IL-2经过树突状细胞负载后,能更有效地诱导T细胞的增殖和细胞毒效应(P＜0.05).结论通过基因转染技术,可以将IL-2锚定到肿瘤细胞来源的exosomes上,获得的Ex/GPI-IL-2能够诱导CTL对肿瘤细胞产生更强的诱导杀伤效应,为以exosomes为基础的肿瘤免疫治疗提供了新的途径.
목적 제비백세포개소2(IL-2)묘정적종류세포래원적exosomes역묘,탐토기체외유도세포독성T림파세포(CTL)적항종류효응.방법 구건함당기화린지선기순(GPI)신호태서렬여인IL-2적융합기인적pEGFP-N1-IL2gpi질립,건립은정표체GPI-IL-2적T24세포계(T24/GPI-IL-2).채용초속화자당제도밀도리심법제취Ex/GPI-IL-2(즉T24/GPI-IL-2세포배양액중제취적exosomes),병대기진행형태학관찰화분자표지검측.통과혼합림파세포실험화세포독실험,관찰Ex/GPI-IL-2대T세포적증식화유도살상효응.결과 성공구건중조질립pEGFP-N1-IL2gpi,건립료은정표체GPI-IL-2단백적세포계.Ex/GPI-IL-2위직경약30～90nm적류원설형소낭포,표체세포간점부분자1(ICAM-1)、열휴극단백70(HSP70)、MAGE-1화GPI-IL-2등면역상관단백.Ex/GPI-IL-2경과수돌상세포부재후,능경유효지유도T세포적증식화세포독효응(P＜0.05).결론 통과기인전염기술,가이장IL-2묘정도종류세포래원적exosomes상,획득적Ex/GPI-IL-2능구유도CTL대종류세포산생경강적유도살상효응,위이exosomes위기출적종류면역치료제공료신적도경.
Objective To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.Methods To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecuule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2. Results The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70,intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P ＜0.05). Conclusions GPI-IL-2 gene-modified tumor cells can make the exosornes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder trnssitional cell tumors.