Research On Building And Mechanism Of Tumor Blood Vessels In The Brain Glioma Model

The first part: research on cell fusion of the tumor vascularization in thehuman brain glioma model with dual-color fluorescent protein tracerObjective: Tumor vascularization was considered to be an important phenomenon of biology in the process of tumor angiogenesis and development and an important factor to determine the fate of diseases.It had been confirmed that the human brain glioma stem progenitor cells could participate in tumor vascularization in vivo and in vitro,but specific mechanism was unknown.It’s purposed to explore that cell fusion between the human brain glioma stem progenitor cells and host cells was an important way of tumor vascularization.Methods: The author established the human brain glioma stem progenitor cells SU3-RFP expressed red fluorescent protein(RFP) with transgenic technology and host cells expressed green fluorescent protein(GFP) in the orthotopic transplantation of human brain glioma model.With fluorescence microscopy and fluorescence confocal scanning laser microscopy, we could observe blood vessels and vessel tissue of the normal host brain tissues and transplanted tumor tissues with slicings.Results: In normal brain tissues, it’s visible to all levels of blood vessels as same as injection of vascular contrast with tabletting methods.According to the organization,it could be determined the morphological features of blood vessels approximately.In tumor tissues, tabletting methodit was visible to the red tumor blood vessels, green host blood vessels, yellow fusion blood vessels of tumor cells and host cells with tabletting methods.With fluorescent confocal scanning laser microscopy,it could be analyzed the organizational structure of the blood vessel walls.Both endothelial cells and smooth muscle cells in the tumor tissues it was observed fusion cells of the green host cells and red tumor cells.Conclusion: In the genetically modified fluorescent protein tracer glioma model,it could be observed host blood vessels,tumor vascularization and fusion vessels of of tumor cells and host cells in tumor tissues during glioma vascularization.In the fusion blood vessels, it could be distinguished red tumor cells and green host cells in Mosaic blood vessels,at the same time,it could be observed the fusion cells in the host tissues.It was suggested that cell fusion was an important way of tumor angiogenesis.The second part:studies in the vascularization of tumor cells fusion of bone marrow mesenchymal stem cells and glioma stem progenitor cellsco-culture in vitroObjective: It could be obtained bone marrow mesenchymal stem cells(MSCs)expressed green fluorescent protein(GFP) from nude mice.The author cultured mesenchymal stem cells in vitro and identified its biological characteristics.Then with transfectied red fluorescence protein(RFP) gene of the human brain glioma stem progenitor cells SU3- RFP co-culture in vitro,it was investigated cell fusion of the two kinds of cells promoted action of tumor vascularization.Methods: MSCs from transfectied green fluorescent protein(GFP) gene nude mice were cultured in vitro, it was observed and identificated with CCK-8, colony experiments,chromosome,Rt-PCR,immunocytochemistry and flow cytometry technology.Co-cultured transfected red fluorescent protein(RFP) gene human brain glioma stem progenitor cells SU3- RFP and MSCs from transfected green fluorescent protein(GFP) gene nude mice in vitro, it was observed cell proliferation characteristics with fluorescence microscopy.With CCK-8, Rt-PCR,immunocytochemistry,flow cytometry technology and transdifferentiation experiments,it was studied the biological characteristics and tumor vascularization of fusion cells.Results:In vitro continuous subculture 5 generations,high proliferation activity of MSCs was remained unchanged basically.MSCs expressed CD105 and did not express nestin/CD31.Its biological and genetic characteristics were stable.With MSCs andSU3-RFP cells co-culture,The number of RFP+/GFP+ yellow cells increased significantly.With flow cytometry instrument filter purification,RFP+/GFP+yellow fused cells were accounted as 83.7%±4.5%.The proliferation characteristics were consistent in a long time.With Rt-PCR,immunohistochemistry and flow cytometry technology,it showed yellow cells were fused cells rather than two cells ’ fluorescence overlap and could transdifferentiated into endothelial cells and promoted tumor vascularization in vitro.Conclusion: The host bone marrow mesenchymal stem cells MSCs keeped stable biological and genetic characteristics in the first five generations, with the glioma stem progenitor cells SU3-RFP co-culture in vitro,fusion reactions were occurred and the fusion cells could promote tumor vascularization.