Biological Characteristics Of Malignant Tumor Vascular Endothelial Cells

Part 1 Sorting and identification of the characteristics of stem cells in CD133~+and CD133~-tumor cellsObjective:To investigate sorting and stem cell properties CD133~+tumor cells and CD133~-tumor cells.Methods:Fisrstly,FCM detected the expression of CD133~+cells in A549,PLC,HT-29 and PCC.CD133~+and CD133~-cell,sorted by anti-CD133microbeads,were detected the expression of CD133.Then,sphere formation assay,MTT assay and colony formation assay detected the stem cell properties between CD133~+and CD133~-cells.Finally,nude mice tumorigenicity assay detected the tumorigenicity between CD133~+and CD133~-cells in vivo.Results:FCM results demonstrated that A549,PLC,HT-29 and PCC expressed CD133.After sorting by anti-CD133 MACS kits,FCM results demonstrated that CD133~+cells from A549,PLC,HT-29 and PCC expressed high level of CD133,while CD133~-cells did not express CD133.Sphere formation assay indicated that CD133~+cells formed more microsphere than CD133~-cells.MTT assay indicated that CD133~+cells took more cell proliferation ability compared to CD133~-cells.Colony formation assay indicated that CD133~+cells took more colony formation ability compared to CD133~-cells.Nude mice tumorigenicity assay indicated that CD133~+cells took stronger tumorigenicity compared to CD133~-cells.Conclusion:1.CD133~+and CD133~-cells were successfully sorted.2.The results demonstrated that CD133~+cells took stem cell properties,while CD133~-cells did not have the characteristics of stem cells.Part 2 CD133~+and CD133~-tumor cells give rise to tumor endothelial cellsObjective:To investigate CD133~+and CD133~-tumor cells give rise to endothelial cells(h CD105~+cells).Methods:Firstly,FCM detected the expression of h CD105 in CD133~+and CD133~-cells.Then,after induction of endothelial cell or Xenografts,FCM detected the expression of h CD105 between CD133~+and CD133~-cells.FCM detected the expression of h CD105 between CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs after anti-human CD105 magnetic beads sorting.Next,q RT-PCR assay and immunofluorescence assay detected the expression of h CD105 in CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs.Endothelial Dil-ac-LDL uptake assay and Matrigel tube formation assay detected the endotheial function in CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs.Finally,FISH assay detected the source of CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs.Results:FCM results demonstrated that CD133~+and CD133~-cells did not express h CD105.After endothelial cell induction or Xenograft,FCM results demonstrated that CD133~+and CD133~-cells expressed h CD105.After anti-human CD105 magnetic beads sorting,FCM results showed that CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs expressed about99%of h CD105~+cells.q RT-PCR results indicated that CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs expressed m RNA of h CD105.Immunofluorescence suggest that CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs expressed the membrane surface protein h CD105.Endothelial Dil-ac-LDL uptake assay indicated that CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs had endothelial cell function.Matrigel tube formation assay indicated that CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs had endothelial cell function.FISH assay demonstrated that CD133~+i ECs,CD133~-i ECs,CD133~+x TECs and CD133~-x TECs had gene duplication.Conclusion 1.Compared to CD133~+cells,CD133~-cells also had the potential to give rise to tumor endothelium.2.i ECs and x TECs sorted by MACS had endothelial cell function.Part 3 Application of transgenic mouse models of investigation in tumor vascular endothelial cellsObjective:To investigate tumor vascular endothelial cell through transgenic mouse model(Pvillin-Cre/p CAG-i DTR-EGFP).Methods:Firstly,we constructed and identified of transgenic mice.Then,mouse colorectal cancer was induced and identified by AOM/DSS method.Then,immunofluorescence assay detected the expression of EGFP and c-myc in m CD105~+cells.FCM detected that expression of m CD105 in mice colorectal cancer tissues.After anti-mouse CD105 magnetic beads sorting,FCM detected the expression of m CD105 in m CC-TECs(mice Colorectal Carcinoma Tumor Endothelial cells).q RT-PCR assay and immunofluorescence assay detected the expression of m CD105 in m CC-TECs.Finnaly,endothelial Dil-ac-LDL uptake assay and Matrigel tube formation assay detected the endotheial function of m CC-TECs.Results:Identification of transgenic mice was successful.We successfully induced the colorectal cancer of transgenic mice.Immunofluorescence assay result indicated that CD105~+cells in the mice co-expressed EGFP and oncogene c-myc.FCM results showed that mice colorectal cancer tissues expressed m CD105.After anti-mouse CD105 microbeads sorting,FCM results showed that m CC-TECs expressed high level of m CD105.q RT-PCR assay indicated that m CC-TECs expressed m RNA of m CD105.Immunofluorescence assay suggest that m CC-TECs expressed the membrane surface protein m CD105.Dil-ac-LDL uptake assay indicated that m CC-TECs had endothelial cell function.Tube formation assay indicated that m CC-TECs had endothelial cell function.Conculsion:1.In primay tumors,tumor vascular endothelial cells derived from tumor cells.2.Mice priamy tumor cells had the potential to give rise to tumor endothelium.3.m CC-TECs obtained by MACS had endothelial cell function.Part 4 Characteristics of tumor vascular endothelial cellsObjective:To investigate the characteristics of CD133~+x TECs,CD133~-x TECs and m CC-TECs.Methods:Invasion assay and wound healing assay detected the invasive ability of CD133~+x TECs,CD133~-x TECs and m CC-TECs in vitro.Nude mice tumorigenic assay detected the tumorigenicity of CD133~+x TECs,CD133~-x TECs and m CC-TECs.HE staining detected the tumor characteristics of CD133~+x TECs,CD133~-x TECs and m CC-TECs.Results:Invasion assay results suggested that CD133~+x TECs,CD133~-x TECs and m CC-TECs had the potential of invasiveness.Wound healing assay results suggested that CD133~+x TECs,CD133~-x TECs and m CC-TECs had the potential of migration.Nude mice tumorigenic assay suggested that CD133~+x TECs,CD133~-x TECs and m CC-TECs had the potential of tumorigenicity.HE staining results suggested that tissues derived from CD133~+x TECs,CD133~-x TECs and m CC-TECs were consistent with tissues derived from tumor cells.Conclusion:CD133~+x TECs,CD133~-x TECs and m CC-TECs,which expressed endothelial cell marker,had similar tumorigenic poperties with tumor cells.