Study On The Effect Of The Recombimation RGD-containing Peptide From Wild Silk Fibroin On Cell Adhesion And Proliferation

Silk fibroin is a natural protein synthesized and secreted by silkworms,including silk fibroin(Bombyx mori)and wild silk fibrion(Antheraea pernyi,Antherara yamamai,Dictyopoea japonica Moore and Attacus atlas Linnaeus etc.).The current research focuses on the application of silk fibroin,and studies have found that silk fibroin not only has excellent mechanical properties and biodegradability,but also the materials it prepares can well support cell adhesion,spreading,and proliferation.It is good for the differentiation of stem cells.However,the study of wild silkworms(Antheraea pernyi and Antheraea yamamai)involves slightly less.Highly repetitive RGD peptide distributes in the amino acid sequence of Antheraea pernyi and Antheraea yamamai silk fibroin.RGD peptide can be used as recognition sites of integrin and ligands protein.Which promotes integrin binding with ligands,then the extracellular signals pass into cells and activate the relevant functions of cells.Since there are not only RGD-containing peptides have the function of promoting cell adhesion and growth,it is clear whether RGD from Antheraea pernyi and Antheraea yamamai has a similar function as the RGD in extracellular matrix,which is an important extension of the application of wild silk fibroin as a biomaterial.In addition,studies have shown that the substitution of any one of the amino acids in RGD will affect its biological activity,so it was important to reveal whether the integrity of the side chain groups of RGD peptides from wild silk fibroin was different for cells.The role is also crucial.Therefore,In this article,E.coli was used to express the sequence of the RGD-containing short peptide gene of the recombinant clone to protect and re-release its pendant carboxyl group and further we studied the effect of this RGD-containing short peptide sequence on cell adhesion and proliferation.The main work accomplished in this paper has the following aspects:Firstly,the expression vector p GEX-KG containing the RGD peptide sequence(GSGAGG-RGD-GGYGDGSS)that has been constructed in the laboratory was transformed into E.coli for expression,and the protein was purified by GST affinity chromatography and purified.After the fusion protein was digested by Thrombin,the target peptide(-RGD-)4 was released.The molecular weights of the peptides of interest obtained by SDS-PAGE electrophoresis and mass spectrometry were basically in agreement with the theoretical molecular weights,indicating that the designed peptide sequences were correctly expressed.Secondly,after carboxyl protection of the side chain group of the target peptide(-RGD-)4,FTIR,Raman,and nuclear magnetic resonance analysis showed that the carboxyl group of the side chain of the target peptide has been protected and a new ester bond was formed.Thirdly,analysis by FTIR and Raman spectroscopy showed that the protected peptide(-RGD-)4 of the pendant carboxyl group was successfully grafted onto the silk fibroin membrane and the protective group could be easily removed;after it was labeled with FITC,the target peptide(-RGD-)4 was used to verify that the target peptide was grafted and evenly distributed on the surface of silk fibroin film by fluorescence spectra and fluorescence photographs;the maximum grafting unit area was 0.485 mg/cm2 as determined by BCA(carboxy-protected(-RGD-)4).Fourthly,different concentrations(0.1/0.2/0.5 mg/cm2)of polypeptide(-RGD-)4 and side group carboxy-protected polypeptides(-RGD-)4 were cross-linked on silk fibroin membranes(the protective groups were retained and one group was deprotected after grafting).After inoculation of BMSCs and HUVECs cells in each group of materials,their effects on cell adhesion and proliferation were tested.The results showed that each group of grafted(-RGD-)4 materials increased the adhesion and proliferation of cells,and when the cells adhered to the silk fibroin membrane with a 0.5 mg/cm 2(-RGD-)4,showing extensive rate,the fastest cell proliferation.At last,compared with the unprotected carboxyl group,the retention of free carboxyl group of(-RGD-)4 significantly increased the adhesion and proliferation activity of BMSCs,but had no significant effect on the cell adhesion and proliferation of HUVECs.