| Background : In today,s society,perpheral nerve injury is particularly high.For longer than 2cm defect injury there is no ideal solution,in recent years with the development of bio-engineering and stem cells,there is new hope in treat it.Seed cells like Schwann cells have been the studied “bottleneck”,because of their own shortcomings,autologous and allogeneic Schwann cells have not been large-scale used.how to prove induced Schwann cells have a crucial function of Schwann cells, Schwann cells have powerful autocrine and paracrine functions, which may be one of the mechanisms exert biological effects, so if they can prove that the induced secretion of Schwann cells can truly Schwann cells secrete some functional protein can be provided that further study late experimental theory.Objective: In this study,the research group intends to combine with previous work experience,isolated from human umbilical cord blood mesenchymal stem cells were identified, amplified, cryopreservation, and induced to differentiate in vitro induced by artificial chemical solution oriented Schwann cells. Prepared by enzymatic digestion human Schwann cells. Were collected mesenchymal stem cells, Schwann cells, cell culture supernatants Schwann cells, extract the protein, using two-dimensional electrophoresis technology compares three cell secretory protein differences and similarities between the query protein with a significant difference function.Methods: According to our group before,first use erythrocyte sedimentation using hydroxyethyl starch, and then using centrifugal extraction of human lymphocyte separation medium between primary mesenchymal stem cells, cell surface markers was measured by flow cytometry and morphological identification and measurement methods its purity and meet the requirements to the next experiment, the third generation of stem cells taken before our group to optimize the use of inducedartificially induced in vitro methods. Prepared by enzyme digestion human Schwann cells. The three cells(mesenchymal stem cells, Schwann cells, Schwann cells) using the "progressive over law" over to the serum-free culture, three cell culture supernatant was collected, filtered with 0.22 um filter to remove residual cells, add after 1m M concentration of protease inhibitors into the ultra-low temperature freezer spare. Extract broth in secreted proteins, the use of two-dimensional electrophoresis and mass spectrometry techniques to find significant differences protein of mesenchymal stem cells than Schwann cells less protein and Schwann cells than mesenchymal stem cells and more protein match, inquire about their protein function and to classify.Results:1)2-D pattern with good resolution and repeatablility,protein-point average per gum has about three thousand, each match rate is 96%,97%,98%.2)HUCBMSCs before induction and protein differences SCs have 290 points after induction class SCs and SCs only 161 protein spots, mesenchymal stem cells after the class description between Schwann cells secrete more similar aspects of proteomics Schwann cells in.3) in accordance with the screening criteria for screening out of 12 differentially expressed proteins were identified by mass spectrometry, and ultimately identified seven protein4) select two kinds of proteins in these seven points in protein immunoblotting verify and validate the results of the basic two-way consistent with the results of electrophoresis and mass spectrometry.Conclusion:1)Use hydroxyethyl starch and human lymphocyte separation medium can be extracted prinary mesenchymal stem cells.2)Inter-human umbilical cord blood mesenchymal stem cells secreted proteins in terms closer to the real Schwann cells.3)The experiment successfully identified the seven protein spots analysis of secreted proteins is further evidence of induced Schwann cells and normal Schwann cells have a lot of similarities, but it also shows that there is a gap induction program still need further improvement. |
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