Establishment And Pathological Observation Of Nervous System Tumor Model For Nestin-positive Reporter Mouse

BACKGROUNDIncidence of human primary central nervous system tumors, which are composed of intracranial tumors(85%) and intraspinal tumors(15%). is about2to5/100.000. Glioma. a most common intracranial tumor, accounts for about40%intracranial tumors. Malignant astrocytoma (glioblastoma) accounts for50%to60%of gliomas. Incidence of intracranial tumor in children also is very high, just below the leukemia, accounting for the second in malignant tumors of the children. In addition to glioma. medulloblastoma is the most common in the intracranial tumor of children.Nervous system tumors can exhibit the following symptoms:1.Surrounding brain tissue compressed or damaged by tumor caused local neurological symptoms, such as epilepsy, paralysis, visual field defect.2. Intracranial lesions cause the symptoms of increased intracranial pressure, such as the performance of headache, vomiting, and papilledema.Nestin. a type of intermediate filament protein, specifically expressed in neuroepithelial stem cells, is one of the characteristic markers for neural stem cells. Nestin-GFP transgenic reporter mouse is the most commonly tool mouse which is used to track neural stem cells. In nestin-GFP transgenic reporter mouse, the reporter gene GFP was inserted in the downstream of nestin promoter and regulated by the nestin gene. Nestin-GFP transgenic reporter mouse can be used to label and track differentiation and migration of neural stem cell.A great progress has been achieved in the field of nervous system tumors. However, the origin and evolution of nervous system tumors are still not well understood. Most patients with nervous system tumors can not be diagnosed at an early stage. It is very difficult to get specimens of nervous system tumor at the early stage. Therefore, establishment of mouse models for nervous system tumors is very helpful to find the early markers for nervous system tumors and study the molecular mechanisms for origin, evolution and progression of nervous system tumors. A nervous system tumor model for nestin-positive mouse provides the important tool to study the origin and evolution of nervous system tumor.Here, we first introduced a nestin-GFP transgenic reporter mouse in our study and observed the characteristics of nestin positive cells in brain and pancreas. Then, we try to induce some kinds of nervous system tumor in nestin-GFP transgenic reporter mice with ENU. Finally, we produced a medulloblastoma model of nestin-positive mouse and preliminarily study the evolution for medulloblastoma.Methods:1. Characteristics of nestin positive cells in brain and pancreas in nestin-GFP transgenic reporter mice(1) The strategy for generation of nestin-GFP transgenic reporter mice;(2) Dynamic changes of nestin positive organ in embryos or early stage of nestin-GFP transgenic reporter mice by whole-body fluorescent imaging system;(3) Characteristics of nestin positive cells in brain of the adult nestin-GFP transgenic reporter mice by immunohistochemical staining.(4) Characteristics of nestin positive cells in brain of the adult nestin-GFP transgenic reporter mice by immunofluorescence.2. Types and morphological changes of tumors induced by ENU in nestin-GFP transgenic reporter mice.(1) Tumors were induced transplacentally by ENU ENU was administrated intraperitoneally at8doses respectively (50mg/kg,83 mg/kg,167mg/kg,200mg/kg,233mg/kg,250mg/kg,267mg/kg and300mg/kg.weigh) into nestin-GFP transgenic reporter mice at the end of pregnancy. One hundred and ten offspring were produced and feeded alone according to sex distribution. These mice were observated carefully to examine sleep, appetite, gait, body temperature, spirit, body weight, hair color.(2) Tumors were induced intraperitoneally by ENUENU was administrated intraperitoneally at a dosage of200mg/kg weigh and feed alone at each cage. Symptoms of these mice including sleep, appetite, gait, body temperature, spirit, body weight, hair color, were observated carefully. The mice had cclinical symptoms or near death were killed and dissected for further analysis. All specimens were fixed, dehydrate, embedded in paraffin slice, and stained with HE. Neoplastic lesions were found by gross examination and histological characteristics under the light microscopy.3. A medulloblastoma model of nestin-positive mouse was established.