Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neurons By Functional Peptide Self-assembled Nanofiber Hydrogels

Objective:Design containing IKVAV/RGD functional self-assembled peptide hydrogel was co-cultured with bone marrow mesenchymal stem cells to observe cell growth status,cell compatibility,cell adhesion and cell differentiation into neuronal cells.Method:1.SD rats?Sprague Dawley?aged 3-4 weeks were isolated and cultured BMSCs?bone marrow mesenchymal stem cells?by whole bone marrow adherence method and BMSCs surface antigen was identified by flow cytometry.2.Design containing IKVAV?I-isoleucine,K-lysine,V-valine,A-alanine,V-valine?/RGD?R-arginine,G-glycine,D-aspartic acid?polypeptide is synthesized by Solid-phase peptid synthesis?SPPS?,The purity was purified by high performance liquid chromatography?HPLC?and the average molecular weight was measured by mass spectrometry?MS?.The self-assembled hydrogel was prepared by using an equal dose DMEM with 1 wt%amphiphilic polypeptide containing IKVAV/RGD,and the internal structure of the self-assembled hydrogel was observed by transmission electron microscopy?TEM?.3.CCK-8 method was used to detect the proliferation/toxicity of co-cultured cells containing different concentrations of IKVAV-RGD self-assembled hydrogel and bone marrow mesenchymal stem cells;IKVAV-RGD self-assembled peptide hydrogel and bone marrow mesenchymal stem cells were co-cultured?three-dimensional system culture?,and bone marrow mesenchymal stem cells were seeded in PLL?poly-amino acid?package.,cell growth under the surface of the cover glass?two-dimensional system culture?,Calcein-AM/PI for detection of dead and live cell staining reagents.In addition,nuclear staining was used to detect cell adhesion properties of self-assembled hydrogels at different time points;Subsequently,the experiments were divided into three groups:IKVAV/RGD group,IKVAV group,PLL?polylysine?group.After 7 days of co-culture,the expression levels of MAP-2protein and GFAP-related protein after cell differentiation were detected by cell immunofluorescence.result:1.Separate and obtain the third generation BMSCs in a shuttle shape,Flow cytometry showed that the cells were highly expressed CD 105 positive and CD45negative was 90.88%,while the ratio of CD 90 positive and CD 34 negative was94.14%.2.Amphiphilic polypeptide design sequence:^RGDK-\_IKVAVAK-/KGGGGAAAAK-C₁₆H₃₁O,Purification and detection of the purity of the peptide by HPLC was 95%.The molecular weight of the synthetic IKVAV-RGD amphiphilic peptide was determined by MS to be M.W.:2191.72,which was consistent with the actual theoretical molecularweight.TEMresultsshowthattheIKVAV-RGDamphiphilic self-assembled hydrogel consists of nanofibers,which are dendritic and intersect with each other.The nanofibers are 2 to 5 nm in diameter and 150 to 500 nm in length.3.The enzyme-labeled instrument measured the absorbance of CCk-8 at a wavelength of 450 nm.The control group A and the experimental group BCDE group showed an increasing trend in 1,3,and 7 days,and the E group had slightly lower absorbance than the control group A;After 1d,3d and 7d,Calcein-AM/PI staining was performed respectively.The living cells and dead cells increased with the increase of cell culture time,and the number of living cells also increased significantly.There was no significant difference in dead cells;1000 cells/cm2,2h,4h were inoculated.After 6 h,the amphiphilic IKVAV-RGD peptide group had the strongest adhesion.4.After 7 days of BMSCs inoculation in IKVAV-RGD hydrogel,the expression of MAP-2 neurons in the IKVAV-RGD group was higher than that in the IKVAV group,while the expression of GFAP in astrocytes was decreased.Conclusion:1.IKVAV-RGD amphiphilic polypeptide can increase cell adhesion;2.The IKVAV-RGD amphiphilic polypeptide has the ability to induce differentiation of BMSCs into neuronal cells more than the IKVAV amphiphilic polypeptide.