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Construction and Purification of KGF-2 and TAT-KGF-2 with Effects on Proliferation and Differentiation in hUC-MSCs

Author: RongJing
Tutor: WangYiFei
School: Jinan University
Course: Genetics
Keywords: Human umbilical cord mesenchymal stem cells Human keratinocyte growth factor CPPs Differentiation MAPK pathway
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 24
Quote: 0
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Abstract


Objective: To establish from human umbilical cord in the effective separation of cultures between the charge and quality of stem cells (of MSCs, mesenchymal stem cells), and can be carried out in vitro amplification of Passage; simultaneously construct and expression, and purification of human keratinocytes cell growth factor 2 (keratinocyte growth factor 2, KGF-2) and the penetrating type KGF-2, to study the proliferation of human umbilical cord MSCs (hUC-MSCs, human umbilical cord MSCs); study KGF-2 cell adipogenic osteogenic adipogenic differentiation, while conducted a preliminary study of its mechanism of action. Methods: The tissue explants adherent method in vitro the isolated hUC-MSCs; build the pET28a-KGF-2 and TAT-pET28a-KGF-2 recombinant plasmid, and transformed into E. coli Rosetta (DE3), optimization of the target protein expression conditions and soluble purified; MTT assay on cell proliferation in the identification and activity of the purified protein detection; Western blot detection kit KGF-2 protein on the cell alkaline phosphatase ALP and calcium deposition into bone markers, Western blot analysis by real-time quantitative PCR detection of KGF-2 protein adipogenic markers PPARγ2 and GPDH; MAPK pathway of ERK1 / 2 and p38 pathways change. Results: 1, tissue explants adherent method in 10-15 days may vitro amplification of the passage, and has the the MSCs surface antigen labeled hUC-MSCs induced in vitro can be divided into fat cells and osteoblasts. Successfully constructed recombinant plasmid pET28a-KGF-2 and of TAT-pET28a-KGF-2, through the optimization of expression conditions for efficient, soluble KGF-2 expression and recombinant protein TAT-KGF-2. 3, the high concentrations of KGF-2 and recombinant protein TAT-KGF-2 can be generated hUC-MSCs proliferation effect, and TAT-KGF-2, maximum cell proliferation rate to the desired protein concentration higher than that of the KGF-2 recombinant protein. 4, a certain concentration of KGF-2 recombinant protein can effectively promote osteogenic differentiation, but had no significant effect on adipogenic differentiation. Found in the study of its mechanism, KGF-2 activation of ERK1 / 2 pathway, this pathway is inhibited, KGF-2 contributing suppressed bone differentiation. Conclusion: The successful separation of the can body rumor on behalf of hUC-of MSCs; access to high soluble expression, and activity of KGF-2 and TAT-KGF-2 recombinant protein, its able to promote hUC-of MSCs of proliferation; including KGF-2 recombinant protein promote osteogenic differentiation, the role is likely to be achieved through the activation of ERK1 / 2 pathway.

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