Stichopus Japonicus Polysaccharide Synergistically Enhance The Functionality Of BMP-2 To Induce Osteoblastic Differentiation

Bone morphogenetic protein(BMP-2)is an acidic glycoprotein which can promote bone formation and osteoblast differentiation..and the only local growth factor that can induce bone formation alone.However,the half-life of BMP-2 is very short,the diffusion speed is also fast,and it is easy to be decomposed by protease.These shortcomings result in less chance of BMP-2 binding to the receptor on the surface of cell membrane,and the role of promoting cell differentiation is limited to a certain extent.It has been reported that sulfated polysaccharides,such as heparin,chondroitin sulfate combined with BMP-2,could prolong the half-life of BMP-2 and promote the differentiation of osteoblasts.In this experiment,the crude extraction and purification of SP were carried out,and the effects of SP combined with BMP-2 on the osteogenic differentiation activity of cultured MC3T3-E1 cells were studied.The molecular weight and monosaccharide composition of SP were determined by HPLC.MC3T3-E1 cells were used as model to determine the ability of SP to promote bone activity in combination with BMP-2.Cell activity of MC3T3-E1 cells were determined by MTT method.Alkaline phosphatase(ALP)activity of MC3T3-E1 cells were determined by BCIP/NBT tissue staining;Calcium node and the content of osteocalcin were detected by alizarin red staining?Von Kossa staining and Elisa;The binding ability of BMP-2 to cells was detected by immunofluorescence;The expression of Runx-2,related gene BMP-2,Col1,OCN of osteoblast differentiation related protein in MC3T3-E1 cells was detected by Western Blot method and RT-PCR method.SCP were extracted and separated,and two components of SP-1,SP-2 were obtained.the molecular weight of which were 199.256 kDa?5.21 kDa.SP-1,which mainly consisted of Man?GlcA?GalN?Glc?Gal and Fuc.SP-2 mainly consists of Man?GlcN and Gal.ALP activity was determined by BCIP/NBT tissue staining.Compared with the effects of BMP-2alone,SP-2 combined with BMP-2 at 5 ?g/mL could significantly enhance the activity of ALP;Alizarin red staining,Von Kossa staining and Elisa staining also showed that 5 ?g/mL SP-2 and 50 ng/mL BMP-2 synergistic effect increased the formation of mineralized matrixand osteocalcin in the late stage of osteogenic differentiation compared with BMP-2 alone.IF method was used to analyze more BMP-2 binding to cell surface receptors when the concentration of SP-2 was 5 ?g/mL,and the binding was more stable.Western Blot and RT-PCR experiments demonstrated that SP-2 combined with BMP-2 at 5 ?g/mL could promote the expression of BMP-2,Runx-2,Col1 and OCN in BMP-2/Smad signaling pathway.SP-2 combined with BMP-2 promoted the differentiation of MC3T3-E1 cells by regulating BMP-2/Smad signaling pathway,and the secretion of early,middle and late differentiation marker ALP,Runx-2,Col1,OCN,and promoted the calcification of the cells in the late stage of differentiation.These results suggest that SP-2 can promote the differentiation of MC3T3-E1 cell and has the potential of developing and utilizing new functional foods and provides a new way of thinking for the prevention or remission of osteoporosis fracture and so on.