| BackgroundGlioma is a common intracranial tumor,which accounts for 50%-60%of the total.The malignant gioma,accounting for 81%of central nervous system malignant tumors,including glioblastoma multiforme(GBM),for which the average survival period is 14 months,and which is almost always a fatal disease for patients.Despite the continuous improvement of diagnostic tools and treatment methods,the survival period of malignant glioma has not been significantly improved.The reason is that there are many causes and the pathogenesis is still not very clear.As a tool to study diseases,animal models play a huge role in the study of tumor pathogenesis,treatment effects and preclinical experiments.There are more and more types of brain glioma models,but most of them are mouse brain glioma models,which cannot fully meet the requirements of preclinical treatment or drug screening on animal models:in particular,their characteristies are very different to human glioma disease.So we need a more suitable animal model for the development of human glioma.Recently,it has been found that Syrian hamster is an effective model to evaluate the anti-tumor effect of tumor targeting adenovirus and human inflammatory cytokines.At present,the immune-targeted treatment of glioma have not been studied,and the advantage of Syrian hamster in immunotherapy makes it the first choice model animal in this research direction.In addition,the Syrian hamster has a short reproductive cycle,a large body size which is conducive to experimental operation,susceptibility to the viruses that can induce tumors,and similar anatomical and physiological characteristics to human beings,which makes it a potential model animal.Therefore,the establishment of a new Syrian hamster model of gliomas is of great significance for the study of the pathogenesis of gliomas,the development of new therapeutic methods and preclinical drug trials.ObjectiveWhereas Tp53 and RAS pathway abnormalities are common molecular changes in GBM,In this study,a Syrian hamster glioma model was induced by infection of the brain of animals carrying homozygous or heterozygous deletion of Tp53 gene with a lentivirus that carries the oncogene KRASG12D,then the models were examined by pathology and molecular biology methods to evaluate tumor type and malignant degree,comparing glioma model established by this approach to human glioma.This study provides a new method of glioma model building and an animal model closer to human glioma disease for future clinical treatment and research of glioma.MethodsIn this study,we establish a model with lentiviral injection of brain tissue,after the operation,we monitor changes in body weight,analyze the imaging images of tumor formation and the survival time,and score the disease to study animal tumor models preliminarily.Then,the tumor type and malignant degree were evaluated by morphological observation and pathology of hamster lesion brain.The specific research methods are as follows:(1)Constructing plasmid of KRASG12D.The second generation of lentiviral vector plasmids with biological tags such as Luciferase gene and green fluorescent gene(EGFP)were used as the skeleton into which were inserted the target oncogene KRASG12D to construct the main vector plasmid,named FUB-KRASG12D.(2)Packaging virus with KRASG12D.The plasmid constructed in method 1 was used as the main carrier plasmid to package the FUB-KRASG12D lentivirus with a second generation lentivirus packaging system,and the virus titer was detected after concentration.(3)Establishing the glioma animal model.In this study,there are four animal groups:A,B,C,and D,group A is the control group,perform empty vector lentivirus injection surgery,B,C,D are experimental groups,perform KRASG12D lentivirus injections surgery,hamster genotype of group A and group B for wild type(WT),the group C for lack of Tp53 gene hybrid type(p53+/-),group D for Tp53 homozygous deletion(p53-/-),each group has seven hamsters.At the age of 3 weeks to 4 weeks,they were subjected to brain surgery by injecting lentivirus,and after the surgery,they were observed by monitoring ofphysiological function over time.(4)Analysing Tumor model.The brains of the Syrian hamsters were dissected after they developed corresponding symptoms or after 60 days for asymptomatic animals,and brain tissue was analysed,including gross observation,pathological detection,and molecular detection.Results(1)The KRASG12D vector plasmid was successfully constructed and confirmed by sequence comparison analysis after sequencing.The KRSG12D vector plasmid and empty vector plasmid were used as the main plasmid for the production of lentivirus.After concentration,the KRASG12D vector virus titer was 1.28 × 107pfu/ml and the empty vector virus titer was 8.72 × 106pfu/ml by flow analysis.(2)After the surgery of virus injection in brain,the body weight change curve showed that group A and group B had the largest average body weight,followed by the group C,and the group D had the smallest average body weight,and the corresponding survival curve of each group also showed the same trend.The survival rate was 100%in group A and group B;57.4%in group C with an average survival time of 47 days;14.2%in group D with an average survival time of 40 days.Using the lentivirus vector with luciferase reporter gene allows animals to be tested every 7 days with live imaging to check to detect fluorescent expression in tumor.Each group will have different fluorescence expression quantity depending on the growth of the malignant tumors.group A and group B had the lowest fluorescence expression quantity,while there was no overall difference in fluorescence expression between the group C and group D,although the increase of fluorescence expression in the group D was slightly larger.(3)From the 30th to the 50th day after the operation,the hamsters showed more serious symptoms of glioma,including tardiness,tiredness,tremor,hemiplegia and dying state.The morpholo gical and patholo gical analysis of the brain tissue of Syrian hamsters showed that compared with the control group,the brain tissue with disease of had different degrees of deformation,such as abnormal bulge,different sizes of left and right hemispheres,blackening at the injection site,necrosis observed in the longitudinal section.The results of the pathological analysis showed that the tumor tissue had a large area of necrosis and hemorrhage,and there were abundant microvessels in the tumor tissue.The edge of tumor tissue was fuzzy and showed infiltrative growth.At the junction of brain tissue and tumor,tumor cells could be seen to invade brain parenchyma.Tumor tissue had a large cell density,round or irregular nuclei,more mitotic phases,less cytoplasm and there was local infiltration of lymphocytes and macrophages.Conclusion(1)The expression of KRASG12D and deletion of Tp53 can induce Syrian hamsters to develop gliomas;(2)KRAS gene mutation alone cannot induce the development of glioma disease in Syrian hamsters(3)From 30 days to 50 days after the operation,the hamster showed serious symptoms of glioma.The comprehensive average time was 42 days to build hamster glioma model.(4)The tumor tissue of hamster brain has a series of glioma characteristics such as rich in blood vessels,large-area necrotic lesions,irregular cell shape,mitotic phase,heterogeneous nucleoplasm,and so on,which is similar to the pathological state of human brain glioma. |
PDF Full Text Request