MAGE1纳米蛋白疫苗的研制及其抗肿瘤效应

Preparation and antitumor immunity of nanoemulsion-encapsulated MAGE1 protein vaccine

目的制备纳米乳剂包裹基因工程MAGE1(melanoma antigen1)抗原,研究其生物学特性及抗肿瘤免疫效应。方法采用界面聚合法,制备包裹MAGE1抗原的纳米乳剂(NEMAGE1),测量粒径、包裹率、载药量。检测乳剂包裹对小鼠DC摄取抗原能力的影响。用NEMAGE1免疫小鼠,运用酶联免疫斑点法(ELISPOT)和细胞毒性杀伤实验(LDH)检测NEMAGE1激活机体细胞免疫反应的状况,并观察纳米蛋白疫苗对荷瘤小鼠的治疗作用。结果成功制备粒径为(15±5)nm的纳米乳剂,包裹率为91%,载药量0.091gPL,4℃放置6个月后性质稳定。DC摄取NEMAGE1的能力明显强于对游离蛋白抗原的摄取。ELISPOT和细胞毒性杀伤实验显示,NEMAGE1可以诱导机体产生针对MAGE1的CTL,特异性杀伤表达MAGE1的肿瘤细胞。荷瘤小鼠的治疗实验显示,NEMAGE1对表达MAGE1的肿瘤有明显疗效,其效果优于单独使用游离MAGE1蛋白。结论纳米乳剂具有较高包裹率和稳定性。纳米乳剂包裹的肿瘤特异性抗原可以有效激活机体产生抗肿瘤免疫效应,是具有临床应用前景的新型抗肿瘤疫苗。

Objective To prepare nannoemulsion-encapsulated MAGE1 （NE MAGE1 ） and to prove its antitumor immunity in mouse. Methods NE MAGE1 was prepared by the interfacial polymerization method. The shape and size of NE MAGE1 were shown by electron microscopy. The encapsulation rate, drng-carrying capacity, stability were tested by Sephedex-G100 gel filtration. The uptake of NE MAGE1 by murine BMDC was detected by FCM. C57BL/6 mice were intraperitoneally immunized with NE MAGE1, free MAGE1, blank NE and PBS as negative controls. A second vaccination was performed 2 weeks later. The mice were killed 2 weeks after the booster, and splenecytes were pooled. The antitumor immunity was detected by enzyme-linked immunospot assay（ELISPOT） and cytotoxicity assays. Then the therapy efficacy of NE MAGE1 was detected among the mice which had bearing B16-MAGE1 melanoma. Results The mean size of NE MAGE1 was （15±5） nm, the encapsulation rate was 91%, the drug content was 0.091 g/L. NE MAGE1 has a good stability after stored in 4℃ for 6 months. The resuits of FCM showed that DC can take in more NE MAGE1 than free MAGE1.ELISPOT and cytotoxicity assays showed the immunization using NE MAGE1 stimulated splenocytes secreting IFN-γ. Cytotoxicity assays showed the B16-MAGE1 lysis of CTL from mice immunized with NE MAGE1 was greater than that from mice immunized with free MAGE1. The tumor mass formed in the mice immunized with NE MAGE1 （0.29 g） was markedly smaller than those immunized with free MAGE1 （0.42 g）. The tumor inhibiting rate as high as 62.83% of the NE MAGE1 was obtained, while free MAGE1 canonlyattachalowereffectas46.10%（P〈0.05）. Conclusion Nanoemulsion has novel characters, and NE MAGE1 can greatly enhance the potency of MAGE1 protein vaccines, and generate specific antitumor immurnity against MAGE1 expression tumors.