The Effect Of CIB1on The VEGF-induced Differentiation Of MSCs Into Blood Vessels

Purpose:1. We aimed to isolate, culture and identify the human umbilical cord mesenchymalstem cells;2. We aimed to explore the effect of CIB1on the VEGF-induced differentiation ofMSCs into blood vessels.Method:1. Get primary hUCMSCs from mature newborn umbilical cord with tissue adherentassay. Cell phenotype and mitotic cycle were detected with flow cytometry. Potent ofdifferentiation was identified by evaluation of differentiation to adipocytes.2. In this experiment, we transfect hUC-MSCs by the CIB1lentiviral vector. Thisexperiment was studied on the hUC-MSCs,4groups were involved: normalhUC-MSCs induced group; CIB1lentiviral vector-transfected hUC-MSCs inducedgroup, this group was also divided into NC infection induced group, pGV-siCIB1infection induced group; and the control group. At the beginning of the experiment,we tried to decide the most appropriate infection MOI under the rational design ofexperiments.Observed the proliferation of hUC-MSCs after CIB1lentiviralvector-transfected by MTT. Detected status of mitotic cycle in each group with flowcytometry.Matrigel angiogenesis experiments and Dil-ac-LDL absorptionexperiments were used to detect the ability of each group differentiation into theblood vessels, and also we identificated for cells vascular endothelial cell markersCDH5, FLK-1and CD34with immunofluorescence techniques.Results:1. We successfully solutated and extracted hUC-MSCs. The cell was almost found inthe previous stage of differentiation by the flow cytometry, it seems that the cell hasgood differentiation ability. Using flow cytometry, we found that cells expressedmesenchymal cellular markers CD29(92.62%), CD44(88.83%), CD105(91.87%),CD130(92.45%); low expessions CD45(1.46%) and HLA-DR (2.06%). At the sametime, this cell can be induced into adipose cell by oil red〝O〞staining, and thecell-specific expression of cell markers LPL and Leptin by Qrt-PCR.2. We successfully got the CIB1lentiviral vectors of infection MOI, the best MOI wasfound to be20. We infected cell cycle and cell survival activity after transfected,found that the proportion of infected G2/M phase the NC infected cells infectedgroup was significantly lower than pGV-siCIB1. The cell was arrested in G2/M phase by RNAi down regulate the CIB1express. MTT datas showed the survivalactivity pGV-siCIB1infected cells was much more decreased, while the NC infectiongroup did not take place obviously. After the induction condition into endothelial cellsfor21days, it was shown that the lumen of vascular endothelial cell-like structuresignificantly increased, to21days almost the entire field under the microscopecovered, which is similar to that in the three-dimensional gel matrix lumen structure.And the Dil-ac-LDL absorption significantly increased compared to the former. Whilethe surface of vascular endothelial cell markers CD34, CDH5and FLK-1appearspositive after induction. It was not only the same as vascular endothelial cell inmorphology, but also the cell activity and cell surface marker expression areconsistent with the them.The pGV-siCIB1group which doesn’t show the markers ofvascular endothelial cell after21days′inducd. There was neither the lumenstructure, small vessel endothelial cells nor expressed markers CD34, CDH5orFLK-1. The absorption of Dil-ac-LDL was also not increased. Experimental resultsshowed the NC group after inducd was consistent with the results of normalhUC-MSCs after induction, it was proved that CIB1played an important role in theprocess hUC-MSCs differentiate into vascular endothelial cellsConclusion:1. The humanbeing umbilical cord mesenchymal stem cells are successfully obtained,isolated and confirmed, which is proper for the next experiment.2. The CIB1plays a very important part in VEGF-induced MSCs differentiation intovascular molecules channels.