VEGF₁₆₅ Expressing Bone Marrow Mesenchymal Stem Cells Differentiate Into Hepatocytes Under HGF And EGF Induction In Vitro

Part1The culture and biological characteristics of bone marrow mesenchymal stem cell and amplification of VEGF165plasmidAIM:1.To explore the optimal methods of isolating, culturing, proliferating, and freezing of bone marrow mesenchymal stem cell (BMSCs) in vitro, and investigate their biological properties.2.To amplificate and identificate VEGF165-pCMV6-AC-GFP plasmid.METHODs:BMSCs were purified by gradient centrifuge and adhesive culture methods. And being cultured and expanded in flasks. Using Flow cytometry and immunocytochemistry to identify BMSCs. Using optical microscopy and scanning electron microscopy to observe cell morphology and growth characteristics. Drawing the growth curves of1,3,5-generation BMSCs and3-generation BMSCs cultured with10%,15%,25%FBS. BMSCs were stored with10%DMSO in liquid nitrogen or-60℃refrigerator long term, and their survivability was determined.RESULTS:The BMSCs in cultured were uniform spindle-shaped in appearance. Flow cytometry showed that BMSCs had obvious expression of CD29(93.41%±3.2%,n=3) and CD44(91.55%±4.1%), rather than the expression of CD45(3.95%±2.9%) and CD34(3.36%±3.6%). Immunocytochemistry showed that BMSCs were positive for CD29and CD44, while negative for CD45and CD34. BMSCs had active proliferative capacity and had similar S-shape growth curves.1-3d was incubation period during which cells growed slowly, and entered a rapid growth after3d,7-8d was platform period during which the proliferation rate was leveling off.15%FBS and1-generation BMSCs had the best proliferation capacity. The cell viability of recoveried BMSCs after cryopreservation was more than90%, there were no change in the cell morphology and proliferation capacity compared with those in cryopreservation. BMSCs could be used in follow-up research. The purity and concentration of extracted VEGF165-pCMV6-AC-GFP plasmid was1.83,0.75ug/ul respectively, and can be applied to cells transfection in the next research.CONCLUSION:The methods of isolating, culturing, puring, expanding, and freezing of rabbit BMSCs are feasible. Th BMSCs can be cultured, expanded and cryopreserved stably in vitro and maintain their original biological properties. We have successfully amplificated high-purityVEGF165-pCMV6-AC-GFP plasmid, and can be applied to cells transfection in the next research. Part2Construction of VEGF165-pCMV6-AC-GFP gene-modified BMSCsAIM:1.To provide a viable method for BMSCs gene transfection by comparing the methods of liposome2000and electroporation, and establish the optimal transfection conditions.METHODs:1. Rabbit BMSCs were amplificated in vitro. The3-generation BMSCs were transfected with Lipofectamine2000and electroporation respectively. Cell morphology and GFP expression were observed. Growth curves were drawed and transfection efficiency was compared by flow cytometry.2.Optimizing transfection parameters, by selecting different transfection buffer (Opti-DMEM, DMEM containing serum and D-Hanks solution), by selecting different voltage (250V,280V,310V), by selecting different amount of plasmid (20ug,50ug,70ug) VEGF165-pCMV6-AC-GFP was transfected into BMSCs. Cell morphology and GFP expression were observed and transfection efficiency was compared by flow cy tome try.3. BMSCs were transfected under optimal parameters and selected by200ug/ml G418. VEGF165gene levels were detected at different times by RT-PCR.RESULTS:GFP fluorescence of BMSCs transfected by Lipofectamine2000was less than electroporation(P<0.05), the transfection efficiency was5.12%,17.3%respectively. The growth curve of BMSCs transfected by Lipofectamine2000was in the reduction type, while the growth curve of electroporated BMSCs remained S-shape. The transfection efficiency of Opti-DMEM, DMEM containing serum and D-Hanks solution was15.27%,10.53%,13.91%respectively. The cell viability of250V,280V and310V was75.6%,60.12%,42.59%respectively. The transfection efficiency of20ug,50ug and70ug was14.10%,27.44%,40.57%. As increased the voltage and plasmid amount, the numbers of dead cells also increased. Using200ug/ml G418select20days, the transfection efficiency was up to70.46%. The VEGF gene was tested by RT-PCR in BMSCs electroporated after48h, and there was a significant increase selected by G418after20days (P<0.05). After selection, there was a small decrease in expression with prolonged time, but there were no significant differences between post-selection afte14days and21days (P>0.05).CONCLUSION:Compared with Lipofectamine2000, electroporation is more applicable to transfection for BMSCs, which maintain cell morphology and proliferation. The best parameters of electroporation is2×106cells/ml,50ug plasmid, voltage280V, capacitance1000Uf, infinite resistance, once pulse. And electroporated BMSCs selected by G418can achieve higher transfection efficiency. Part3VEGF165Gene-modified BMSCs differentiate into hepatocytes under HGF and EGF induction in vitroAIM:To investigate the ability of VEGF165gene-modied BMSCs to differentiate into hepatocytes when cultured with hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in vitro.METHODs:Rabbit BMSCs were isolated, cultured and identified, then electroporated with VEGF165-pCMV6-AC-GFP plasmid. G418was used to select transfected cells and the efficiency was up to70%. The groups were divided as follows:Group A was electroporated with pCMV6-AC-GFP plasmid+HGF+EGF and Group B was electroporated with VEGF165-pCMV6-AC-GFP plasmid+HGF+EGF. On day10and20using light microscopy and scanning electron microscopy to observe cell morphology and surface structure, with immunohistochemistry, immunofluorescence and western-blot to detect the specific signs of liver cell:alpha-fetoprotein(AFP) and albumin (ALB), Real-time PCR to detect the level of ALB gene.RESULTS:After14days induction, BMSCs were induced into short spindle and polygonal. Polygonal cells increased with time prolonged, and hepatocyte-like cells formed colonies. Immunocytochemistry and immunofluorescence showed that the expression of AFP was positive and ALB was negative in Group A, while both AFP and ALB were positive in group B on the10th day. The expressions of AFP and ALB in both groups were positive on the20th day. Western-blot showed that the expression of ALB was earilier and more in group B than group A, whlie the expression of AFP decreased with prolonged time in group B and was less than group A on the20th day. Real-time PCR showed that the quantity of the ALB gene was increased with prolonged time in both groups. However, there was no significant difference between group A and B on day10and20.CONCLUSION:VEGF165-pCMV6-AC-GFP plasmid modified BMSCs still had the ability to differentiate into hepatocytes. The VEGF165gene promoted BMMSCs to differentiate into hepatocyte-like cells under the induction of HGF and EGF, and reduced the differentiation time. These results have implications for cell therapies.