Study On Fluorescent Tracing Roles Of GEP In Green Nude Mice

Part I The expression of EGFP in the brain tissues of "green nude mice"【Objective】 To analyze the expression levels of enhanced green fluorescent protein(EGFP) in brain tissues of EGFP transgenic mice for providing the basis of green nude mice brain transplantation.【Methods】 The EGFP m RNA expression level in various organ tissues/cells and different parts of the brain of Foxn1 nu.B6-CAG-EGFP|SU nude mice were analyzed by semi-quantitation RT-PCR method. Using fluorescence microscopy, the green fluorescence in main organ tissues of whole body and 12 brain coronal frozen tissues were direct observed to survey the pattern of EGFP expression. Further, the EGFP expression in different parts of brain was detected by quatitative PCR(q PCR), fluorescence spectrophotometer assay and immunohistochemistry. After intracranial inoculating in green nude mice with glioma stem progenitor cells SU3-RFP which expressed red fluorescent protein expression, the transplanted tumor in mice brain were observed by in vivo inmaging and fluorescence spectrophotometer.【Results】 The level of EGFP expression varied among main organs tissues/cells and different parts of the nude mice brain analyzed by RT-PCR. The green fluorescence could be observed in 12 coronal frozen sections of brain with intensity difference under the fluorescence microscope. Compared with cerebral cortex tissue, the EGFP expression levels in cerebellum, hippocampus and olfactory bulb tissues were higher significantly(p <0.05), showed by q PCR and fluorescence spectrophotometer assay. Meanwhile, the immunohistochemistry results showed that there were some cells which expressed EGFP strongly in the cerebellum, hippocampus and olfactory bulb tissues. Intracranial inoculating in green nude mice with SU3-RFP, the red xenograft was observed under the green brain backgroud by in vivo imaging and fluorescence spectrophotometer assay. It indicated that the red and green bi-color fluorescent tracer model was established successfully.【Conclusions】 Our results show that EGFP is positive expressed in the brain tissues and cells of green fluorescence nude mice, which indicates that the brain may prove useful for human cancer transplantation research as other organs with high EGFP expression.Part II Preliminary study on malignant transformation of host cells from bone marrow in dual color fluorescence tracer xenograft model【Objective】 To investigate the malignant transformation of the host bone marrow stromal cells in tumor microenvironment using the dual color fluorescence xenograft model.【Methods】 Based on the green fluorescent tracer marrow rebuild model, the red and green fluorescent tracer xenograft model was established by inoculating glioma stem progenitor cells which expressed RFP in the brain of nude mice. The tissues were isolated from the xenograft and cultured in vitro, and then separated monoclonal green fluorescence cells(named as ih BTCBM) which could be serially passaged. The biological characteristics of the cells were analyzed, which consists of drewing the cell growth curve with CCK-8 method, analyzing the chromosome karyotype used conventional method, inoculating ih BTCBM cells subcutaneously in nude mice to observe its tumorigenicity, and detecting the expression of Sca-1, CD29 and CD44 with immunofluorescence staining methods.【 Results 】 The above collected GFP+ cells(ih BTCBM) were performed monocloning, which possessed phenotype of cancer cells when cultured in vitro. The cells were confirmed unlimited proliferative capacity in vitro cultivation by CCK-8 assay. The chromosome karyotype analysis showed that the cells were aneuploidy. Inoculated subcutaneously in nude mice, the tumorigenicity rate of ih BTCBM was 100%(6/6). The results of immunofluorescence staining showed that ih BTCBM cells could express bone marrow mesenchymal stem cells marker proteins Sca-1, CD29 and CD44.【Conclusions】 By the EGFP fluorescent tracing, the ih BTCBM cells were separated from the xenograft tissues in double-color fluorescent tracer model. The cells could be serially passaged and might be malignant transformation of host cells developmented from the bone marrow mesenchymal stem cells.