The Effect Of Ad-GFP-NICD Transfection On The Arteriogenesis Of Human Umbilical Cord Derived-Mesenchymal Stem Cells

Objective:(1)To isolate, culture and identify the human umbilical cord-derived mesenchymal stem cells (hUCMSCs) and transfect hUCMSCs with the adenovirus vector expressing human Notch-1 intracellular domain (Ad-GFP-NICD). To observe the activation effect of Ad-GFP-NICD transfecting on the Notch signaling of hUCMSCs and provide basis for the relationship of Notch signaling and differentiation of hUCMSCs to arterial endothelial cells.(2)To investigate the effect of Ad-GFP-NICD transfecting on the proliferation, cell cycle and the differentiation toward endothelial cells of hUCMSCs and elucidate the mechaniam of hUCMSCs promoting arteriogenesis regulated by Notch signaling.Methods:Part 1 Collect the aseptic umbilical cord and isolate and culture the hUCMSCs by tissue adherent method. To observe the morphologic changes of adherent cells and identify hUCMSCs by flow cytometry.Part 2 Transfect hUCMSCs with Ad-GFP-NICD according to the optimal multiplicity of infection(MOI) of early experiment and observe the expression of green fluorescent protein(GFP), then the total genomic DNA was extracted and normal PCR was used to measure the expression of NICD gene; at 12h,24h,48h and 72h after transfection, the proliferation capability and the changes of cell cycle were detected by MTT and flow cytometry respectively; meanwhile, the total RNA was extracted at different times and the Real time PCR method was used to measure the expression of Hey2 gene in the downstream of Notch signaling pathway, the arterial marker gene ephrinB2 and venous marker gene EphB4; the extracellular matrix(ECM) gel experiment was used to measure the formation of tubular-shape structure. The hUCMSCs transfected with Ad-GFP were used as control.Results:(1) The isolated and cultured hUCMSCs by tissue adherent method were long and spindle-shaped with a typical clone-like growth style. After transferring of culture, the cells were school of fish-shaped with more polarity.(2) The results of flow cytometry showed that expression of CD44, CD13 and CD71 on the isolated and cultured hUCMSCs were all positive while that of CD34 was negative.(3) After transfecting hUCMSCs with Ad-GFP-NICD by 100MOI (MOI=plaque forming unit/cell), the expression of GFP strengthened gradually and peaked at 48h, meanwhile, the NICD gene can be amplificated from the cells genomic DNA of the Ad-GFP-NICD group while that of Ad-GFP group and untransfected group were negative.(4) The results of MTT showed that, compared with the Ad-GFP group, the proliferation and survival capability of hUMMSCs enhanced significantly at 12h and 24h after transfection with Ad-GFP-NICD (P<0.05), and reached the peak at 48h (P<0.01).(5) The results of cell cycle showed that the cell percentage in S phase, compared with the Ad-GFP group, increased gradually at 24h and 48h after tansfection with Ad-GFP-NICD (P<0.01), while that in G0/G1 phase decreased markedly than the control group (P<0.01).(6) The Real time PCR results showed that the expression of Hey2 gene was up-regulated at 12h,24h,48h and 72h after transfection with Ad-GFP-NICD than that of the Ad-GFP group (P<0.01), moreover, the change of Hey2 gene expression presented a dynamic process, namely it peaked at 24h, decreased at 48h and increased again at 72h. The expression of ephrinB2 gene (arterial marker gene) was up-regulated at 24h and 72h after transfection with Ad-GFP-NICD than that of Ad-GFP group (P<0.01), while that at 48h decreased than the Ad-GFP group, showing the dynamic changes similar to the expression of Hey2 gene. The expression of EphB4 gene (venous marker gene) has no difference at 12h,24h and 48h after different transfection, but only at 72h after transfected with Ad-GFP-NICD, the expression of EphB4 increased than that of Ad-GFP group (P<0.01).(7) The results of ECM gel experiment showed that the hUCMSCs transfected with Ad-GFP-NICD can form the tubular-shape structure, while the same result was not found in Ad-GFP group.Conclusions:(1) Ad-GFP-NICD improves the proliferation capability of hUCMSCs and accelerates the cell division.(2) Ad-GFP-NICD may activate the Notch-Hey2 signaling pathway of hUCMSCs, up-regulate the expression of arterial marker gene ephrinB2, improve the formation of tubular-shape structure in vitro. The result implies that Ad-GFP-NICD can induce hUCMSCs to differentiate towards arterial endothelial cells.