质粒介导的血管抑制和免疫强化协同诱导肝癌细胞凋亡的研究

目的 探讨真核表达质粒介导的血管抑制和免疫强化治疗诱导小鼠种植性肝癌肿瘤细胞凋亡的效果和机制.方法 制备、纯化小鼠内皮抑制素真核表达质粒(pSecES)和小鼠白细胞介素12(IL-12)质粒(pmlL-12).小鼠股部肌肉内接种H22细胞建立肝癌种植瘤模型,分成4组,接种部位分别多次注射pSecES、pmIL-12、pSecES+pmlL-12和pcDNA3.1质粒裸DNA,观察种植瘤的形成和重量;肿瘤组织切片CD31免疫组织化学染色检测肿瘤微血管密度,HE染色观察肿瘤浸润淋巴细胞,并用TUNEL检测肿瘤细胞的凋亡.结果 与pcDNA3.1对照组相比,注射pSecES,pmIL-12组小鼠肝癌种植瘤生长缓慢,血管减少,后者并可见瘤内大量淋巴细胞浸润.两组肿瘤细胞凋亡增加,pSecES、pmIL-12和pcDNA3.1组凋亡细胞数分别为(11.3±4.1)、(14.6±3.2),(1.4±1.3)个/高倍视野,pSecES+pmIL-12组凋亡细胞数为(19.9±5.5)个/高倍视野,明显高于单一治疗组.结论 真核表达质粒介导的血管抑制和免疫强化治疗可通过抑制肿瘤血管形成,促进瘤内淋巴细胞浸润,协同诱导肿瘤细胞凋亡,较好的抑制了小鼠种植性肝癌的生长.

A study on the apoptosis of hepatoma cells synergistically induced by plasmid-mediated anti-angiogen-esis and immunopotentiation therapy

Objective To study the effect and mechanism of apoptosis of implanted hepatoma cells in mice induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. Methods Mouse endostatin eukaryotic plasmid (pSecES) and mouse IL-12 (intorleukin 12) eukaryotic plasmid (pmlL-12) were extracted and purified from E.coli. H22 hepatoma cells were inoculated into the leg muscles of 32 mice. The mice were divided into four groups with pSecES, pmIL-12, pSecES+pmIL-12 and pcDNA3.1 naked plasmid DNA injected into the tumor cell implantation sites of each group. Tumor formation and their weights were evaluated. Microvessel density, numbers of the infiltrating lymphocytes in the tumors and the apoptosis of tumor cells were assayed by microscopical examination of the CD31 and HE stained slides of the tumors and TUNEL assay. Results The tumors of those with pSecES or pmIL-12 injections grew slower and with less microvessel density, more lymphocyte infiltration and with more apoptosis tumor cells compared with those with pcDNA3, l injections. There was much more tumor cell apoptosis in the pSecES+pmIL-12 group (19.9±5.5 per 400 × microscope field, P < 0.05) than that in any other single plasmid injection group (400 × microscopic field: pSecES 11.3±4.1, pmIL-12 14.6±3.2, pcDNA3.1 1.4±1.3). Conclusions Tumor cell apoptosis of the implanted hepatoma in mice can be induced by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocyte infiltration, by which the growth of the implanted hepatoma was inhibited.