Study On The Endothelial Progenitor Cells Modified With Gene And Three Dimensional Culture In Vitro

Endothelial Progenitor Cells (EPCs) are endothelial precursors that have an enhanced potential for differentiation into the endothelial cell lineage. EPCs are recruited into injured tissues and contribute to vasculogenesis and angiogenesis by directly incorporating into vessel walls or by secreting a variety of angiogenic growth factors. Subsequently, EPCs transplantation has been widely used in treatment of non-curable ischemic diseases and construction of tissue engineered vessels. To overcome the drawbacks of conventional cell transplantation methods and improve the quality of EPCs grafted into the injured sites. A variety of biosynthetic scaffolds have been developed as EPCs carrier for cells in vitro expension and in vivo tansplantation.In our study, EPCs obtained from rat bone marrow mononuclear cells(BMMNCs) were cultured in special medium. The constructed recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor(TFPI) and GFP was packed into TFPI retrovirals. and infected EPCs successfully. Biological compatibility of the poly (D, L-lactide) (PDLLA) and the RGD modified PDLLA porous microspheres scaffolds were assessed by in vitro culture of EPCs and cervical cancer cells. The main content of this research are shown as follows. To establish an efficient and stable method for obtaining endothelial progenitor cells from rat bone marrow. Bone marorw mononuclear cells were isolated from rat bone marrow of femurs and tibias by Histopaque-1083 density gradient centrifugation. Then were cultured in endothelial basal medium(EGM-2) supplemented with vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, ascorbic acid. The form of colonis of EPCs was observed under inverted microscope. The function of EPCs were identified through uptaking DiL-acLDL and binding FITC-UEA-1. The expressions of surface markers (CD133,flk-1,CD31,v-WF) were analyzed by immunofluorescence and Fluorescence-activated cell sorting.The adhesive cells formed clusters after 4 days plating. These colonies proliferated rapidly and exhibited spindle-shaped morphology. Twelve days after plating most colonies had reached near confluence and differentiated into cobblestone-shaped cells. The majority of the cells expressed endothelial markers of CD31 and v-WF After 14 days culture by immunofluorescence detection. Furthermore, These cells were positive for Dil-acLDL incorporation and UEA-1 lectin binding after 14 days culture. Fluorescence-activated cell sorting (FACS) detection demonstrated that the expression of the early markers of EPCs (CD133,Flk-1) increased gradually after 3d,7d culture.Then decreased after 14 days culture. The expression of the late markers of EPCs (CD31) was detected highly increased after 14d culture.To construct the recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor and GFP in rat endothelial progenitor cells (EPCs). Full length TFPI cDNA obtained from pIRES-TFPI by PCR amplification was digested with EcoRI and Xhol restriction enzymes and subsequently inserted into pMSCV-IRES-GFP expression vector to create the recombinant bicistronic retroviral vector pMSCV- TFPI- IRES -GFP encoding both TFPI and GFP. The recombinant plasmid was identified with restrictive endonuclease digestion and DNA sequencing. The recombinant plasmid was transfected into 293T and the supernatant containing packaged recombinant retroviral particles was collected and used to infect the EPCs isolated from rat bone marrow. TFPI mRNA was measured by RT-PCR and the amount of TFPI protein secreted from the transfected cells was determined by ELISA. GFP expression in the infected cells was analyzed by fluorescent microscopy and fluorescence activated cell sorting (FACS). Restriction endonuclease mapping and DNA sequencing confirmed the in-frame insertion of TFPI cDNA into the constructed vector-pMSCV-TFPI-IRES-GFP. Both RT-PCR and ELISA analysis demonstrated increased TFPI expression in the EPCs infected with TFPI retroviral. FACS analysis demonstrated that the transduction efficiency of EPCs with TFPI retroviral in vitro was over 90%. pMSCV-TFPI-IRES-GFP could be effectively expressed in cultured EPCs and may provide a useful tool for further study on the application of TFPI in the prevention of restenosis.Solvent-evaporation method was modified using sucrose as an additive to form large porous microspheres of PDLLA polymers.Then porous PDLLA microspheres were modified by RGD via a method of hydrolysis and grafting-coating to improve their compatibility to cells. Surface morphology of PDLLA microspheres before and after hydrolysis was visualized by scanning electron microscopy. The SEM images of PDLLA microspheres revealed that there was a highly open porous structure between the surface and the internal.There are more porous on the hydrolysis microspheres than untreated microspheres.Modified microspheres were characterized for surface chemistry by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR). FT-IR spectra exhibited characteristic peaks of amide at 3430cm-1 and 1630cm-1, XPS spectra shows the N 1s (atomic orbital 1s of nitrogen) region of the surface of the microspheres.Biological Compatibility of the control PDLLA and the RGD modified porous PDLLA microspheres were assessed by in vitro culture of endothelial progenitor cells. The morphology and distribution of cells on the microspheres were observed under SEM,inverted fluorescence microscopy after cultured for 1d,7 d. Cell viability was measured by MTT assay.RGD modified PDLLA microspheres exhibited stronger ability to promote EPCs attachment and proliferation after 24 h,3d,7 d culture compared to unmodified microspheres. Morever, EPCs on RGD modified microspheres maintain the function of uptaking DiL-acLDL and binding FITC-UEA-1. Scanning electron microscopy also revealed copious amount of extra-cellular matrix production by EPCs.Therefore, the RGD-coated PDLLA microspheres can be potentially used as the injectable cell microcarriers for EPCs in the treatment of Ischemic disease and tissue engineering vessels.