| Objective: To research the basic biological characteristic of hUC-MSCs. To explorethe optimum condition to extract HHK and observe its biocompatibility withhUC-MSCs. Adopting different methods to induce hUC-MSCs differentiating toSCs,then to explore the role HHK has played on the process and find the optimummethod of them so as to provide theoretical grounds for the application of hUC-MSCsinto the repair of peripheral nervous and spinal cord injury.Methods: The hUC-MSCs were isolated from human umbilical cord by digested withcollagenase,and their growing status and morphologic characteristic was observed byoptical microscope and atomic force microscope. The growth curve of P3generationand the cell proliferation rate of P1-P8were measured by MTT assay.The growthcycle of P1-P6cells and the cell phenotype were determined by Flow cytometry. TheHHK was extracted by reduction method and co-cultured with hUC-MSCs, to observethe role it has played on the proliferation and chemotaxis against hUC-MSCs. Itseffect of seeding and adherence to hUC-MSCs shall be observed by coating cultureflask with HHK solution of different concentration. Dividing the hUC-MSCs to groupA、B、C、D、E、F at random, then they were cultivated by respectively adopting themethod of composite induced factor plus HHK, composite induced factor,co-cultivation plus HHK, co-cultivation，HHK, and Common culture medium. After9days, its morphology and ultrastructural changes can be observed by HE colorationand AFM, the specific protein expression of divided cells(S-100and GFAP) can beobserved by immune cell fluorescence, at the same time, the positive of S-100proteinexpression and apoptosis rate of divided cells were observed by flow cytometricquantitative detection.Results: High purity of hUC-MSCs is obtained by digested with collagenase and thecell cycle of P1-P6generation and the growth vitality of P1-P8generation shows noobvious difference. FCM analysis reveals that CD105、CD90、CD73、CD54、CD44、CD13、CD29were highly expressed on the surface of cells, but CD34、CD14、CD45and HLA-DR were negative.HHK is successfully extracted by reduction method and the results show that HHK of proper concentration can improve cell proliferationvitality, promote cell migration, accelerate the adherence of cells wall and increase theanchoring strength. The divided cells, directional induced by SCs, gradually turnedinto a long spindle shape, with thin bumps at two poles. And more bumps has beenfound in nucleus surface by atomic force observation. Adopting the method of cellimmuofluorescences, the obvious enhance of hUC-MSCs’s expression of S-100protein and GFAP protein after induction can be observed. FCM has detected theresult that hUC-MSCs’s expression S-100protein has increased, cell apoptosis ratehas decreased. Among the six groups, the first four group has been seen inducingdifferentiation of different degree, which can not been seen in the later two group. Thedifferentiation efficiency ranking is as follows: cell factors group is higher thanco-cultivation group, HHK group is higher than then without HHK group; The cellapoptosis rate of co-cultivation group is lower than cell factors group, HHK group islower than then without HHK group. The result also shows that the differentiationefficiency is in a positive correlation with cells apoptosis rate, while HHK can reducethe cells apoptosis rate and improve the inducting differentiation efficiency.Conclusions: The experiment has successfully separated, cultivated and identifiedhUC-MSCs in vitro. HHK extracted by ways of reduction method is of goodbiocompatibility, can improve hUC-MSCs proliferation vitality, and promote cellmigration and adhesion. HHK joint with compound factor is the best inductionprotocols, which can greatly improve efficiency of hUC-MSCs’s differentiation toSCs and reduce the apoptosis rate. |
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