The Proteomics Analysis Of Human Dental Follicle Cells And Human Periodontal Ligament Fibroblasts With Label-free Technology

Human dental follicle cells( h DFCs) from dental sac, are the ancestral cells of periodontal ligament-oriented periodontium, while human periodontal ligament fibroblasts,(h PDLFs), which assuming responsibility for the extracelluar matrix metabolism of periodontium, are major daughter cells of h DFCs. For the high incidence of periodontitis as well as the accompanied teeth losing, periodontal tissue regeneration concentrated on periodontal attachment has always been an attctive work. As we know, h DFCs and h PDLFs are histologically homologous and biologically continuous, which are in discrepant proliferative and differentiative stage. h PDLFs inherited some features from h DFCs. For example, we consider both of them biologically heterogeneous, and have the fibroblast- like cell morphology and the ability of collagen formation and reconstructio n. In the last few years, researches which were related to h DFCs transforming into h PDLFs focused on stem cell differentiation and methods to regenerate periodontal ligament with attachment. Many of those in vivo or in vitro experiments paied attention to the effects of single gene, protein, cytokine or pathway on osteogenesis and fibrosis of h DFCs. Some researchers even analyed the gene expression veriation after fibrosis differentiation with High-Throughput Technology. However, the information on the changes of total celluar metabolism or proteins expression after h DFCs differentiating into h PDLFs so far is very poor. In the era of post-genomic, profiles of protein expression will primely reflect the functional similarities and differences between dental follicle and periodontal ligament. Exploring the differences before and after cell differentiation in protein level will provide us insight into which and why proteins are significantly changed. Thus, at protein level, these exploration will benefit our study on h DFCs–fibroblast transdifferentiation mechanism. Therefore, by virtue of Label- free protein quantification technique, this study concentrated on protein pro files as well as differentially expressed proteins of h DFCs and h PDLFs, and find enssencial proteins participating in h DFCs–h PDLFs transdifferentiation and to provide clues for clinically regeneralizing periodontal tissue with truth attachment.Aims:1. To isolate and culture h DFCs and h PDLFs respectively, followed by cell identification and protein sample preparation. 2. To obtain the cell total protein profiles of h DFCs and h PDLFs and analyse the similarity. 3. To analyse the proteins of h DFCs and h PDLFs significantly differentially expressed, and then test with q PC R technique. Also to interpretate the cell metabolism differences between h DFCs and h PDLFs.Material and Methods:Experiment 1: h DFCs and h PDLFs were isolated and cultured, the origination of cells were identified. For the total protein extracted from h DFCs and h PDLFs, the protein abundance were assessed preliminarily with SDS-PAGE electrophoresis.Experiment2: The peptide fragments in the preprocessed pretein samples of hDFCs1, h DFCs2, h PDLFs1, h PDLFs2 were used for mass spectrometry with LTQ orbitrap Velos liquid chromatograph/mass spectrometer accompanied by daian ultramate 3000 nano-UPLC operating system. To obtained the original protein data of h DFCs and h PDLFs, qualitative and quantitative ana lysis software maxquant(1.5.0.12) were applied, meanwhile referring to the human protein data base V3.87. Biological repeatability and correlation analysis were culculated for the homogeneous samples. For nonhomologous samples of h DFCs and h PDLFs, protein proportion, relative amount and Gene ontology(GO)analysis were calculated.Experiment 3: Combined with primary protein identification data base, the differentially expressed analysis was taken for h DFCs and h PDLFs protein profiles. For those proteins significantly differential expressed, GO analysis, KEGG analysis, and q PCR verification for some selected prroteins were carried out.Result:1. Immunofluorescence stain suggested that the h DFCs and h PDLFs have the bio logical character of fibroblast, both of which express vimentin positively while cytokeratin negatively. At the same time, SDS-PAGE proved that no extremely high expressed proteins were found in h DFCs or h PDLFs. 2. We got 906 proteomes from the four samples of h DFCs1, h DFCs2, h PDLFs1, h PDLFs2 by chromatography- mass spectrometry, and the repeatability and correlation of homogeneous samples were high. Analysis of protein proportion, protein amount and GO conformed that h DFCs and h PDLFs had similar protein profiles. 3. 22 proteins between h DFCs and h PDLFs were found to be statiscally different expressed(p<0.05,FC>2). In detail, 7 proteins up regulated in h PDLFs compared with h DFCs, while 15 down regulated. The up regulated protein were DRAP1, ECI1, EIF3 G, HSP90B1, NIT1, PPP1 CB, STX7, while the down regulated were AKR1A1, ALCAM, C DC42, CTGF, CYR61, DDX1, DDX17;DDX5, DDX39 A, F2, FKBP4, FST, GDI1;GDI2, MAT2 A, RPL18 A, TOMM6. q PCR showed that the m RNA variation of FST、C TGF、STX7、NIT1 between h DFCs and h PDLFs were consistent with the results of protein quantification, however no statistical differences was found for C TGF. Moreover, GO analysis revealed an enhanced function of protein synthesis and secretion after h DFCs transforming into h PDLFs.Conclusion: The similarity and continuity of cell morphology and biological function between h DFCs and h PDLFs were mainly reflected in similar protein profiles. For the distinct differentiated degree and the continued differences of microenvironments once differentiation, quantitative analysis determined that 22 proteins were differentially expressed. The up or down expressed proteins obtained in this research might be molecular manifestation that h DFCs had a higher proliferation and differentiation capacity than h PDLFs, on the other side h PDLFs were much more specialized in collagen metabolism. Moreover, they may also provide clues for studying h DFCs—h PDLFs transformation as well as periodontal regeneration.