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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。
OBJECTIVE: To construct the cationic liposome plasmid DNA complex CL-IL15 by cationic liposome (CL) and pcDNA3.1-IL15, and to observe its therapeutic effect on anti-mouse B16-F10 lung metastases and to explore its mechanism . Methods: The hIL-15 eukaryotic expression vector was constructed and the optimal entrapment efficiency of liposome-plasmid DNA complex was determined. The complex was transfected into CHO-K1 cell line and West-ern blott was used to detect IL -15protein expression was detected by MTT assay. The proliferation of CTLL-2cells was detected by MTT assay. The lung metastasis model of B16-F10 mouse melanoma was established. Times, a total of 6 times, after the end of treatment 24 hours after the observation of tumor lung metastasis; LDH release (lactate dehydrogenase release method) to detect the cytotoxic effect of spleen lymphocytes, frozen section immunofluorescence observed NK cells on the tumor tissue infiltration. Results: The hIL-15 expression vector was constructed successfully and the mass ratio of the hIL-15 to cationic liposomes was 1: 5. The entrapment efficiency of hIL-15 was better than that of the cationic liposomes. The biological activity of secreted IL-15 protein; CL-IL15 complex treatment of mouse tumor lung metastasis model can significantly reduce the number of lung metastasis of the tumor, increase the cytotoxic activity of spleen cells to tumor cells (P <0.05), increase the number of NK cells The proportion of infiltration in tumor tissue. CONCLUSION: CL-IL15, a cationic liposome plasmid DNA complex, can effectively inhibit lung metastasis of B16-F10 in mice. Its mechanism may be related to IL-15-induced killing of tumor cells by splenocytes and activation of natural killer cells On the mechanism of tumor invasion and other related.