Liposome Mediated Green Fluorescent Protein Transfect Into Human Endothelial Progenitor Cells Derived From Cord Blood

Background and ObjectiveThe endothelial progenitor cells (EPCs) is a kind of precursor cell that can differentiate into vascular endothelial cells. They can be derived from marrow, peripheral blood and cord blood also. In 1997, Asahara and colleagues published that purified CD34+ hematopoietic progenitor cells from adults can differentiate ex vivo to an endothelial phenotype. And it is proved that EPCs are related with angiogenesis. After this, it is found that basic fibroblast growth factor and vascular endothelial growth factor can induce stem cells into endothelial cells in vitro and show expression of various endothelial markers. For the role of EPCs played, the assumption of transplant EPCs into human's body to treat diabetes retinopathy (DR) that provide a new idea.EPCs transplantation research has been a more popular topic, but EPCs transplantation is still in the experimental stage, only for the treatment of ischemic disease, repair of vascular injury and tissue engineering. Recently, Au and colleagues found in adult peripheral blood endothelial progenitor cells in the 3 weeks will be degraded, and in umbilical cord blood endothelial progenitor cells to maintain the normal function of more than four months. As a result, EPCs derived from cord blood can be a great source of seed cells.In order to confirm the effect of transplantation for DR and vascular lesions treatment, it is necessary to mark EPCs before they transplant into body for tracking transplanted EPCs. For the reason that markers in human body will stay a long time, and the marker of EPCs after transplantation into animals, it is required a label which will be able to long-term expression of host cells, low toxicity and easy to observe in the transplanted EPCs.Green fluorescent protein (GFP) is one of markers which is most commonly used in biology and histology. GFP is found in a kind of jellyfish named Aequorea Victoria earliest by Chalfie and colleagues when they researched luminous phenomena of jellyfish. Because of their fluorescence stability, to facilitate detection of living cells without damage, etc., have been used as a marker gene (Marker Gene) widely applied to various fields of biology.In recent years, as the development of molecular biology and genetic engineering, foreign genes into eukaryotic cells growing through transfection of host cells become increasingly more common method of marking. This article will introduce a measure that liposomes (Lipofectamine2000)-mediated green fluorescent protein transfered into endothelial progenitor cells. The aim is to explore a new approach to tag EPCs for transplantation and provide basis for the future experimental and clinical application. At the same time, it is hoped that the transplantation EPCs marking through this method will be used to demonstrate the role of vascular lesions clearly, to solve ischemia and hypoxia of retinal and to eliminate the pain of patients on physical and psychological.Methods1. Isolation, induction and culture of endothelial progenitor cells from cord bloodCollected 40ml umbilical cord blood from newborn under sterile conditions and preserved with 5.6ml Sodium Citrate. MNCs were isolated from cord blood by Percoll, and then induction of EPCs with VEGF, bGFG and other factors. Culturing in Medium 199 (M199) with 20% Fetal Calf Serum (FCS). After 3 days, replaced M199 half in quantity and then replaced all of M199 every 3 days.2. Liposome-mediated transfection of human endothelial progenitor cells from cord blood with green fluorescent proteinTransfect cells in 14th day after the start to culture. Setting up three groups: blank group, control group and transfected group. There was no pEGFP-N1 and Lipofectamine2000 in blank group, the control group only contained pEGFP-N1 and the transfected group used liposome called Lipofectamine2000 mediated pEGFP-N1 which carrying green fluorescent protein gene into cells. Before transfection, sucked M199 from wells of transfected group and washed cells twice with DMEM which contains no serum and antibiotic. In two EP tubes, dilute DNA and Lipofectamine2000 to DMEM separately. Incubate for 5 min at room temperature. Mix two dilutions gently and incubate at room temperature for 20 min to allow DNA- Lipofectamine2000 complexes to form. Add the DNA- Lipofectamine2000 complexes directly to each well of the plates containing cells and mix gently. Incubate for 5 hours at 37℃in couveuse and then replaed M199 and replaced medium 3 days once. Sucked M199 from wells of control group and wash cells twice with DMEM. Add DNA dilution to wells.3. Changes of fluorescence intensity expressed in cellsFive hours after transfection, replaced M199 contains serum, growth factors and antibiotics in control group. And then, observed expression of fluorescence with the laser scanning confocal microscope in wavelength 488nm. Decompose cells after transfection at 24h, 48h, 72h, 96h and then collect them. Determinate green fluorescent protein ray absorption value (OD) released from cells to reflect fluorescence intensity expressed in cells.4. Changes in number of cells before and after transfectionSix hours after transfection, digest cells and compared with ray absorption value of transfected group non-transfected group (control group) cells using by MTT method to reflect differences in the number of cells.5. Determination of transfection rateDetermine transfection rate using by flow cytometry in 48 hours after transfection. 6. Data Analysis Calculate data by SAS9.1 software. Data has statistical significance when P <0.01.ResultsThere is no expression in blank team and control team at 24h after the start of transfection, and transfection froup had already shown the expression of green fluorescent at same time. At 48h after the start of transfection, fluorescence intensity ratio was higher than 24h. And then, at 48h, 72h and 96h the fluorescent intensity ratio gradually weakened. The number of transfected cells has decreased. Transfection rate is 21.6%±1.3%.Conclusions:1. Liposome can be mediated green fluorescent protein transfect into endothelial progenitor cells derived from cord blood.2. Green fluorescent protein can be expressed in primary endothelial progenitor cells.3. Cells decrease in the number after transfection.