Design Of Recombinant Human Fibronectin Peptide(rhFN) And Its Mechanism Of Regulating HPDLSCs To Promote Osteogenic Differentiation

Objectives: Biomaterials are the most basic part of bone tissue engineering.It is the goal of medical tissue engineering to obtain ideal repair materials by simulating the microenvironment of bone repair through bionic technology.Fibronectin(FN),as an important component of extracellular matrix(ECM),can communicate with cells through the integrin on the cell surface,thereby regulating the fate of stem cells,which has significant significance for bone defect repair and bone regeneration medicine.However,natural fibronectin comes from blood.Because of its unstable extraction process,high immunogenicity,complex functions and difficult to clarify the mechanism,large-scale medical applications are limited.In this study,a novel recombinant human fibronectin peptide(rh FN)was designed and obtained by using integrin α5β1,a communication tool of stem cell osteogenic differentiation,as a breakthrough point.It can specifically adsorb growth factors and promote the osteogenic differentiation of stem cells,which can further construct scaffolds for osteogenic tissue engineering.Methods:(1)Use online software such as Prot Param,Prot Scale,TMHMM,Signa IP 4.1Server,Net NGlyc,Net Phos 3.1 Server to determine the hydrophilicity/hydrophobicity of human fibronectin peptides,protein transmembrane domains,protein amino acid sequence signal peptides,and protein sugars.Online prediction of physical and chemical properties such as basement sites and phosphorylation sites.The protein structure was modeled using I-Tasser software.The Z-DOCK method was used to perform molecular docking with integrin α5β1 to predict its binding ability.(2)According to the preference of E.coli,the p ET20b-rh FN recombinant plasmid was constructed by the whole gene synthesis after codon optimization,and transformed into E.coli expression system for recombinant expression and purification.Through the selection and optimization of expression strains and expression conditions,an engineered strain BL21(DE3)with high expression of rh FN was obtained.Ni-NTA Sepharose affinity chromatography column and cationic affinity chromatography column purification.And carried out the 15 L fermentation tank scale-up process research.(3)Using ECV304 cells and h PDLSCs as model cells,CCK8,crystal violet staining,cytoskeleton staining,cell scratch experiment,and tube formation experiment were used to compare the effects of rh FN and full-length human fibronectin on the proliferation,adhesion and migration of two cell lines.(4)SPR tested the affinity between rh FN and growth factors(GFs).At the same time,the effect of b FGF,rh FN,b FGF and rh FN combined on the lumen forming ability of ECV304 cells was compared through the experiment of luminal formation.The osteogenic differentiation experiment was used to compare the effects of TGFβ3,rh FN,and the combination of TGFβ3 and rh FN on the osteogenic differentiation of h PDLSCs(observation indicators include: alkaline phosphatase(ALP)staining and ALP activity determination,WB detection).(5)In view of the existing research shows that integrin α5 has been proved to play an important role in the osteogenic differentiation of stem cells,while the role of integrin β1in the process of osteogenic differentiation of stem cells is rarely reported.In this study,we constructed and screened integrin β1 lentiviral interference plasmids.By interfering with the expression of integrin β1,we explored the role of integrin β1 in rh FN and/or TGFβ3 inducing and promoting the osteogenic differentiation of h PDLSCs.Results:(1)The analysis of the peptide domain of human fibronectin showed that the protein has no transmembrane region and signal peptides.It contains transmembrane cytokine receptors,adhesion molecules,growth factors and other binding domains at positions 21-290.At the same time,the protein has Multiple phosphorylation sites.The prediction of the secondary structure of the fibronectin peptide shows that the protein contains a large number of random coils,and this structure constitutes a specific functional site and enzyme active site of the protein.The interaction prediction between human fibronectin peptide and integrin α5β1 was analyzed by ZDOCK docking software,and the results showed that the intercepted human fibronectin peptide forms a relatively stable complex with integrin α5β1.(2)The p ET20b-rh FN recombinant expression vector was constructed,and two engineering strains BL21(DE3)/rh FN and Ply Ss/rh FN were successfully obtained.At the same time,the expression conditions of the recombinant protein rh FN were optimized,and the results showed that IPTG inducers with different concentrations were not selective,the best induction temperature was 37℃,and the best induction time was 4h.HPLC results showed that the purity of rh FN reached 96%.The molecular weight of rh FN determined by LC-MS/MS was 30.36 k Da,which was consistent with the theoretical molecular weight,and the amino acid sequence coverage was 89.3%.Circular dichroism spectroscopy studies the secondary structure of recombinant protein rh FN,and the results show that the proportion of random curls in the secondary structure of rh FN is the highest,which is consistent with the predicted value.Through the expansion of 15 L fermentation tank,the maximum output of rh FN reached 570mg/L,and the wet weight of bacteria reached 60.0 g/L.(3)rh FN has strong binding ability with b FGF and TGFβ3 respectively.rh FN(30 μg/ml)did not affect the proliferation of ECV304 cells,but promoted the adhesion(P <0.001,vs FN)and migration(P <0.05,vs FN)of ECV304 cells.At the same time,rh FN has the ability to promote ECV304 cell lumen formation,and when rh FN is used in combination with b FGF,its effect is significantly better than that of single use.(4)rh FN promoted the proliferation of h PDLSCs within a certain concentration range(P<0.01,vs Control),and at the same time promoted the adhesion(P <0.001,vs FN)and migration of h PDLSCs(P < 0.001,vs FN).In addition,the ALP staining and activity test results showed that rh FN induced 7 days to significantly increase the ALP activity of h PDLSCs(P <0.01),and when rh FN was combined with TGFβ3,the ALP activity of h PDLSCs was significantly higher than when used alone.WB results showed that rh FN and TGFβ3 induced,the expression of α5β1 protein in h PDLSCs increased(P <0.001).After interfering with the expression of integrinβ1,the results of ALP staining showed that the combination of rh FN and TGFβ3 induced a decrease in the osteogenic differentiation ability of h PDLSCs.Conclusion: In this study,bioinformatics was used to design and function prediction of fibronectin peptides.A new recombinant human fibronectin peptide(rh FN)was successfully constructed using the E.coli expression system,and fermentation amplification and protein identification were carried out.HPLC of rh FN Purity>95%.rh FN has the ability to recruit growth factors and is mediated by α5β1 integrin,which significantly promotes the osteogenic differentiation of h PDLSCs.Recombinant protein rh FN can be used as an extracellular matrix biomaterial for bone tissue repair engineering.