The Preparation Of Modified IGE-1 Containing Collagen Binding Domain And The Effect Of CBD-IGF-1 Composite PLGA Conduit In The Repair Of Nerve Injury

Peripheral nerve injury,as a common disease,is different from other tissue injuries;its regeneration is slow and usually incomplete.Only less than half of the patients with nerve injury recovered good to excellent motor or sensory function.Nerve injury and repair is the focus of interdisciplinary research in the field of neuroscience and tissue engineering.And axonal oriented directional regeneration is the key to nerve function recovery.The nerve conduit plays an important role in the directional regeneration of axon.As a famous material of neural tissue engineering,PLGA is widely used because of its good histocompatibility and controllable degradation.However,the rate of nerve regeneration and recovery of nerve function is still lower than that of autologous nerve graft.PLGA catheter requires cytokine to provide more favorable microenvironment for nerve regeneration,such as cell adhesion molecules and neurotrophic factors.IGF-1 is a single peptide chain containing about 70 amino acids,belonging to insulin family.IGF-1 can promote growth and development of human body,proliferation and differentiation of nerve cells,repair of trauma,and integration of structure and function.In the nervous system,IGF-1 can promote neurogenesis,synapse formation,neuroprotection,proliferation and anti-apoptosis.At the same time,it also has the function of promoting axon regeneration and remyelination.However,its limited target specificity,and short half-life inhibit its application as therapeutic agents.To overcome these obstacles,we fused the collagen binding domain(CBD)of collagenase to human IGF-1 protein.CBD can enhance the retention time of the drug in the damaged area.And then Type I collagen was cross linked to the PLGA catheter.Type I collagen loaded on the PLGA catheter can enhance the cell adhesion of the catheter and provide the target binding site for CBD-IGF-1.ObjectiveThe purpose of this study was to modify human insulin-like growth factor 1 using gene recombination technology,overcome the shortcomings of rapid clearance,avoid the serious side effects caused by long-term repeated overuse,and provide a new treatment for peripheral nerve injury with the help of neural tissue engineering.MethodThe study fused IGF-1 to the collagen binding domain(CBD)through gene recombination technology and expressed target proteins in microbial expression system.And then affinity test with collagen and biological ability were tested in vitro.Biological ability included proliferation effects,neural differentiation effects and the ability to promote the expression of other growth factors.And then,loaded on PLGA,the effect of promoting nerve repair in vivo was tested in rat sciatic nerve injury model.ResultsThe recombinant protein was obtained using gene recombination technique and microbial expression system.The recombinant protein was identified as CBD-IGF-1 by sodium dodecyl sulphate polacrylaminegel electrophoresis and Western blot.The results of collagen-binding assay showed that collagen binding ability of CBD-IGF-1 is significantly better than that of commercialized IGF-1(p<0.05).And with the increase of drug concentration,the collagen binding rate was getting higher and higher.CBD-IGF-1 could effectively promote the proliferation of Schwann cells and PC12 cells.Compared with the cytotoxicity of IGF-1 at lower concentration,CBD-IGF-1 could effectively promote cell proliferation at 1000 ng/ml.CBD-IGF-1 Showed a role for neurodifferentiation similar to IGF-1(p>0.05).CBD-IGF-1 could promote the expression of IGF-1 receptor and NGF in Schwann cells,and inhibit the synthesis of Nogo-A protein in PC12 cells.Compared with IGF-1,the ability of CBD-IGF-1 was less effective in the initial stage,but it was stable and lasting(p<0.05).The surface characteristics analysis showed that the contact angle of PLGA was significantly reduced after loading with collagen and CBD-IGF-1,which meaned that the hydrophilicity was increased(p<0.05).After successfully constructing model of rat sciatic nerve defect,distal and proximal nerve stumps were bridged using a PLGA catheter with collagen and CBD-IGF-1.The results of gait analysis,electrophysiology and histomorphometry illustrated that PLGA loaded with collagen and CBD-IGF-1 could effectively promote nerve regeneration,and CBD-IGF-1 was more effective in promoting nerve regeneration in vivo than IGF-1(p<0.05).Conclusion1.CBD-IGF-1 can be successfully obtained through gene recombination and microbial expression system.2.The binding ability of CBD-IGF-1 is significantly higher than that of IGF-1.CBD-IGF-1 can promote the proliferation rate of Schwann cells and PC12 cells.CBD-IGF-1 can promote the neurodifferentiation of PC12 cells.CBD-IGF-1 can promote the expression of IGF-1 receptor and NGF in Schwann cells,and inhibit the synthesis of Nogo-A protein in PC12 cells.3.CBD-IGF-1 has better function of promoting nerve regeneration in vivo than IGF-1.4.The PLGA catheter carrying collagen and CBD-IGF-1 are effective combinations to promote nerve regeneration.Innovation point1.The study fused the collagen binding domain(CBD)of collagenase to human IGF-1 protein,which enhanced the binding ability and stability of IGF-1,and optimized the biological properties of IGF-1.2.PLGA catheters loaded with collagen and CBD-IGF-1 are potential neural tissue engineering materials.