Construction And Purification Of KGF-2 And TAT-KGF-2 With Effects On Proliferation And Differentiation In HUC-MSCs

Objective:To establish the methods of isolation and expansion of mesenchymal stem cells(MSCs) derived from human umbilical cord; To construct and purify protein keratinocyte growth factor 2(KGF-2) and TAT-KGF-2, then study their effects on proliferation of human umbilical cord MSCs(hUC-MSCs); To investigate the effect on adipogenic and osteogenic differentiation induced by KGF-2 and its mechanism.Methods:Tissue culture was used to obtain hUC-MSCs;Construct recombination plasmid pET28a-KGF-2 and TAT-pET28a-KGF-2,express protein in Rosetta(DE3) E.coli, optimize the expression condition and purify the protein; After identification and activity detection, MTT assay was employed to determine the effect on proliferation of hUC-MSCs;The changes osteogenic differentiation was characterized by western blot and assay kit,and adipogenic differentiation was detected by Realtime PCR;Using western blot to identify the changes of ERK1/2 and p38 path.Result:1. We can get hUC-MSCs during 10-15 days by tissue culture method. The cells can expand in vitro and possess surface antigen of MSCs, it also could be induced into adipocyte and osteoblast.2. Successfully get recombination plasmid pET28a-KGF-2 and TAT-pET28a-KGF-2, through optimizing expression conditions, we obtain effectively expressed and soluble proteins.3. Recombined protein KGF-2 and TAT-KGF-2 of high concentration can promote proliferation of hUC-MSCs, and protein TAT-KGF-2 requires higher concentration.4. Recombined protein KGF-2 can enhance osteogenic differentiation with certain concentration, however, no effect on adipogenic differentiation. In the study of mechanism,we found that KGF-2 can activate ERK1/2 path, when the path was inhibited, the enhancement of osteogenic differentiation was also suppressed.Conclusion:The hUC-MSCs were get from the human umbilical cord; Then obtained recombined protein KGF-2 and TAT-KGF-2 which were soluble and activated. The proteins can promote proliferation of hUC-MSCs; And KGF-2 can enhance osteogenic differentiation of cells through activation of ERK1/2...