Amphiphilic Peptide Self-Assembly Gel Induces Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neurons

Objective::To investigate the growth of bone marrow mesenchymal stem cells in gels containing IKVAV amphiphilic peptides and gel-induced differentiation of bone marrow mesenchymal stem cells.Methods:1.Bone marrow mesenchymal stem cells are isolated and cultured by whole bone marrow adherence.The morphology of the cells was observed under an inverted microscope.Flow cytometry was used to detect CD29 antigen,CD34 antigen,CD45antigen and CD90 antigen on the cell surface.The cells were induced to differentiate by the medium and the cells were observed under a microscope to differentiate into adipogenic and cartilage directions.2.Design thee experimental group IKVAV?isoleucine-lysine-valine-alanine-proline?and the control group EQS?glutamic acid-glutamine-serine?amphiphilic peptide.The 10 mg/m L solution containing the IKKVAV amphiphilic peptide was added to an equal volume of DMEM/F12 for several seconds and then self-assembled into a three-dimensional gel and the internal structure of the three-dimensional gel was observed by transmission electron microscopy?TEM?.Multipotential culture of IKVAV amphiphilic peptide gels and bone marrow mesenchymal stem cells,stained with live-cell assay reagent Calcein-AM/PI double-labeled,observed in fluorescence microscopy of bone marrow mesenchymal stem cells in IKVAV amphiphilic peptide gel Internal survival.1×106 cells/m L BMSC suspension and zero-point gel were mixed to form a gel?three-dimensional culture system?and 1×106 cells/m L of bone marrow mesenchymal stem cell suspension was inoculated into amphipathic peptides.The surface of the rubber-coated cover wave sheet?two-dimensional culture system?was cultured in a complete culture medium.The IKVAV amphiphilic peptide gel was used to detect the mesenchymal stem cell proliferation/toxicity by CCK-8 assay.3.After the cell-containing culture solution is mixed with the polypeptide solution,self-assembly occurs to form a three-dimensional culture system.Experiments were divided into 3 groups:Group A:Bone marrow mesenchymal stem cell culture?blank control group?;Group B:Three-dimensional culture system formed by EQS gel co-cultured with cells?experimental control group?;C:IKVAV gel and cells together the cultured three-dimensional culture system?experimental group?was cultured.The expression levels of MAP-2 protein,GFAP protein,and NSE protein in bone marrow mesenchymal stem cells of each group were detected by Western blotting.The expression of GFAP protein and NSE protein in bone marrow mesenchymal stem cells were detected by immunofluorescence.Results:1.Inverted microscope observation of bone marrow mesenchymal stem cells showed a long spindle shape,arranged in a swirling state.The original three generations of bone marrow mesenchymal stem cells flow cytometry results showed that bone marrow mesenchymal cells high expression of CD29 antigen and CD90antigen,not expression or low expression of CD34 antigen and CD45 antigen.Bone marrow mesenchymal stem cells appear lipid droplets after 25 days of induction by adipogenic medium,stained with oil red O and become red;after 24 days of induced culture with chondrogenic induction medium,after staining with aliski blue,it was observed that the intracellular acid mucopolysaccharides in the cells stained pale green.2.In the experimental group IKVAV?glutamic acid-glutamine-serine?peptide design sequence:C₁₆H₃₁OA₃G₄D₂-IKVAV;control group EQS?glutamic acid-glutamine-serine?peptide design sequence:C₁₆H₃₁OA₃G₄D₂-EQS.Electron microscopy showed that the IKVAV amphiphilic peptide gel was composed of multi-dimensional nanofibers.The diameter of the nanofibers was 3-6 nm and the length was 50-500 nm.The molecular weight of the IKVAV amphiphilic peptide measured by mass spectrometry was 1438.71,which was in accordance with its theoretical value;HPLC analysis the purity of the amphiphilic peptide of IKVAV was analyzed to be 95.74%.Calcein-AM/PI double staining showed that the bone marrow mesenchymal stem cells were cultured with the gel for 1 day,3 days,and 5 days later,using calcein-AM/PI?Viable cell staining?Staining was performed on the 1st,3rd,and 5th days of culture.The number of living dead cells was counted under a fluorescence microscope.As a result,it was found that the number of viable cells increased significantly with time,and then it died was not obvious change in cells.The CCK enzyme labeling showed that the proliferation of bone marrow mesenchymal stem cells in the three-dimensional system formed by the co-culture of bone marrow mesenchymal stem cells and gels was higher than the two-dimensional system formed by the co-culture of the gel.3.Western-Blot method was used to detect the relative amounts of target protein in group A,B and C.One-way analysis of variance was used to show that the expression levels of NSE protein,MAP-2 protein and GFAP protein in group C were much higher than those in group A and B.Statistical analysis was performed between group A and group B,and the difference between the three groups was statistically significant?P<0.05?.The cells expressing the NSE protein in the fluorescent dual-labeled bone marrow mesenchymal stem cells exhibited red fluorescence,and the cells expressing the GFAP protein exhibited green fluorescence.In group C,approximately 50%of the red fluorescence expressing NSE protein was detected,and approximately 30%of the green fluorescence expressing GFAP protein was detected.However,the expression levels of GFAP protein and NSE protein in group A and B were detected to be low.The expression levels of NSE protein and GFAP protein in group C were significantly higher than those in group A and B.Statistical analysis showed that the difference was statistically significant?P<0.05?.Conclusions:Amphiphilic peptide self-assembly gels induce bone marrow mesenchymal stem cells to differentiate into neural cells.