Cell Tracking and imaging gene expression in the brain

描述(申请人提供)：大脑的代谢障碍和遗传性疾病可以通过移植神经干细胞来治愈。这些细胞可以迁移和分化为神经元、星形胶质细胞或少突胶质细胞，从而可以传递治疗性基因，甚至取代大脑中的缺陷细胞。干细胞治疗发展的一个主要挑战是非侵入性地监测这些捐赠者细胞的迁移和消失的表达。绿色荧光蛋白和荧光素酶仅适用于浅层器官，而单纯疱疹病毒胸苷激酶消失需要与阿昔洛韦配体结合，而阿昔洛韦不会越过血脑屏障。这些技术的灵敏度高，但空间分辨率非常有限。使用磁共振成像可以在体内检测到标记有氧化铁颗粒的干细胞。这些细胞还可以被标记有慢病毒多巴胺2型受体，并可以通过PET成像来监测它们在体内的基因表达。这项提案的总体目标是评估磁共振成像(MRI)和正电子发射断层扫描(PET)在细胞跟踪和脑内基因表达成像方面的效用。这一假说将通过以下特定目标进行验证：目标1：使用高分辨率体内磁共振成像，连续监测标记有临床批准的超顺磁性氧化铁(SPIO)粒子的C17.2神经前体细胞在小鼠大脑中的迁移和植入。目的：使用突变的多巴胺2型受体(D2R-80A)和3-(2‘-[18F]-氟乙基)螺环酮(FESP)配体，用显微PET成像技术研究供体C17.2神经前体细胞的基因表达。目的：利用体内磁共振和正电子发射计算机断层扫描(PET)技术，研究C17.2细胞迁移与D2R受体基因表达的关系。 神经C17.2祖细胞将与慢病毒D2R-80A受体和临床批准的氧化铁颗粒进行转导。C17.2细胞表达来自克隆整合位点的β-半乳糖苷酶基因，该基因将被用作独立的供体细胞标记。这些细胞将被移植到成年小鼠的海马体内和新生小鼠的脑室中。这些细胞的迁移将使用自旋回波和梯度回波磁共振成像进行连续监测。D2R受体的GONE表达之后将与FESP配体结合，并将使用微型PET进行成像。MRI和PET图像的相关性将在体内和体外使用解剖标记物和β-半乳糖苷酶的免疫组织化学染色进行。

DESCRIPTION (provided by applicant): Metabolic disorders and genetic diseases of the brain can potentially be cured by transplantation of neural stem cells. These cells can migrate and differentiate into neurons, astroglia or oligodendrocytes and thereby can deliver therapeutic genes or even replace defective cells in the brain. A major challenge towards development of stem cell therapy has been to non-invasively monitor the migration and gone expression of these donor cells. Green fluorescence protein and luciferase are suitable only for superficial organs while herpes-simplex virus-thymidine kinase gone requires binding to acyclovir ligands that do not cross the blood brain barrier. The sensitivity of these techniques is high but spatial resolution is very limited. Stem cells labeled with iron oxide particles can be detected in vivo using magnetic resonance imaging. These cells can also be labeled with a lentiviral dopamine type 2 receptor and can be imaged by PET to monitor their gene expression in vivo. The overall goal for this proposal is to evaluate the utility of magnetic resonance imaging (MRI) and positron emission tomography (PET) for cell tracking and imaging gene expression in the brain. This hypothesis will be tested with the following specific aims: Aim 1: To serially monitor migration and engraftment of C17.2 neural progenitor cells tagged with clinically approved super paramagnetic iron oxide (SPIO) particles using high resolution in vivo MR imaging in a mouse brain. Aim 2: To image gene expression in donor C17.2 neural progenitor cells in the mouse brain using the mutant dopamine type 2 receptor (D2R-80A) and 3-(2'-[18F]-fluoroethyl)-spiperone (FESP) ligand with micro PET imaging. Aim 3: To co-register migration of C17.2 cells with gene expression of the D2R receptor using in vivo MR and PET imaging. Neural C17.2 progenitor cells will be transduced with the lentiviral D2R-80A receptor and with clinically approved iron oxide particles. C17.2 cells express the beta-galactosidase gene from a clonally integrated site which will be used as an independent donor cell marker. The cells will be transplanted intracranially in the hippocampus of adult mice and in the ventricles of neonatal mice. Migration of these cells will be monitored serially using spin echo and gradient echo MR imaging. Gone expression of the D2R receptor will be followed by its binding with FESP ligand and will be imaged using a micro PET. Correlation of MRI and PET images will be performed in vivo and in vitro using anatomical markers and with immunohistochemical staining for the beta-galactosidase activity.