磁螺菌靶向肝癌乏氧区联合动脉栓塞抗肿瘤效果及机制

Anaerobes can target and interfere with tumor hypoxic microenviroment,however,the antitumor mechanisms of currently used anaerobes haven't been fully understood,further researches are necessary for traceable effective anaerobes and clarifying their antitumor mechanism. In current study, Magnetospirillum with magnetotaxis and anaerobic property are exploited for targeting the hypoxic zone within rabbit VX2 liver cancer, and will be traced by MRI combining with 18F-fluoromisonidazole PET-CT. The tumor MR images overlay with PET-CT images by using image post-processing technique to generate a magnetic hypoxic subregion image indicating the tumor targeting capability of Magnetospirillum. Imaging differences will be compared between different treatment groups (+/- Magnetospirillum; artery or vein injection group). The fused imagings will be matched and compared with H&E, immunohistochemisty staining and Gram staining of the continuous tumor tissue slices in order to further validate the tumor targetting and the distribution of Magnetospirillum within tumor hypoxic zone. Magnetospirillum can settle and grown within tumor, compete nutrition with cancer cells to destroy them in hypoxic regions,and therefore inhibit angiogenesis and epithelial-mesenchymal transition (EMT) due to hypoxia. Magnetospirillum targeting hypoxic area combing with artery embolization will destroy the transplanted VX2 liver cancer. Differences of tumor inhibition ratio, hypoxic extent and scope, microvessel density, hypoxic stress genes or proteins and EMT marker between groups will be detected by various methods such as MRI scans, [18F]FMISO-PET-CT scan, hypoxia proteomics, immunohistochemisty, quantitative PCR. The effects and molecular mechanisms inhibiting liver cancer by Magnetospirillum will be explored.

厌氧菌可靶向干扰肿瘤乏氧微环境，但目前所用厌氧菌抗肿瘤机制尚未完全明确，有必要深入研究可示踪、有效的菌种并阐明其作用机制。本研究利用磁螺菌（一种趋磁菌）的趋磁性和厌氧特性，靶向兔VX2肝癌乏氧区。行MRI及18F-氟米索硝基PET-CT乏氧显像，经图像后处理产生磁性乏氧区图像，确定磁螺菌靶向性及在有/无磁螺菌组间、动脉/静脉给菌组间的差异。将影像同肿瘤H&E、免疫组化、细菌革兰氏染色对照，进一步验证磁螺菌肿瘤靶向定位及瘤内分布。磁螺菌在乏氧区内繁殖，干扰乏氧微环境，竞争营养以破坏乏氧区癌细胞，抑制其引发的血管生成及乏氧导致的上皮间质转化（EMT），同动脉栓塞结合，治疗兔VX2肝癌。通过MRI、PET-CT乏氧显像、免疫组化、乏氧相关蛋白质组学、定量PCR等手段检测各组间肿瘤抑制率、乏氧程度变化、微血管密度、乏氧相关基因及蛋白质表达及EMT标志物的组间差异，探索磁螺菌抑制肝癌效果及分子机制。

本项目首先对磁螺菌进行培养、鉴定及特性观察，然后将磁螺菌用于对兔VX2移植性肝癌的抗肿瘤治疗研究中。研究了磁螺菌靶向肝癌的影像、病理、安全性及疗效，也初步探讨了磁螺菌靶向抗肿瘤的相关机制，研究了血管生成、肿瘤相关细胞免疫及上皮间皮转化等方面机制是否参与磁螺菌的抗肿瘤机制中。本课题成功培养了具有趋磁性和厌氧特性的磁螺菌，并通过电镜等方法观察到菌体内具有成串排列的磁小体，该细菌在磁场引导下可以沿着磁力线方向移动。将磁螺菌通过静脉途经注入荷VX2移植性肝癌的新西兰大白兔体内，经过影像、大体标本与病理对照，发现磁螺菌可以靶向移动到VX2移植性肝癌坏死乏氧区内，并在局部形成脓肿，与磁共振信号及革兰氏染色显示的细菌分布相吻合。为进一步强化磁螺菌在肿瘤内的趋向性，我们采用磁粒子吸附磁螺菌的方式，观察到经过这种处理后，肿瘤内部更易于形成局部脓肿，但不影响荷瘤兔生存，该干预安全有效。同对照组相比，采用磁螺菌治疗的荷瘤兔肿瘤缩小，肿瘤受到明显抑制。通过对比，磁螺菌治疗组肿瘤血管生成相关的基因蛋白表达（CD31、VEGF、HIF-a） 均未见明显差异，表明磁螺菌抑制肿瘤生长并不是通过抑制肿瘤血管生成途经，治疗组和对照组间的E-cadherin及N-cadherin的表达也未见明显差异，表明磁螺菌抑制癌细胞的作用同上皮间质转化关系不大。通过对Arg-1、TNF-a及iNOS的mRNA表达进行定量测定后发现磁螺菌治疗组iNOS表达显著增高，因此我们推测磁螺菌通过干扰肿瘤乏氧微环境，破坏癌细胞生存条件，同时激活M1型巨噬细胞，激活了肿瘤杀伤机制，释放诱导型一氧化氮合酶，是机体抗肿瘤、抗微生物双重反应机制。