Tf-USPIO纳米微粒通过神经干细胞配体介导吸收及其体外磁共振成像研究

发布时间：2018-01-26 17:28

本文关键词： 神经干细胞转铁蛋白受体磁共振成像超小顺磁性氧化铁　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景神经干细胞（neural stem cell, NSC）是一种能够自我增殖、自我更新、并能够分化成神经元和胶质细胞的干细胞，可以用作修复神经功能损伤的种子细胞，但神经功能改善与干细胞移植之间的相关性还未完全明确。动物实验采用无创性活体动态示踪技术有助于活体评价干细胞移植的疗效，，是干细胞治疗走向临床前必须解决的问题。磁共振分子影像学因其高组织空间分辨率、无硬性伪信号、无辐射等优点而得到越来越多的应用。目前，超小顺磁性氧化铁（USPIO）是最常用的体内外细胞磁共振标记物，其存在着明显的缺点就是随着细胞的传代而出现信号递减，这限制了其不能长期动态的观察，但利用磁共振报告基因转染细胞方法可以达到无创动态观察移植的细胞变化。目的 1.合成Tf-USPIO纳米微粒以及建立稳定表达TfR的神经干细胞。 2.检测TfR神经干细胞吸收Tf-USPIO能否在磁共振上成像，为下一步动物活体实验提供基础。方法 1.利用慢病毒转染神经干细胞，通过流式细胞仪筛选出稳定表达TfR的神经干细胞，运用WB检测其表达水平。将Tf与USPIO进行偶联，合成Tf-USPIO纳米微粒，通过DLS方法测出USPIO和Tf-USPIO的直径分别为36±0.7nm，43.5±0.08nm。 2.将Tf-USPIO与USPIO分别于TfR神经神经干细胞共培养，通过普鲁士蓝染色、电镜以及ICP方法检测细胞中的铁离子含量，并用CCK-8试剂检测其对干细胞的毒性，利用7.0T磁共振检测其成像结果。结果 1.慢病毒成功的转染神经干细胞，通过流式细胞仪器筛选出稳定表达的神经干细胞，并用WB证实了TfR的过表达；通过偶联的方法合成了稳定的Tf-USPIO微粒。 2. Tf-USPIO与USPIO分别与TfR神经干细胞孵育后，普鲁士蓝染色、电镜及ICP检测铁离子浓度证实Tf-USPIO比USPIO在TfR神经干细胞中吸收的明显增多（P0.05）,CCK-8结果显示二者对神经干细胞的毒性较小（P0.05）,磁共振成像结果亦证实Tf-USPIO吸收量更多。结论 1.成功的构建了TfR神经干细胞系，合成出稳定的Tf-USPIO纳米微粒。 2.Tf-USPIO特异性的与TfR神经干细胞结合，并且可在磁共振上成像，可以作为一种磁共振对比剂。 3.Tf-USPIO在体外可以作为TfR神经干细胞的特异性示踪剂，为下一步活体成像实验研究奠定基础。
[Abstract]:Research background Neural stem cells (NSCs) are stem cells capable of self-proliferation, self-renewal, and differentiation into neurons and glial cells. It can be used as seed cells to repair neurological damage. However, the correlation between neural function improvement and stem cell transplantation has not been completely clear. In animal experiments, the use of non-invasive dynamic tracer technique is helpful to evaluate the efficacy of stem cell transplantation in vivo. Mr molecular imaging has been applied more and more because of its high tissue spatial resolution, no hard pseudo-signal, no radiation and so on. Ultrasmall paramagnetic iron oxide (USPIO) is the most commonly used magnetic resonance marker in vitro and in vivo. This limited its long-term dynamic observation, but the method of MRI report gene transfection could be used to observe the changes of transplanted cells in a non-invasive manner. Purpose 1. Synthesis of Tf-USPIO nanoparticles and establishment of neural stem cells stably expressing TfR. 2. Detecting whether TfR neural stem cells can absorb Tf-USPIO on magnetic resonance imaging can provide the basis for animal experiment in vivo. Method 1. Neural stem cells were transfected with lentivirus. Neural stem cells expressing TfR stably were screened by flow cytometry, and the expression level was detected by WB. TF was coupled with USPIO. Tf-USPIO nanoparticles were synthesized and the diameters of USPIO and Tf-USPIO were 36 卤0.7 nm and 43.5 卤0.08 nm, respectively, measured by DLS. 2. Tf-USPIO and USPIO were co-cultured in TfR neural stem cells. The iron ion content was detected by Prussian blue staining, electron microscopy and ICP. The toxicity of stem cells was detected by CCK-8 reagent and the imaging results were detected by 7.0T magnetic resonance imaging. Results 1. Lentivirus was successfully transfected into neural stem cells. The stable expressed neural stem cells were screened by flow cytometry, and the overexpression of TfR was confirmed by WB. Stable Tf-USPIO particles were synthesized by coupling method. 2. After Tf-USPIO and USPIO were incubated with TfR neural stem cells, Prussian blue staining was used. Electron microscopy and ICP showed that the absorption of Tf-USPIO in TfR neural stem cells was significantly higher than that of USPIO (P0.05). The results of CCK-8 showed that the toxicity of the two to neural stem cells was less than that of P0.05, and magnetic resonance imaging also confirmed that the absorption of Tf-USPIO was more. Conclusion 1. TfR neural stem cell lines were successfully constructed and stable Tf-USPIO nanoparticles were synthesized. 2. Tf-USPIO specifically binds to TfR neural stem cells and can be used as a magnetic resonance contrast agent. 3. Tf-USPIO can be used as a specific tracer for TfR neural stem cells in vitro.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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