Proteomic Profiling Of Human Placenta-derived Mesenchymal Stem Cells Upon Transforming LIM Mineralization Protein-1 Stimulation

Human placenta-derived mesenchymal stem cells(h PDMSCs) are the progenitor cells existing in the placenta tissue, a kind of pluripotent stem cells, with the capacity to self-renew as well as the ability to generate differentiated cells. Recent study indicates that h PDMSCs can be differentiated to osteoblasts in particular environment. Hence,h PDMSCs as seed cells for bone tissue engineering can play an important role in the remodeling of mature bone area, especially in the construction of bone tissue engineering. Moreover, most of the newly discovered that LIM mineralization protein-1(LMP-1) is an important regulating factor of bone formation promotion; it also plays an important modulatory role in osteoblast differentiation, maturation and bone formation. Therefore,development of new ways in bone tissue engineering, which is using h PDMSCs as seed cells and adding LMP-1 as osteogenic factors, is of great necessity in theoretical basis and favorable clinical prospects.Before constructing of tissue engineering bone, we designed a study using a proteomic approach combined with adenovirus-mediated gene transfer of LMP-1 to identify LMP-1-induced changes in h PDMSCs on proteome level to determine a global effect of LMP-1 on h PDMSCs.Moreover, we have generated proteome maps of undifferentiated h PDMSCs and LMP-1 induced h PDMSCs.The research details are as follows:1. Culture and identification of the placenta-derived mesenchymal stem cellsThe surface markers(antigen positive: CD29 、 CD44 、 CD73 、CD90 、 CD105 、 CD117) of h PDMSCs were determined by flow cytometry. Moreover, there is no expression of CD33、CD34、CD45 in the surface markers of h PDMSCs, and the conclusion is basically the same as reported at home and abroad of cell surface markers of h PDMSCs. Experimental results proved that the h PDMSCs were well capable of differentiating into osteocytes and adipocytes.2. Construction of recombinant plasmid vector as human LMP-1 and the expression of LMP-1 in h PDMSCSThe main purpose of this part is to construct the gene recombinant plasmid vector as human LMP-1, and make it be expressed in h PDMSCS continuously and efficiently. We transiently overexpressed the human LMP-1 gene in h PDMSCs using the adenoviral expression system based on gateway technology and investigated the regulation of h PDMSCs proteins during their differentiation induced by LMP-1. We then used the Western Blot detection to expression the LMP-1 gene in h PDMSCs, and then detected the expression of different groups osteocalcin and type II collagen by RT-q PCR methods. Results showed the LMP-1 can be helpful to enhancement the osteogenetic differentiation in cells.3. The differentiation effect of LMP-1 on h PDMSCs by Proteomic analysis researchThis step is mainly adopting the strategy of comparative proteomics,to analyze the osteogenetic function effect of the LMP-1 from the protein level system, as well as the impact on the global h PDMSCs osteogenic differentiation. The result of proteomic analysis revealed 22 spots with at least 2.0-fold changes in expression and 15 differently expressed proteins were successfully identified by MALDITOF-MS. The expression of some identified proteins was confirmed by further Western blot analyses.Our results will play an important role in better elucidating the underlying molecular mechanism in LMP-1 included h PDMSCsdifferentiation into osteoblasts. All these results may be useful to elucidate the precise molecular mechanism of h PDMSCs osteogenic differentiation. As the subsequent, building bone tissue engineering that using h PDMSCs as seed cells and the LMP 1 as inducing osteogenesis factors provides the scientific basis.