(1) Nestin-GFP transgenic reporter mice were crossed with Ptch-/+mice to produce nestin-GFP-/+/Ptch-/+mice.(2) Identification of genotypes for Ptch gene by genomic polymerase chain reaction.(3) Morphological changes of brain were observed in nestin-GFP-/+Ptch-/+mice.Results1. Characteristics of nestin positive cells in brain and pancreas in nestin-GFP transgenic reporter mice.(1) Whole-body fluorescent imaging in nestin-GFP transgenic reporter mice showed that nestin was mainly expressed in the cerebrum, cerebellum and olfactory bulb. And, nestin expression of the cerebrum and cerebellum decreased significantly over three weeks after birth. However, nestin expression of olfactory bulb was not significantly changed even over three weeks after birth.(2)Whole-body fluorescent imaging in nestin-GFP transgenic reporter mice also indicated that nestin was strongly expressed in the pancreas at different developmental stages of mice.(3) Immunohistochemistry and immunofluorescence results showed that nestin positive cells were mainly located in ventricle ependymal cells and choroid plexus cells in the brain of nestin-GFP transgenic reporter mice. Interestingly, a small number of nestin positive cells were scattered in cortex, medulla, hippocampus and cerebellum of the brain.2. Types and morphological changes of tumors induced by ENU in nestin-GFP transgenic reporter mice.(1)Many types of tumor were induced by ENU in nestin-GFP transgenic reporter mice, including1cerebral cavernous hemangioma at about8months,22lung adenomas at about11months.24lung adenocarcinoma at about11months,5lymphoma at about7months (2Burkitt-like lymphoma,3Diffuse Large B-cell-like lymphoma) and1ovarian tumor at about14months.(2)Tumor formation rate in nestin-GFP transgenic reporter mice induced transplacentally by ENU at dose of50mg/kg,83mg/kg,167mg/kg,200mg/kg,233mg/kg,250mg/kg,267mg/kg and300mg/kg was respectively22.2%,23.1%,35.3%,38.9%.70.6%.85.7%,87.5%,87.5%.(χ2=25.300, P<0.001)(3)Thymic lymphoma was induced intraperitoneally by ENU in adult nestin-GFP transgenic reporter mouse at a dosage of200mg/kg. And. tumor formation rate was46.2%.3. A medulloblastoma model of nestin-positive reporter mouse was established.Nestin-GFP-/+/Ptch-/+mice were successfully established by crossing nestin-GFP transgenic reporter mice with Ptch-/+mice. Medulloblastoma was developed about10months later in nestin-GFP-/+/Ptch-/+mice. Whole-body fluorescent imaging in nestin-G-/+Ptch-/+mice showed that nestin was strongly expressed in medulloblastoma developed in the mice. Abnormal proliferation of external granular layer (EGL) cells was found in the cerebella of mouse at the early stage of medulloblastoma formation.4.Morphological dynamic observation of medulloblastoma model of nestin-positive reporter mouse at different stagesWe found that firstly the cells of the lateral ventricle proliferated and had strong nestin expression at the early stage of medulloblastoma formation, and then external granular layer(EGL) cells proliferated and had strong nestin expression at the middle stage of medulloblastoma formation, finally tumor cells had strong nestin expression after tumor formation at the later stage of medulloblastoma formation in medulloblastoma model of nestin-positive reporter mouse.Conclusion1. Most of nestin positive cells located in subependymal ventrical zone (SVZ) of the brain and a few nestin positive cells scattered in other parts of the brain. Nestin-GFP transgenic reporter mice can be applied to trace and analyze neural stem cells.2. Lung adenomas, lung adenocarcinoma, lymphoma, brain cavernous hemangioma, ovarian tumor can be induced by ENU. No nervous system tumor can be induced chemically with ENU.3. A medulloblastoma model of nestin-positive mouse was successfully established. Nestin was strongly expressed in medulloblastoma developed and abnormal proliferation of external granular layer (EGL) cells in the cerebella may be the early changes of medulloblastoma formation.4.There was proliferation of neural stem cells in the lateral ventricle initially at the early stage of medulloblastoma formation, and then proliferation of neural stem cells in the lateral ventricle led to the proliferation of external granular layer(EGL) at the middle stage of medulloblastoma formation, and finnly the proliferation of cerebellar external granular layer(EGL) led to the tumor formation at the later stage of medulloblastoma formation.