Construction Of Double Fluorescent Labeled Glioma Cell Lines And Optimization Of Giloma Animal Models

Background:Glioma is the most common primary intracranial tumor in the central nervous system,which accounts for 81%of malignant brain tumors,Although the incidence is low,its mortality is extremely high.With the development of glioma research,a variety of new ideas of treatment have been put into preclinical and clinical practice applications,but the effect is minimal,successful animal experiments but poor clinical evaluation experience shows that the animal model does not reflect the true physiological characteristics of the patient's tumor,It is urgent to establish reliable and stable glioma orthotopic transplantation model for glioma research.Objective:To construct glioma fluorescence stable cell lines,and to establish a novel model of orthotopic xenograft tumor in nude mice which can monitor tumor growth in real time,and compare three different glioma orthotopic models.Methods:Three differents glioma cell lines(U87,U251,C6)were selected as the research object.The plasmid vectors containing Luciferise gene and Green fluorescent protein(GFP)gene was constructed.The vector was packaged with lentivirus by using lentivirus kit and human embryonic kidney cell(293FT)and extracted to influence glioma cells.Most glioma cells were labeled with GFP and Luc fluorescence after influencing by lentivirus.After screening by flow cytometry,three glioma cell lines stably expressing GFP-Luc fluorescence were obtained.Then the relationship between luminous intensity and cell number was detected by living luminescence imaging system,and the results were analyzed by one-dimensional linear regression.Cells with GFP-Luc fluorescence were as experimental groups and the cells without fluorescence were as control groups.In order to evaluate the biological characteristics of GFP-Luc fluorescence cells not influenced by the fluoyescent gene,the proliferation,cycle,metastasis and invasion ability of fluorescent cells and control cells were evaluated by CCK-8 test,cell cycle test,transwell tumor invasion and migration experiment.Then three kinds of GFP-Luc modified glioblastoma cells were implanted into the right Caudate nucleus of athymic nude mices by stereotaxic device respectively to establish thotopic tumor model.Intracerebral tumor growth was monitored in real time by small animal bioluminescence imaging(BLI)system.Hematoxylin-eosin staining(HE)was used to evaluate the pathological features and tumorigenicity of each cell line in the brain of nude mice.Result:1.The vector with GFP and Luciferise gene was packaged successfully with lentivirus.The lentivirus was used to infect three glioma cell lines C6,U87 and U251.GFP was expressed in about 90%glioma cells under fluorescence microscopy;2.Flow cytometry was used to screen the cells with fluorescence and the cells remain without fluorescent signal attenuation after continuous 6-8 generation.Cells stable expressing fluorescence were obtained.The cells with fluorescence were named as U87-GFP-Luc,U251-GFP-Luc,C6-GFP-Luc;3.The results of in vivo bioluminescence imaging system showed that the intensity of Luc showed a good correlation with the number of fluorescent cells.The R2 of U87-GFP-Luc,U251-GFP-Luc and C6-GFP-Luc were 0.9957,0.9920 and 0.9782,respectively;4.The results of cell function showed that,there were no statistically significant differences between the control group and the experimental group in the proliferation,cell cycle distribution,migration and invasion abilities(P>0.05);5.HE staining results showed that,U87-glioma nude mice model:boundaries of tumor tissue was clear,HE sections showed obvious neovascularization,tumor formation rate was 100%.The median survival period was 25 days in nude mice;U251-glioma nude mice model:the boundary between tumor and normal brain tissue was unclear,HE sections showed visible cell necrosis group,The tumor formation rate was 90%,median survival time was 29 days;C6-glioma nude mice model could be seen obvious nuclear atypia,tumor formation rate was 70%,the median survival period was 31 days.Conclusion:1.The biological characteristics of glioma cells were not significantly affected by lentivirus infection;2.Compared to traditional glioblastoma cells,GFP-Luc-transfected human glioblastoma cells were more beneficial to the study of glioblastoma;3.U87-glioma nude mice model:tumor formation rate was 100%,the groth of tumor was stable in intracranial,without extracranial metastasis and had moderate experimental cycle,sections showed obvious neovascularization and the model is suitable for glioma vascular formation and anti angiogenic therapy research;4.U251-nude mice model had high tumor formation rate(90%).The survival time of this model was not very stable,however,the tumor growth and pathological characteristics were similar to that of human glioma;5.C6-nude mice tumor formation rate lower slightly(75%),a large number of model appeared extracranial tumor growth which suggesting that this cell line may not be suitable for planting in the nude mice.