Experimental Study On Induction And Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells Into Vascular Endothelial Cells

Objective 1.To discuss the experimental induction and differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells.2.To study the effect and significance of induction and incubation time on NO secretion of vascular endothelial cells.3.To discuss the feasibility of replacing rat with rabbit as the donors of bone marrow mesenchymal stem cells and receptors of autologous cells transplantation.Method 1.Selecting the New Zealand white rabbits at 3-4months to perform anesthesia operation.After the anesthesia was completed,using marrow puncture needle to insert into the femoral bone marrow cavity at the puncture point.Then to extract 3ml bone marrow fluid samples into centrifuge tube and mix it into bone marrow suspension.After centrifuging 5min,to remove the upper layer of fat and tissue fluid and collect the cells at the bottom of the tube.2.Primary culturing the harvested cell.To resuspend the harvested cells in PBS and add it to Percoll separation solution for centrifugation.Then to absorb the middle layer of milky white cells with thickness of 0.2cm and resuspend them with nutrient solution after washing.Counting cells and adjusting the cell concentration so that to inoculate cells in a plastic culture dish and culture in the incubator.To change fluid regularly and remove unadherent cells.3.Inoculating and culturing cells after the primary culture was completed.When the cell confluence reached 80%,discarding the nutrient solution and cleaning them three times.Then add 0.125% trypsin and observe the morphological changes of cells under the light microscope during digesting.To terminate the digestion when the cell gaps are enlarged and the cells are irregular.Collecting and centrifuging the cell suspension,then to resuspend cells in a culture medium containing cyan-streptomycin and inoculate and culture cells in a 1:2 ratio.4.Directionally inducing and differentiating bone marrow mesenchymal stem cells into vascular endothelial cells.To choose the third generation cells with stable cell traits and good growth status and clean them after the nutrient solution was discarded.Then to add the endothelial cell induction medium containing cyan-streptomycin,VEGF and b FGF,and regularly change the liquid.To continuously observe the changes of cell morphology under microscope.5.Authenticating with FCM(Flow cytometric method).To detect the expression of CD31 and v WF by using FCM in the induced culturing third-generation vascular endothelial cells.After the cells were digested and centrifuged,to resuspend cells in PBS.After the cell concentration was adjusted,to add the cells into 3 flow tubes and mark as 123.To respectively add PBS,CD31,and v WF into 123 tubes and then to identify with the machine.6.Testing NO secretion.To take the 5d,10 d and 15 d supernatant according to Griess reagent instructions and use EXCEL to draw the standard curve.The absorbance of the measured sample was substituted into the calculation of NO secretion and was shown as the mean plus or minus standard deviation.The paired sample t was used to compare the results,t=26.574?t(df)0.05,and the difference was statistically significant with P < 0.05.Result1.(1)The primary cells adhered to the wall and grew after 4h inoculation.The growth of cell colony was observed after 48 h.The cells are fusiform and triangular,and the clustered cells are meshed and swirled after 10-12 d.The inoculated cells grew rapidly and are arranged evenly in a long spindle after 5-6d.After adding endothelial cells induced culture medium for 48 h,it was observed that the cells were transformed into short fusiform under microscope.The cell clusters grew and were arranged in a fish-like pattern after 10 d.The endothelioid cells showed like a paving stone,and the cell length increased and the morphology was flat after 20 d.(2)Flow cytometry detection results showing that the positive rate of endothelial-like cells CD31 is 97.3% plus or minus 0.8% and the positive rate of v WF is 95.7% plus or minus1.1%.(3)The testing results of NO secretion volume from the 5d,10 d and 15 d supernatant showing that the volume of NO secretion in the 5d supernatant was(31.25±2.67)?mol/L,in the 10 d supernatant was(65.88±3.64)?mol/L and in the 15 d supernatant was(86.31±3.93)?mol/L.2.The paired sample t test was used to compare the results of the three NO secretion volume of vascular endothelial cells obtained from induced differentiation groups.According to it,The difference was statistically significant(P<0.05).NO secretion volume of 5d<volume of 10d<volume of 15 d.3.The same VEGF+b FGF inducing method was used in the experiment and rabbit vascular endothelial cells were successfully obtained.The results were consistent with the experimental study of rats.Conclusion1.Rabbit bone marrow mesenchymal stem cells can be induced and differentiated into vascular endothelial cells by VEGF and b FGF.2.During the inducing and differentiating,the activity of vascular endothelial cells increased gradually with culture time in 15 days which make the NO secretion volume increased.3.The rabbit can replace rat as the donors of bone marrow mesenchymal stem cells and receptors of autologous cells transplantation.Mesenchymal stem cells can be induced and differentiated into vascular endothelial cells which can provide seed cells for autologous cells transplantation.