The Research Of HUCMSCs Transfected By VEGF Gene Were Induced To Neuronal-like Cells

ObjectiveTransfect VEGF to hUCMSCs, detects the change of ability of the transfected hUCMSCs indction-differentiating to neuroid cells.MethodsDivide the fourth generation purified hUCMSCs into four groups, cell groups of routine culture; make transfection of the test group without transfecting plasmid; transfect PEGFP-N3 plasmid group; transfect GFP-VEGF plasmid group, use Lipofectamine method to transfect VEGF in PEGFP-N3 into hUCMSCs. Then use Real-time PCR to detect VEGF expression, taking GAPDH as internal reference, make analysis of relative expression quantity. Western blot testing VEGF protein expression, take GAPDH as internal reference, make analysis of protein relative expression quantity, observe the influence of VEGF expression on hUCMSCs. Use the orient revulsion, takingβ-mercaptoethanol as inducer, directionally induce the transfected hUCMSCs into nerve cells, and observe the change of ability of the transfected hUCMSCs to induce to neuroid cells with viewpoint of cytomorphology and using the nerve marking (NSE, Nestin, GFAP) test method.Results1. The result of RT-PCR shows the expression quantity of VEGF in hUCMSCs-VEGT cells is distinctive from the other three groups of cells p< 0.01, however, compared with it, the un-transfected cells, vainly transfected cells, transformed empty plasmid cells have no clear difference.2. Western blot test result shows the VEGF protein expression quantity in the un-transfected cells, vainly transfected cells, transformed empty plasmid cells and transfected VEGF cells has changed clearly; the VEGF expression quantity of the untransfected cells, vainly transfected cells, transformed empty plasmid cells and transfected VEGF cells has clearly risen(p<0.01).3. Cytomorphology observe after induction, use the orient revulsion, takingβ-mercaptoethanol as inducer directionally induce the transfected hUCMSCs into nerve cells, the un-transfected cells taking the shape of long shuttles or form of fiber cells; having been induced, the sytomophology appears distinctly changed the cells contracting, gradually transform to nerve cells, induced after 6h, most of cells are transformed into typical neuroid cells, having been induced for 24h the cells are contracted in the shape of come, triangle or irregular shape, the performance of refracting light is good, the ends of some long apophyses form the shape of long cone or filiform.4. Pass through immune histochemistry experiment, identify the nerve cells differentiated through inducing, complete the test of the marks of NSE, Nestin and GFAP respectively, discover the chromosome of NSE and Nestin has positive result, having fluorescence, GFAP chromosome and the control group have no positive result, quantitative counting analysis, NSE positive cells are about (43.25±4.1)%, Nestin positive cells are (38.63±3.6)%.ConclusionsVEGF induced into hUCMSCs can be expressed stably, after inducing, clear change of neuroid cells occurs to hUCMSCs-VEGF and the ability of hUCMSCs transfected by VEGF to induce and differentiate to neuroid cells is not affected distinctly.