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

Silk fibroin is a natural protein synthesized and secreted by silkworms,mainly composed of silk fibroin and sericin.It has excellent mechanical properties and biocompatibility and is now widely used in biomaterials,especially in tissue engineering.A large number of studies have shown that silk fibroin can better support the adhesion,proliferation or differentiation of various cells,while the research on silk fibroin(the silkworm and tussah)is slightly less.However,the highly aminogenic RGD sequence is distributed in the amino acid composition of the wild silk fibroin.The RGD short peptide is also a tripeptide present in various extracellular matrices,which has been shown to be related to cell adhesion and has the function of regulating cell behavior.However,not all proteins or polypeptides with RGD short peptides have the function of regulating cell function,and the functions of different cells are not completely the same.Studies have found that arginine substitution,glycine deletion or asparagine instead of aspartic acid greatly reduced the activity of RGD sequence,indicating that the integrity of the RGD tripeptide side chain is also crucial for its activity.In order to understand whether RGD-containing silk fibroin has a special role in regulating cell function and its influencing factors,this paper biosynthesized peptides from wild silk fibroin containing RGD and studied the adhesion of peptides to skin fibroblasts.The effect of the RGD tripeptide side group on cell adhesion proliferation was studied with emphasis.The main work done in this paper has the following aspects:(1)The expression vector pGEX-AY4 containing the gene encoding the RGD peptide gene(GSGAGG-RGD-GGYGDGSS)which our laboratory had been constructed was transformed into Escherichia coli to induce expression,and the fusion protein was purified by GST affinity resin.The peptide of interest(-RGD-)4 was released by thrombin digestion.The expression products were identified by polyacrylamide gel electrophoresis and mass spectrometry.The results showed that the molecular weight of(-RGD-)4 was consistent with the theoretical molecular weight,indicating that the designed RGD sequence was correctly expressed.(2)Covalently grafting onto the polyester film by(-RGD-)4 side group-NH2,-COOH and-OH,using Fourier transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS)Coomassie blue staining and spectrometer element scanning(EDS)qualitative analysis of grafting.FTIR and XPS results indicate that an ester bond and a new amide bond are formed in each group respectively after after the grafting reaction;Coomassie blue staining showed that the(-RGD-)4 peptide appeared on the polyester film after the graft reaction.EDS showed that nitrogen was present on the polyester film after the graft reaction.(3)The graft amount,arginine(Arg)content and-COOH content on the surface of polyester film(-RGD-)4 were quantitatively determined.The maximum grafting amounts of the(-RGD-)4-OH graft group,the-COOH graft group and the-NH2 graft group were 0.356,0.364 and 0.342 mg/cm2,respectively.The maximum Arg content of the polyester film on the(-RGD-)4-OH graft group and the-COOH graft group was 0.044 and 0.076 mg/cm2,respectively,(-RGD-)4-NH2 grafted polyester film surface-COOH maximum content is 0.075mmol/cm2.When the same amount of grafting(0.342 mg/cm2)was controlled for three groups,the addition amount of the-OH grafting group,the-COOH grafting group and the-NH2 grafting group(-RGD-)4 for the surface grafting of the polyester was measured.They were 0.38,0.55 and 0.4 mg/cm2,respectively.(4)The effect of(-RGD-)4 on cell adhesion was detected by cytometry and CCK8.The results showed that when(-RGD-)4 was directly added to the medium at a concentration of 0.06 mM,the cell adhesion rate was remarkably increased.Grafting of the(-RGD-)4 peptide on the surface of the polyester significantly increased the adhesion of skin fibroblasts.When the surface of polyester was grafted with the same amount(-RGD-)4 by different principles,the grafting of-OH group was the most favorable for cell adhesion,followed by the grafting of-NH2 group,and the grafting adhesion rate of-COOH group was the lowest.The cell adhesion of-OH grafting group increased with the increase of grafting amount.When the grafting amount was 0.1 mg/cm2,the adhesion rate increased significantly,and the adhesion rate was the highest at 0.15 mg/cm2.(5)The morphology of the cells was observed by inverted fluorescence microscopy and the effect of(-RGD-)4 on cell proliferation was examined by CCK8.The results showed that(-RGD-)4 also promotes the proliferative activity of skin fibroblasts,when the surface of polyester was grafted with the same amount(-RGD-)4 by different principles,the grafting of-OH group was the most favorable for cell proliferation,followed by grafting of-NH2 group,the-COOH group had the lowest degree of proliferation.The proliferation of cells in the-OH grafting group increased with the increase of grafting amount.When the grafting amount was 0.15 mg/cm2,the degree of proliferation was remarkably increased.