| Bone-related diseases and bone injury seriously affect healthy life and work of modern people,while the process of bone repair is slow and the medical costs is large,which is one of the most difficult problems that needs to be solved.With the development of molecular biology,gene therapy,cell therapy and treatment of growth factors have gradually become therapeutic hotspots.Growth factors are especially important and have become the focus of research on the treatment of fractures and bone injuries in the whole world.It is important to find a growth factor that accelerates the bone repair process and reduces the cost of treatment.Studies have shown that IGF-1 as a growth factor can regulate bone growth during growth and development.Three types of E peptides formed by alternative splicing at the C-terminus of IGF-1 have the functions of regulating bone and muscle development.Bone is the most intuitive indicator of the bodysize,therefore,the expression of the three types of IGF-1 E peptides was detected in seven tissues of large pigs(Large White pigs)and miniature pigs(Bama Xiang pigs).The results showed the expression level of Ea and Ec showed significant difference(P<0.01),indicating that Ea and Ec may be involved in bone growth.According to previous studies,Ec is also known as Mechano growth factor(MGF).MGF is highly expressed in many tissues after damage,participating in the repair process of skeletal muscle,nerve,heart muscle and other tissues.MGF is easy to be purified and synthesized,could function independently of IGF-1 without binding to IGF-1R receptor.Studies have shown that MGF mRNA can be overexpressed in osteoblasts under mechanical overload conditions.In the process of bone injury healing,the MGF mainly involves the proliferation,differentiation and mineralization of osteoblasts,then futher participate in the new bone formation.Many studies about,MGF peptides are concentrated on MGF-40 E peptides and MGF-24 E peptides of human and murine,while that of other species or shorter MGF E peptides have not been found.This study firstly obtained the amino acid sequence of MGF E peptides of different species from the NCBI database,then the sequences were aligned,and four types of representative MGF E peptides(T-MGF-25 E peptide,M-MGF-25 E peptide,T-MGF-19 E peptide,M-MGF-19 E peptide)were finally selected according to the secondary structure difference of the polypeptide and the hydrophobicity of the amino acid.Based on the selected four MGF E peptides,this study first detected their effect on the proliferation of osteoblast MC3T3-E1.The results showed that all the four types of MGF E peptides can promote osteoblast proliferation,of which T-MGF-19 E peptides can significantly block cells in S phase and G2 phase after 72 h,promoting DNA synthesis and cell proliferation(P<0.01),indicating that T-MGF-19 E peptide is more effective in bone repair.Then,the study further investigated the effect of the four types of MGF E peptides on the differentiation ability of MC3T3-E1 cells by detecting the enzyme activity of osteoblast differentiation marker protein ALP.The results showed that MGF-25 E peptide can promote the early differentiation of osteoblasts(7d).MGF-19 E peptide promoted the terminal differentiation of osteoblasts(14d),indicating the T-MGF-19 E peptide has a significant higher promoting cell differentiation ability(P<0.05).At the same time,this study examined the expression level of related factors(OCN,OPN,Col-1,Runx2,OSX)involved in osteoblast differentiation,similar to the ALP activity results,T-MGF-19 E peptide promoted or inhibited the expression of related differentiation genes,but the statistical analysis was not significant(P>0.05).Osteoblast mineralization maturation in vitro is a marker of osteoblasts,indicating that osteoblasts enter maturity.At 21 days,MGF-19 E peptide mineralization significantly higher than that of MGF-25 E peptide,T-type MGF E peptide was higher than that of the M-type MGF E peptide,and the T-MGF-19 E peptide mineralized nodules were the most apparent than the other groups(P<0.01).The above results showed that the T-MGF-19 E peptide can significantly promote the transformation of osteoblasts into bone cells.Due to the results of the four types of MGF E peptides on the proliferation,differentiation and mineralization of osteoblasts at the cellular level,,preliminarily confirmed the T-MGF-19 E peptide have positive effect on the bone repair and osteogenesis.Then,we further verified the bone damage repair ability of T-MGF-19 E peptide in vivo.In this study,three month old New Zealand white rabbit was selected to construct a 1 cm long tibia injury model,which was divided into T-MGF-19 E peptide low-dose and high-dose experimental groups for in vivo verification,the saline group was set as a control group.Both X-ray and HE bone tissue staining showed that T-MGF-19 E peptide had bone damage repair function,the recovery effect of low dose T-MGF-19 E peptide group was better than that of high dose T-MGF-19 E peptide group.The above results analyzed the effects of four types of MGF E peptide on bone injury mechanism and bone damage in rabbits,and proved that T-MGF-19 E peptide can promote bone repair,providing further development theoretical data and utilization of MGF E peptide in the field of bone injury repair. |
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