磁共振报告基因MagA对肺癌早期转移示踪的安全性研究

Early detection and treatment on metastasisis is the key to improve the prognosis of lung cancer patients. When the mammal cells over-expressed MagA gene of magnetotactic bacteria, the tumor imaging effects were significantly improved by regulating of extracellular iron into cells to compose magnetosome. As a MRI reporter gene, MagA could exist a long term through cell division and trace metastasis and invasion of lung cancer cells at an early stage. We successfully constructed a MagA transgenic mice model in the preliminary work, there were no differences in the life cycle and reproduction breeding of transgenic mice comparing to the wild type mice, meanwhile MagA could attenuates the oxidative damage induced by iron overload in transgenic mice. Preliminary experiment also showed that the proliferation capacity weakened in MagA over-expressed lung cancer cells. This project plans to analyze the influence of MagA on the biological behaviores of lung cancer cells such as cell cycle, apoptosis, invasion and migration by using lentiviral plasmid stability transplanted tumor experiments; construct the transplantation tumor mouse model of transgenic mice and observe the tumor growth and metastasis; utilizing the specific tumor metastasis-homing ability of amniotic fluid stem cells, we intend to explore the MagA tracer ways in lung cancer cells using oncolytic adenovirus plasmid-infected amniotic fluid stem cells targeted carrying method, and evaluate the safety and effectiveness of early metastasis of lung cancer cells aiming to provide sufficient scientific basis for clinical application of MagA.

肺癌的转移灶早期发现和治疗是提高患者预后的关键。趋磁细菌中的MagA基因在哺乳动物细胞内过表达能调节磁小体形成，显著增强MRI成像效果。MagA作为报告基因，在体内随细胞分裂而长期存在，可在早期示踪肺癌细胞的转移和侵袭。本课题在前期工作中成功构建了MagA转基因小鼠模型，结果显示其生命周期、繁殖繁育与正常对照小鼠没有显著差异，并且能够缓解铁超载对机体造成的氧化应激损伤。预实验表明过表达MagA的肺癌细胞增殖能力减弱。本课题拟进一步采用慢病毒稳定转染细胞的方法，探讨MagA对肺癌细胞周期、凋亡、侵袭和迁移等生物学行为的影响；构建转基因小鼠异体移植瘤小鼠模型，观察肿瘤的生长和转移情况；利用羊水干细胞特异性的肿瘤归巢能力，应用溶瘤腺病毒质粒感染羊水干细胞靶向运载MagA到达肺癌细胞的途径，对MagA示踪肺癌细胞早期转移的安全性及有效性进行评估，为MagA合理应用提供充分的科学依据。

背景：本研究构建慢病毒稳定表达MagA的A549细胞，体外观察MagA是否改变肺癌细胞的增殖、迁移、侵袭、凋亡等生物学行为。分析MagA表达的肺癌细胞铁代谢情况及总铁含量的表达，初步探讨MagA影响肺癌生物学的潜在机制。构建异体移植瘤小鼠模型，通过对比成瘤大小、体积和转移情况及体内体外MRI成效效果评价其应用于肺癌示踪的安全性和有效性。.结果：细胞生物行为学结果显示MagA表达A549细胞的体外迁移和侵袭能力明显受到抑制，增殖速度减慢，凋亡能力没有明显改变。LV-vector处理A549细胞的接种组瘤体体积显著大于LV-MagA处理A549细胞接种组。MagA慢病毒稳定转染细胞组与其他两组相比，TfR和Tf蛋白表达显著性下降；Fn蛋白表达显著性升高。IRP-1和IRP-2蛋白的表达没有受到MagA的影响。MagA慢病毒稳定转染细胞内总铁含量没有显著性差异；在外源性铁添加的情况下，MagA慢病毒稳定转染细胞内总铁含量与未转染细胞相比显著性升高。MagA慢病毒稳定转染细胞与未转染细胞的培养液相比，细胞内总铁含量没有显著性差异；添加铁剂后，MagA慢病毒稳定转染细胞培养液内总铁含量与未转染细胞相比显著性降低。TfR mRNA表达结果显示，稳定表达MagA的A549细胞内TfR mRNA含量显著低于空白对照细胞组；MagA mRNA表达结果显示，稳定表达MagA的A549细胞瞬时转染pcDNA3.1-TfR质粒后，MagA mRNA表达量相比未转染组降低；反之，转染TfR-siRNA后，MagA mRNA表达量相比未转染组升高。透射电镜观察到表达MagA的瘤体内出现纳米颗粒，聚集成簇，也有散在的纳米颗粒存在。铁剂添加后，MagA表达的A549细胞R2值和R2∗值都显著性升高；而没有外源性铁参与的情况下，MagA表达的A549细胞与对照组A549细胞相比，没有显著性差异。在铁剂补充的状态下，MagA表达的肿瘤组织的R2值显著高于对照侧肺癌组织。.结论：成功构建稳定表达MagA的A549细胞；MagA能够抑制A549细胞的增殖、迁移和侵袭能力，但凋亡不受影响；MagA表达的肺癌肿瘤组织生长速度减慢；MagA能够在mRNA和蛋白水平抑制TfR的表达；MagA表达的肺癌肿瘤组织内也发现纳米颗粒的集聚；在体内和体外，铁剂补充下，MagA能够显著增强MRI成像效果。