阳离子脂质体介导IL-15基因治疗小鼠肺转移模型的实验研究

目的:用阳离子脂质体(CL)包裹pcDNA3.1-IL15制备阳离子脂质体质粒DNA复合物CL-IL15,观察其抗小鼠B16-F10肺转移瘤的治疗作用,并初步探讨其作用机制。方法:构建hIL-15真核表达载体,测定脂质体-质粒DNA复合物的最佳包封率;将该复合物转染CHO-K1细胞株,West-ern blott检测体外转染条件下IL-15蛋白的表达情况,MTT法检测细胞转染上清对CTLL-2细胞株的增殖刺激作用;建立B16-F10小鼠黑色素瘤肺转移模型,尾静脉给药,每隔1 d给药1次,共6次,治疗结束24 h后观察各组肿瘤肺转移情况;LDH释放法(乳酸脱氢酶释放法)检测脾淋巴细胞的杀伤作用,冰冻切片免疫荧光法观察NK细胞对肿瘤组织的浸润。结果:成功构建hIL-15表达载体,并验证其与阳离子脂质体的质量比为1∶5时,包裹后形成的复合物具有较好的包封率;可以在体外有效转染并表达具生物活性的分泌型IL-15蛋白;CL-IL15复合物治疗小鼠肿瘤肺转移模型,可以显著减少肿瘤肺转移结节数目,提高脾细胞对肿瘤细胞杀伤活性(P<0.05),增加NK细胞在肿瘤组织中的浸润比例。结论:阳离子脂质体质粒DNA复合物CL-IL15可有效地抑制小鼠B16-F10肺转移瘤,其作用机制可能与IL-15诱导脾细胞对肿瘤细胞的杀伤、激活NK(natural killer)细胞对肿瘤组织的浸润等机制有关。

Antitumoral efficacy by systemic delivery of cationic liposome-plasmid interleukin-15 complexes in murine models of lung metastasis

AIM: To investigate the therapeutic effect of the plasmid pcDNA3.1-IL15 complexed with cationic liposome(CL-IL15) in the B16-F10 melanoma lung metastasis model.METHODS: A plasmid with high secretive efficiency of IL-15 was constructed and the optimum mix ratio was determined to formulate cationic liposome-plasmid complex with the optimal encapsulation.The CHO-K1 cell line was transfected by CL-IL15.The secretion of transfected IL-15 gene was detected by Western blot and its biological function was measured through the proliferation response of CTLL-2 cytotoxic T cell line of murine by MTT assay.The C57BL/6 mice were inoculated intravenously(i.v.) with B16-F10 melanoma lung metastasis cells then treated(i.v.) by CL-IL15 in a therapeutic setting to derermine the tumorigenesis and research the corresponding mechanisms.RESULTS: The pcDNA3.1-IL15 plasmid was successfully constructed and the mass-ratio of optimal condition of cationic liposome-plasmid with perfect entrapment was 1∶ 5(plasmid: cationic liposome).Western blot analysis displayed the detection of IL-15 both in the medium and the pcDNA3.1-IL15 transfected cells.MTT assay showed that CTLL-2 cells could proliferate with the medium obtained from CHO-K1 cells transfected by CL-IL15.And the administration of CL-IL15 complexes led to the significant inhibition lung metastasis of malignant melanoma(P<0.05).CONCLUSION: CL-IL15 could inhibit the metastasis of malignant melanoma and the cationic liposome delivered plasmid pcDNA3.1-IL-15 complexes may be an efficient therapeutic strategy for the treating of lung metastasis.And the effective splenic cell-mediated cytotoxicity and the obvious NK cells recruitment may be involved.