磁共振光学分子影像研究脑胶质瘤浸润性生长和干细胞对其靶向诊疗作用

With the development of new technical adjuncts for surgical management of gliomas, such as neuronavigation and functional imaging, a renewed interest in a more radical surgical removal of these tumors has emerged, and it has been hypothesized that more complete resection of tumors may translate into prolonged survival. However, the inherent tendency of glial tumors to widely disseminate within normal brain has to be regarded as a natural limit to this concept. Upon to the findings of the survival of tumor cells within peritumor brain edema in gliomas indicted by our previous study with the novel MRI techniques such as MRS and DTI, previous studies have been already done for the investigation of the innovative exogenous contrast agents composited of fluorescence nanocrystallites and superparamagnetic iron oxide(SPIO) with coupling with transferrin or lactoferrin as targeting molecular probes for optical and MR imaging. Exogenous iron oxide particle such as SPIO based cell-labeling approaches enable cell tracking by MRI with high resolution and good soft tissue contrast in the brain. However, free iron catalyzes radical formation in oxygenated tissues(Fenton's reaction) leading to cell damage, and delivery barriers must be considered, as well as the dilution effect upon cell division is a major drawback for longitudinal follow-up of highly proliferating neural stem cells or glioma cells with MRI. Endogenous reporter proteins for optical imaging such as green fluorescent protein (GFP) are widely used. However, sensitivity as well as resolution for detection of fluorescent proteins are significantly limited by the depth of penetration and scattering of light. MRI offers exquisite spatial resolution for deep tissues; thus, an endogenous reporter of gene expression for MRI would provide an important tool, complementing optical imaging for deep tissue molecular imaging. Stable viral vector-mediated marking of endogenous stem cells with a reporter gene for MRI could overcome the limitations of exogenous administration of contrast material. In this study, we aim to develop the heavy chain of murine ferritin as a novel endogenous reporter for the detection of gene expression by MRI. Expression of both enhanced GFP and ferritin will be tightly coregulated by tetracycline (TET), using a bidirectional expression vector. Glioma and stem cells labeled with TET-regulated gene expression of both GFP and ferritin will be dynamically detected by MRI, followed by independent validation using fluorescence microscopy and histology, as well for assessment of the invasion patterns of gliomas associated with its vascularization. In addition, we will further demonstrate that the stem cells labeled with TET-regulated gene expression possess extensive tropism for glioma and significant migratory behavior and inhibition of glioma growth, which will be used for future detection of disseminated glioma cells and as an innovative delivery method for targeting gene therapy.

脑胶质瘤浸润生长和脱离肿瘤主体的浸润瘤细胞群，是导致目前综合治疗脑胶质瘤不理想或肿瘤易复发的主要原因。目前研发的外源性含铁或钆及其相关的靶向对比剂，由于稀释效应和不能有效通过血瘤和血脑屏障，限制了长时程示踪胶质瘤的浸润生长，也无法达到靶向诊断或成为有效的靶向传递载体，这成为临床诊疗关注的焦点。近期研究提示神经干细胞和骨髓间充质干细胞具有向瘤趋化性和抑制胶质瘤作用。故拟研制内源性红或绿色荧光蛋白和铁蛋白报告基因并携带四环素开关系统，有效控制铁蛋白基因表达，可用于光学和磁共振成像并克服外源性对比剂的缺陷。通过病毒转染双模式报告基因标记瘤细胞和干细胞，用磁共振成像活体观察和微观荧光病理等研究脑胶质瘤的浸润；利用干细胞向瘤趋化性探索标记的干细胞作为胶质瘤的靶向对比剂和靶向传递载体的可行性；活体示踪和荧光病理研究标记的干细胞抑制胶质瘤生长的作用，为临床靶向诊断浸润的胶质瘤细胞群和靶向治疗提供新方法。

我们的研究表明星形细胞具有促进胶质瘤细胞增殖、迁移的作用。我们行CCK8实验及星形细胞与胶质瘤细胞共培养实验发现，无论是用星形细胞条件培养基间接培养还是星形细胞与胶质瘤细胞直接接触的条件下，胶质瘤细胞生长均加快。我们进一步行迁移实验发现，在星形细胞条件培养基的作用下，胶质瘤细胞迁移增多。在体内成瘤实验中，胶质瘤细胞与星形细胞共同注射组较仅注射胶质瘤细胞组相比，前者肿瘤体积、肿瘤重量均明显增大。.胶质瘤的浸润性生长以及由脱离主体胶质瘤的浸润性胶质瘤细胞群所造成的转移，仍然是临床胶质瘤治疗的难点。为改进常规的含铁或者钆及其相关靶向对比剂的血脑穿透力差、代谢快、靶向示踪能力差的问题，本项目制备了多种高效、智能、靶向胶质瘤的细胞，有效的提高了其浸润性生长的示踪能力及治疗效果。主要包括：（1）制备FA-BSA修饰的高分子聚合物磁性胶束作为肿瘤特异性靶向磁共振对比剂，用于肿瘤靶向磁共振成像。体外和体内实验均说明FA-BSA修饰的磁性胶束具有很强的磁共振成像能力，并且能显著增加肿瘤细胞与肿瘤组织对磁性胶束的摄取，可以作为高表达叶酸受体的肿瘤的靶向对比剂；（2）通过自组装法制备了温度敏感脂质体对比剂LTCAs 。马根维显、碘海醇和IR820成功的被装载进脂质体LTCAs的内核。能在近红外光照射下分解产生CO2的NH4HCO3同样成功包裹进LTCAs的内核。体内和体外实验证实与无光照对照组相比，近红外光照射后的MRI/荧光（FI）图像具有更好的对比增强效果，同时，能通过光热治疗较好的抑制肿瘤生长。（3）创新的同时将顺铂和Gd同时与卟啉结合，得到了同时具有化疗光动力治疗及对比剂于一体的分子诊疗剂Gd-Pt-Porphyrins，该分子具有低细胞毒性和良好的生物相容性。