Exploring The Biological Features Of Orthotopic Grafts Of Human Brain Tumors Into Nude Mice

PartⅠEstablishment of Orthotopic Grafts of Human Gliomas into Nude MiceObjective To improve the methods of human glioma xenotransplanted into the brain of nude mice by tumor tissue injected. Methods SHG-44 glioma cells(1×107) were injected subcutaneouly into nude mice, when the solid tumor formed ,it was sharply dissected.Then the xenografts quantified was passed into the brain of nude mice. 30 days later ,all the mice were sacrificed.Slices of intracerebral tumor transplants was stained with hematein-eosin(HE).We study it's histologic characteristics under microscope. Results 3 of the 18 orthotopic grafted nude mice was died earlier .30 days after tumor tissue implanted, all the mice were sacrificed. The brian was cut in frozen section,stained with HE. Under microscope, we can see the orthotopic grafts with invasiveness to normal brain tissue.Take tate of the grafts was 93 %( 14/15 ) .Conclusion Tissue quantitative implantation is operation simple, small wound ,which can save time and easily product model in large quantities .PartⅡEstablishment of Orthotopic Transplantation Model of Human Brain Glioma with High Invasiveness and Overexpression of EGFR in Nude MiceObjective To establish orthotopic transplantation model of human brain glioma in nude mice with characteristics of high invasiveness and overexpression of EGFR .Methods Human glioblastoma multiforme cell line with overexpression of EGFR was cultured in vitro,then tumor cells were injected stereotaxically into the right caudate nucleus of nude mice.The newly formed solid tumor were sharply dissected and solid were passed in the right caudate nucleus of nude mice. Histological characteristics and EGFR protein in the xenografts were determinated by HE staining or immunohistochemical method,respectively.Results The xenografts have been passed continuously in 130 mice for 13 generations in caudate nucleus of nude mice, and the survival time were 19±1.33 day.Histological assay suggests the grafts were high invasiveness and overexpression of EGFR,which were in accordance with the characteristics of human glioblastoma multiforme. The tumor proliferation curve measured at different time points suggests that tumor latent period were shoter(<3 day), rapidly proliferative phase were longer(>15 day),and from the advanced stage to dead point were shorter(<3 day). Conclusion Compared with tumor cells suspension directly innoculated into the caudate nucleus of nude mice,the transplantion of solid tumor tissue take on the advantages of not only injection of plenty of tumor cells,but also with tumor matrix which are necessary for the rapid tumor growth,hence embody the features of high malignant glioma with high invasiveness and overexpression of EGFR.PartⅢEstablishment of Orthotopic Transplantation model of Metastatic Carcinoma Masses of Human Brain in Nude MiceObjective To establish an orthotopic transplantation model of brain metastasis of human lung adenocarcinoma in nude mice and analysize its biological features.Methods Human brain metastatic tumor masses were collected and orthotopicly transplanted into the right caudate nucleus of nude mice. The newly formed xenografts were sharply minced into pieces and re-transplanted. The efficiency of tumorigenicity and the survival time of tumor-bearing mice were observed. HE staining and MRI scanning were performed to investigate the histopathological and imaging features of this metastatic lesion. The expression of tumor marker (CEA) and mucin in xenografts was also investigated by immunohistochemical or Alcian blue/PAS assays.Results The brain xenografs were passed into 65 nude mice to the 6th generation in the right caudate nucleus of nude mice,with average survival time of tumor-bearing mice 47.6±1.8d for primary generation and 38.0±0.9d for 4-6 generation;Histopathologically, the cerebral xenografts were poorly differentiated andenocarcinoma without invasiveness to normal brain tissue. MRI revealed a well-delimitated mass without surrounding edema, which perfectly recapitulated the original tumor. Expression of CEA and acid mucin was positive in the cerebral xenografts.Conclusions The orthotopic xenografts of brain metastasis from human lung andenocarcinoma could faithfully recapitulate the original tumor clinically and pathologically, and provides a reliable animal model not only for studying the biological features but also for experimental treatment of brain metastasis from lung adenocarcinoma.PartⅣPrimarily Investigation on the Human Glioma Stem Cells Transplanted in the Nude Mice with GFP ExpressionObjective To incubate nude mice which could stably express green fluoresent protein(GFP), then human glioma stem cells(GSCs)were transplanted intracranially into NC nude mice with GFP expression. Methods Transgenic C57BL/6J mouse expressing GFP was crossed with NC nude mouse, then the male nude mouse expressing GFP was crossed with the female pubescence mouse to obtain nude mouse with GFP expression, expression of GFP in the skin and organs of nude mice were evaluated under direct observation, by immuno histochemiscal and immunofluorescence assay. Accordingly GSCs were transplanted orthotopically into the brains of nude mice expressing GFP to observe the biological characteristics of the intracranial growth of the transplaned tumor. Results The organs and cells were all obviously expressed GFP in the nude mice propagated to 8th generation. Nonluminous tumour cells were distinguished from the host cell distinctly in the brains bearing tumor- transplanted orthotopically. Microanatomy relationship was clearly showed between the brain cells of host and tumour cells with immunohistochemiscal assay, which stained the tumor cells with species-specific monoclonal antibody(HLA)coupled with red fluorescer(PE) through confocal microscopy. Conclusions The nude mice expressing GFP were obtained by crossing the trangenic mice bearing na?ve immunity with NC nude mice. Orthotopic transplantation of human brain glioma may be used in the investigation on the tumour tissue reconstitution for the identification between the transplantation tumor and host tissue .