FSHβ锚定的外泌体制备及其对卵巢癌细胞诱导杀伤作用

摘要

Objective To explore the preparation of follicle stimulating hormone receptor β (FSHβ) anchored exosomes (Ex) and its effect on induced killing of ovarian cancer cell .Methods Recombinant plasmid pDisplay-FSH was constructed and exosomes was prepared .Morphology of exosomes and related protein expression were detected .Proliferation of T cells induced by Ex /FSHβ and its killing effect on ovarian cancer cell were observed through mixed lymphocyte experiment and tumor cytotoxicity assay .Results Recombinant plasmid pDisplay-FSHβ was successfully constructed and stably transfected into HEK 293 cells .Level of FSHβ in HEK293-FSHβ group was significantly higher than that in HEK293 group and HEK293-N1 group (t value was 17 .221 and 19 .583 , respectively ,both P < 0 .05) after phosphatidylinositol specific phospholipase C (PI-PLC) elution .Western Blotting showed that relative expression quantity of adhesion molecule-1 (ICAM-1) ,CD86 and human leukocyte antigen-DR (HLA-DR) in Ex/FSHβgroup was significantly different from that in Ex /N1 and Ex groups (t value ranged 5 .147 - 7 .886 , all P < 0 .05 ) .T-cell proliferation index in dendritic cell (DC)-Ex/FSHβ group was significantly different from that in DC-Ex/N1 group ,DC-Ex group and DC group (t value was 8 .634 ,9 .773 and 11 .264 ,respectively ,all P < 0 .05) .Killing rate of DC-Ex /FSHβ group was the highest when effect target ratio was 60 :1 .Levels of interferon - γ (INF-γ) ,interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α) in DC-Ex/FSHβ group were significantly different from those in DC-Ex/N1 group ,DC-Ex group and DC group with effect target ratio at 60 :1 (t value ranged 17 .627 - 39 .854 ,all P < 0 .05) .Migration assay indicated that number of cells passing microporous carbon membrane in DC-Ex/FSHβ group was significantly lower than that in DC-Ex/N1 group ,DC-Ex group and DC group (t value was 17 .396 ,16 .517 and 34 .892 ,respectively ,all P < 0 .05) .Conclusion FSHβ can be anchored to exosomes from HEK293 cell by gene recombination technology .DC-Ex/FSHβ can induce cytotoxic T cell to generate strong induced killing effect on ovarian cancer cells ,which provides theoretical basis for exosomes-based tumor immunotherapy .%目的 探讨分析卵泡刺激素受体-β(FSHβ)锚定的外泌体(exosomes,Ex)制备及其对卵巢癌细胞诱导杀伤作用.方法 构建pDisplay-FSH重组质粒并制备exosomes,检测exosomes的形态及相关蛋白分子表达情况.通过混合淋巴细胞实验和肿瘤细胞毒性实验,观察Ex/FSHβ 对T细胞的诱导增殖及对卵巢癌细胞的杀伤作用.结果 成功构建重组质粒pDisplay-FSHβ 并稳定转染至HEK293细胞.经磷酸腺肌醇特异性磷脂酶C(PI-PLC)洗脱后,HEK293-FSHβ 组洗脱液中FSHβ 水平显著高于HEK293组 、HEK293-N1组(t值分别为17.221、19.583,均P<0.05).蛋白免疫印迹法检测显示,Ex/FSHβ 组黏附分子1(ICAM-1)、CD86及人类白细胞抗原-DR(HLA-DR)相对表达量与Ex/N1组 、Ex组比较,差异有统计学意义(t=5.147～7.886,均P<0.05).树突状细胞(DC)-Ex/FSHβ 组T细胞增殖指数与DC-Ex/N1组 、DC-Ex组及DC组比较,差异有统计学意义(t值分别为8.634、9.773、11.264,均P<0.05).效靶比在60:1时,DC-Ex/FSHβ 组杀伤率最高.效靶比在60:1时,DC-Ex/FSHβ 组干扰素-γ(INF-γ)水平 、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量与DC-Ex/N1组 、DC-Ex组及DC组比较,差异均有统计学意义(t=17.627～39.854,均P<0.05).迁移实验显示,DC-Ex/FSHβ 组细胞穿过聚碳质膜微孔的细胞数显著低于DC-Ex/N1组 、DC-Ex组及DC组(t值分别为17.396、16.517、34.892,均P<0.05).结论 基因重组技术可将FSHβ 锚定到HEK293细胞来源的exosomes上,DC-Ex/FSHβ 能够诱导细胞毒性T细胞对卵巢癌细胞产生较强的诱导杀伤作用,这一作用为临床以exosomes为基础的肿瘤免疫治疗提供了一定的理论依据.