双模式编码的慢病毒载体转染C6 Glioma Cells的影像学研究

The aim of this study was to apply modern imaging method to observe the C6 glioma cells occurs, develop, breeding, metabolic mechanism and the spread way of cancer cells in the molecular level. Based on bimodal lentiviral vector transfection technology, we try to develop a bimodal lentiviral vector to monitor deep tissue events using MRI to detect the C6 rats glioma cell myc-tagged human ferritin heavy chain (myc-hFTH) expression and fluorescence imaging to detect enhanced green fluorescent protein (EGFP) expression in the specific C6 rats glioma cell lines (having myc-hFTH) and the corresponding animal models. The both imaging parameters, i.e. the fluorescence intensity of the EGFP for immunofluorescence optical imaging, and the relaxation intensity (T2*, R2* )of the myc-hFTH for MRI, will be compared and analysis. The both imaging characteristics will be matched with the data from the neural histopathological staining and HE dyed slices. Through the qualitative and quantitative analysis and comparison of the results delivered from the C6 rats glioma cell lines with myc-hFTH gene and EGFP expressions in vivo and in vitro, especially the features of the glioma cell nests and the distribution of the new tumor vascular, it is hopeful to further understanding mechanism of gliomas and realizing the development of glioma, being able to have the early diagnosis and differential diagnosis and evaluating gene therapy. It is expected to seek a new type of endogenous MR biological "contrast agents" as well.

本研究目的是应用现代影像学手段在分子水平观察C6胶质瘤细胞发生，发育，繁殖，代谢机制和癌细胞的传播方式。方法是拟以双编码慢病毒载体转染技术，即以编码有增强型的绿色荧光蛋白（EGFP）报告基因和磁共振成像（MRI）的报告基因【携带铁蛋白重链（H-ferritin）】双启动子慢病毒为载体转染C6大鼠胶质瘤细胞为基础，制备特定的C6大鼠胶质瘤细胞株（原癌基因c-myc被标记表达）和相应的动物模型，采用免疫荧光光学成像和MRI技术，对EGFP的荧光强度和myc-hFTH磁共振驰豫效率的进行测定和比较，结合神经病理组织染色和HE染色，定性和定量地观察和分析比较胶质瘤细胞株c-myc基因体内外表达的特征，特别是胶质瘤细胞巢和新生瘤血管生物学分布的分子影像学特征。期望在进一步提高认识胶质瘤发病机制的同时，为实现胶质瘤的早期诊断和鉴别诊断以及评价基因治疗疗效，寻求一类新型的内源性MR生物"对比剂"。

本研究目的在于人铁蛋白重链（human ferritin heavy chain）作为新型的核磁共振显像报告基因的可行性。方法：1）体外实验：利用慢病毒转染大鼠胶质瘤细胞，建立携带有hFTH及EGFP的C6细胞系并利用流式细胞分选技术分选细胞；利用总铁含量分析试验测试细胞铁负载能力；利用CCK-8试验分析细胞增殖能力；利用MRI分析未表达hFTH及表达hFTH的细胞信号值差异。2）体内实验：建立裸鼠皮下及颅内胶质瘤模型，并使用小动物活体成像技术及MRI进行肿瘤活体扫描；利用western blot及组织免疫荧光染色明确hFTH、EGFP及转铁蛋白受体的表达。结果：1）成功建立共表达hFTH及EGFP的C6胶质瘤细胞系。2）表达hFTH的C6细胞铁含量增高，其增殖能力与未表达hFTH的C6细胞无差异（P＞0.05）；3）表达hFTH的细胞、皮下及颅内肿瘤移植物的T2弛豫时间及信号强度均下降（P＜0.05），同时在皮下及颅内肿瘤中均可检测到EGFP的荧光信号；3）表达hFTH的细胞，其转铁蛋白受体的表达量高于未表达hFTH的细胞（P＜0.05）。4）Western blot及组织染色证实hFTH及EGFP在肿瘤移植物中表达。结论：hFTH通过影响转铁蛋白受体的表达成功改变了肿瘤的MRI信号强度。hFTH可作为胶质瘤诊断、基因治疗及研究肿瘤代谢的MR分子影像标记基因。

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