Preparation Of Recombinant Resilin Protein Based Hydrogel And Its Application In Myocardial Tissue Engineering

Myocardial infarction(MI)has a high morbidity and mortality in China.Due to the regenerative capacity of the myocardial cells is extremely limited,the heart cannot make up for the lost myocardial cells by its own repair mechanismafter MI.Fibrosis of the infarcted myocardial tissue and compensatory hypertrophy of the remaining myocardium further lead to the development of heart failure,leading to the death of the patient.Although existing therapeutic methods can alleviate the symptoms of patients to some extent,they cannot fundamentally solve the problem of repair of myocardial injury after ischemia.Thus,negative ventricular remodeling after MI cannot be suppressed,eventually leading to heart failure.The injectable myocardial tissue engineering strategy,in which cells and/or growth factors are injected into the site of myocardial injury to repair the damage using injectable hydrogel materials as carriers,is expected to be a new treatment method in addition to drugs,myocardial revascularization and heart transplantation.In recent years,with the deepening of research on injectable hydrogels for myocardial tissue engineering,researchers have gradually paid more and more attention to elastic materials with good biocompatibility.The results show that the elastic hydrogel material not only can be used as carriers of target substances such as cells and growth factors,also because of its good elasticity and stretchability can provide mechanical assistance to myocardial tissue and improve mechanical microenvironment of infarcted tissue.Therefore,it can improve the local myocardial movement disorder and reduce the stress of the ventricular wall,which is beneficial to the repair of the injured part and the improvement of the heart function.Resilin,an elastin found in insect arthropods,has attracted much attention due to its remarkable elasticity,resilience and fatigue resistance.Since the identification of the first gene sequence for resilin,several recombinant sequences based on the resilin sequence have been designed and constructed,and have been successfully applied in tissue engineering and drug delivery,especially in tissue engineering of blood vessels,vocal cords,cartilage and muscles.In the study of recombinant resilin protein-derived elastic materials,the resilin hydrogel material based on recombinant RLP12 has attracted much attention in recent years due to its excellent mechanical properties and biological activities.RLP12 protein adds a cell binding domain to the elastic sequence ofthe resilin core,thus the hydrogels formed by cross-linking can show excellent cell adhesion and the ability to promote cell proliferation,as well as repair vocal cord injury.However,Whether the hydrogel material derived from the recombinant resilin protein can be used as an injectable vector for stem cell repair of MI is unclear in the field of injectable myocardial tissue engineering research.In this study,we firstly constructed the expression vector of resilin protein by gene engineering,then obtained the recombinant RLP12 protein and rec1-resilin after being induced expression and purified,and prepared the resilin hydrogel material based on the recombinant RLP12 protein by chemical cross-linking method.On those bases,BADSCs labeled with red fluorescent protein were carried by resilin hydrogel and transplanted into rat MI area.Further research was conducted on the ability of repair MI injury,so as to verify the feasibility and effect of resilin hydrogel injection with BADSCs for myocardial injury repair.This paper mainly includes the following two parts:Part I: Preparation of recombinant Resilin protein-derived hydrogel materialsObjective: To prepare recombinant Resilin protein-derived hydrogel and determine its mechanical properties.Methods: Two recombinant resilin protein expression vectors,RLP12 and rec1-resilin,were constructed and induced for expression and purification.The resilin hydrogel material based on recombinant RLP12 protein was prepared by chemical crosslinking method,and its mechanical properties were identified by the oscillating shear rheological properties test.Results: Recombinant resilin protein expression vectors,RLP12 and rec1-resilin,were successfully constructed.Recombinant RLP12 and rec1-resilin protein were obtained respectively after being induced expression and purified.Three recombinant RLP12-resilin hydrogels with different PEG contents were obtained by THP cross-linking.The shear modulus of the oscillating shear rheological properties is 10?15k Pa.Conclusion: Recombinant RLP12 protein and rec1-resilin protein were successfully prepared,the resilin-hydrogel material based on recombinant RLP12 protein was also obtained.Part II: The treatment of myocardial infarction by using Resilin hydrogel as a vehicle of carrying brown adipose tissue-derived stem cell(BADSCs)Objective: To verify the feasibility and effect of recombinant resilin protein based hydrogel as an injectable carrier of stem cells in the repair of MI injury.Methods:BADSCs labeled with red fluorescent protein were transplanted in rat MI area using recombinant RLP12 protein-derived resilin hydrogel as the carrier.After 4 weeks,cardiac function was measured by echocardiography.The MI area,collagen deposition,microvascular density,survival and differentiation of BADSCs in vivo were detected respectivelyby histology,immunohistochemical staining and immunofluorescence staining.Results: The recombinant resilin-protein hydrogel,as the carrier of BADSCs,could significantly improve the survival of BADSCs in myocardial tissue.After 4 weeks,the transplantation could significantly improve the cardiac function,reduce the MI volume,reduce the fiber deposition level and increase the microvascular density in the MI rats.Recombinant resilin-protein hydrogel can improve the distribution level of collagen ? in MI area as well asthe edge area of MI.In addition,the recombinant resilin-protein hydrogel could improve the ability of BADSCs to differentiate into CTNT-positive and A-SMA positive cells in vivo.Conclusion: Recombinant resilin protein hydrogel can be used as an injectable carrier of stem cells in the treatment of MI,which may improve the compliance of myocardial tissue by improving the mechanical microenvironment of the MI site and increasing the distribution of collagen ?.In this study,Resilin hydrogel based on RLP12 protein was used as the carrier of brown adipose derived stem cells to transplant in rat MI for the first time,realizing the effect of inhibiting negative ventricular remodeling and improving cardiac function after MI.This study is not only expected to expand the application of the resilin protein as an elastic materialin myocardial tissue engineering,also provide a new guidance for MI based on injectable hydrogels and stem cells,which is of great significance for future treatment of ischemic heart disease.