The Study Of Hypoxic And High Glucose Preconditioning Of The Umbilical Cord Mesenchymal Stem Cells Transdifferentiate Into Blood Vessel Endothelial-like Cells In Vitro

Objective: TO isolated and proliferated Human umbilical cordmesenchymal stem cells（hUC-MSCs） and Human umbilical vein endotheliacells（HUVECs）, TO investigate the effect of hypoxic and high glucosepreconditioning of the umbilical cord mesenchymal stem cellsTrans-differentiate into blood vessel endothelial-like cells in vitro, Further tocompare endothelia-like cells with HUVECs in biological activity andmorphology, in order to explore whether the hUC-MSCs) which werepreconditioned by hypoxic and high glucose are better to develop the functionof endothelia cells, better to ameliorate the lower-extremity vascular disease ofDiabetes mellitus.Methods：1The umbilical cord was abtained under sterile conditions from full-termfetus of cesarean section （with informed consent of their families）.Then theumbilical cord envelope was cut between the umbilical arterial blood vesselsalong the long axis, and blood vessels were removed. The mesenchymal stemcells were isolated from human umbilical cord Whaton’s Jelly and cultured bytissue adherent culture method; the umbilical vein endothelia cells wereisolated human umbilical cord vein and cultured by digesting with collagenasemethod. Make them proliferating and observed the shape under microscope, toidentify them by flow cytometry or immunohistochemical.2The3th or4th passage of hUC-MSCs in good condition werepreconditioned by COCL₂of different concentrations （50umol/L、100umol/L、200umol/L、300umol/L、400umol/L、500umol/L）, then observed the activityand proliferation of hUC-MSCs by MTT and trypan blue test during thedifferent times （1d、2d、3d、4d、5d）, then test the level of VEGF and bFGF using the method of ELISA in cell supernatant solution from differentconcentrations and different tiems, then the proper concentration of COCL₂will be obtain.3The3th passage of hUC-MSCs in good condition were cultured andproliferated in medium which include the proper concentration of COCL₂,then text by flow cytometry, compared with the not precondition hUC-MSCsin cell-surface markers. And the5th passage of hUC-MSCs were tested thesafety by the method of chromosome.4The3th passage of hUC-MSCs in good condition were preconditionedby the different concentrations of glucose （4mmol/L、8mmol/L、12mmol/L、16mmol/L、20mmol/L）,then observed activity and proliferation of hUC-MSCsby MTT and trypan blue test during the different times （1d、2d、3d、4d、5d）,choose the proper concentration.5The2th passage of HUVECs in good condition were cultured indifferent mediums which on the basis of the components, then draw thegrowth curve, then chose the proper medium to culture.6The3th passage of hUC-MSCs in good condition were preconditionedby the different COCL₂and/or glucose and induced into endothelial-like cellsin the special medium,which comprised of DMEM/F12、VEGF10ug/L、bFGF10ug/L、2%FBS. Cell morphology were observed under microscope. totest the endothelial cells marks by immunohistochemical to compare theprecondition、not precondition with HUVECs, to observed the microscope byHE; to test the ability of moving by the method of cell scratch; to observe theability of forming the vessel by the three-dimensional models; to test thebiological activity by the radioimmunoassay of6-keto-prostaglandin.Results：1The hUC-MSCs isolated from human umbilical cord Wharton’s Jellyand HUVECs isolated from vein vessel by digest of collagenase wereobserved under microscope and occur some changes, such as after5days,wecould see fusiform-like cells,and could see proliferated till after12days;while the hUC-MSCs digested after two days, we can see the adherent cells,and obvious increasing until one week, the shape just like petal andmixture together,and proliferated after two weeks.the hUC-MSCs stronglyexpressed the surface markers of mesenchymal cells CD105,CD29，CD44，anddid not express hematopoietic lineage marks CD14,CD34or CD45,and thisresults can not change with the increasing of the passage; The same as theHUVECs, which strongly express CD31,VWF.2The hUC-MSCs culture in the different medium, which have differentconcentration of COCL₂, the results was the proliferation were correlated withtime and the degree of hypoxia. And the level of bFGF and VEGF werecorrelated the degree of hypoxia, the curve just like a “clock”, meanwhile theactivity of the cell also were closely with the act time and the degree ofhypoxia. Then we can conclude that the proper concentration of COCL₂was200umol/L.3The surface marks of the cell don’t change whatever the differentconcentrations and different times after inducing by COCL₂, and it isconformed to the before of inducing. We also confirm that it is safety toinduced by COCL₂through the method of chromosome.4Through did the test of MTT and drawn the Growth curve afterhUC-MSCs induced by the different concentration of glucose,we can see thatthe proliferation were correlated with time and the degree of glucose, thecurve just like a clock,meanwhile we can gain the proper concentration ofglucose was8mmol/L.5This study we found that the perfect medium to culture HUVECs wasDMEM/F12+VEGF+bFGF after culturing the different mediums throughMTT. It was large found that the proper concentration of glucose orCOCL₂were all can stimulate the proliferation of HUVECs.6In the term of morphology, it is great different between the Huc-MSCswhich were after induced or before induced and HUVECs observed under themicroscope and dye by the HE, after through the immunohistochemical,wecan found that it is more or less expressed the CD31and VWF in the threegroups,that were hypoxia group, glucose group and hypoxia preconditioning 3days–glucose group. and the endothelial-like cells and HUVECs were allhave the function of forming vascular and biological activity, not only thatthey even keep the ability of migration like hUC-MSCs.Conclusion:1The most of MSCs were contained in the Wharon’s Jelly of humanumbilical cord, they can express the surface makrs of CD29, CD44, CD105,however they can’t express the surface marks of CD14, CD34, and CD45,even proliferation the ten passages.2The human umbilical vein can separate the endothelia cells, which canexpress the surface marks of CD31and VWF.3COCL₂can analog the condition of hypoxia in pathology mechanism inbody, and can not change the express of surface makrs, it is safety to inducedby COCL₂through the method of chromosome.4The HUVECs should culture in the special media which contain theVEGF and bFGF, and the hypoxia and high glucose can make the cellproliferation.5The COCL₂and high glucose can influence the curve of theproliferation of the cells, they can also induced into the endothelial-like cellsafter preconditioning with the COCL₂and glucose, which can express thesurface marks of CD31and VWF, they also has the function of formingvascular and biological activity just like endothelial cells.