Evaluation Of The In Vivo Imaging In Glioma-associated Vascularization

Part I Establishment and cytobiological characteristics of enhanced green fluorescent protein(EGFP) expressing GBM cell line Aim:To isolate and culture human GBM in vitro,which was infected with EGFP by lentivirus. Biological characteristics between GBM and EGFP-expressing GBM will be investigated. Meterials and Method:Human GBM were isolated and expansion with cloning cylinders by trypsin/EDTA,then were infected by pseudo- lentivirus carrying EGFP,an efficient gene transfer vehicle for tumor cells and selected by puromycin.The expressions of EGFP of the infected GBM were observed by fluorescence microscopy and detected with flow cytometry(FCM) after 1 month of conventional culture in vitro.Multiple cytobiological characteristics,such as cytomorphology,proliferation,cell cycle,cell invasion abilities,expression of VEGF and the growth rate of subcutaneous xenografts in nude mice were compared between the infected and uninfected GBMs. Result:GBMs could be infected efficiently by pseudo- lentivirus carrying EGFP.We obtain a stable EGFP expression tumor cells, named GBM-EGFP. 1 month after consecutive culture in vitro, green fluorescence can be detected stably and intensely. There were no significant differences between GBM and GBM- EGFP in several cytobiological characteristics.The growth curves of tumors by subcutaneous implantation were similar. The expression of EGFP in GBMs did not interfere the tumorigenicity. Conclusion:GBM- EGFP can express green fluorescence stably without affecting the tumorous characteristics. This could be helpful to investigate the mechanisms of Glioma-associatied recurrence and metastasis.PART II Evaluation of the in vivo imaging in glioma-associated vascularization Aim:To establish the GBM-EGFP tumor model with in vivo imaging by subcutaneously injection, Which can be used to investigate the tumor grouth and glioma-associatied neovascularization continuously and accurately without sacrificing animals.The relationship between tumor growth and neovascularization will also be discussed. Method:To establish the tumor models:25 4-week-old balb/c- nu mice were subcutaneously injected with 1x106 GBM-EGFP,which were randomly devided into 5 groups.The control group consists of another 9 mice,which were injected 1x106 GBM cells.In vivo flurescence imaging were used to at 1w,1.5w,2w,2.5w,3w,3.5w,4w,4.5w,5w after injection to investigate the tumor growth and the neovascularization. Meanwhile,tumor samples were sected with H&E and C D34 IH/IF to observe the tumor histology and.Microvessel density(MVD) is also counted to evaluate the vascular prolifering.The flurenscence intensity of diffident cellular array will be also calculated to estimate the cell number at diffident time,which will help to investigate the relationship between tumor growth and glioma-associated vascularization. Result:Experiment in vitro showed the flurenscence intensity is correlated with the cell number. Stable flurenscence can be detected within the in vivo imaging 7days after injection After 2 weeks the tumor growed rapidly and the flurenscence increased altogether.We observed obvious necrosis in the deep location from the lobulated tumor sample and the H&E staining.CD34 immunohistochemistry showed that primitive vessel formed after 1 week.Sprouting angiogensis can be detected at the edge of necrosis in the middle area of the tumor at 1w.The MVD increased rapidly between 3w-4w and reached the peak at 4w.FCM shows the C D34+ rate is 1.63%. Conclusion:The EGFP tagged glioblastoma with the in vivo imaging is superior for the long-term and dynamic observation o f the tumor growth. These mice modle are suitable for the research about relapse,evaluation of anti-angiogenic therapy.