Construction And Functional Study Of HMW Adiponectin Model Based On Nanospheres

Adiponectin is a cytokine secreted by adipose tissue,which is closely related to diabetes,obesity,and metabolic syndrome.It also plays an active protective role in the course of cardiovascular disease,cancer,non-alcoholic fatty liver and other diseases.Adiponectin exists in the body in various forms such as monomers,trimers,high polymers,and spherical core regions,and the function and action of different forms are not the same.Among them,the high polymer adiponectin formed by polymerization of 18 or more monomers through tail-crosslinking shows the highest biological activity in many aspects.Due to the complex spatial structure of high polymer adiponectin,it has not been able to be obtained in vitro,which greatly limits the related research.In view of this,this study attempts to construct a biologically active HMW adiponectin model in vitro using nanospheres as a carrier.In the study,the adiponectin fusion protein with anchoring function was first prepared;then the polymer materials with both biocompatibility and degradability were selected and prepared into nano-sized microsphere particles with good stability and high dispersion.Finally,the fusion protein was incubated with the nanospheres to aggregate several monomeric proteins to form HMW adiponectin structure through the anchoring of the adiponectin fusion protein tail.Subsequently,its biological activity was verified,and the biological function of HMW adiponectin was further explored.The research has achieved results as follows:(1)The fusion protein and the HMW models of adiponectin were designed innovatatively.The introduction of extensible linking sequences into the fusion protein can make the functional regions of adiponectin more conducive to distribution on the surface of nanospheres.According to bioinformatics analysis,the structure and characteristics of the fusion protein were in line with design expectations.It is feasible to anchor to the surface of nanospheres effectively and exhibit biological activities completely.(2)Four expression systems were constructed to obtain the target protein.In order to obtain the protein with high activity and yield,the four most widely used expression systems were constructed in the study.After selected with different methods,the baculovirus-insect expression system was finally chosen to synthesize the target protein.After detection,the adiponectin and fusion protein showed good biological activity,with the expression levels of 1.8 and 2.3 mg / L,respectively.(3)Preparation of nanospheres and polymer models.Six kinds of polymer materials with excellent biocompatibility and degradability were preferred,and nano-scale microspheres were prepared by solvent evaporation and ultrasonic emulsification methods.Then,several kinds of polymer models were prepared under a variety of compound conditions,and the particle size,zeta potential,and scanning electron microscope were used for detection and selection.Finally,the polymer models that met the test requirements were obtained by using of PLA,PLGA and PHB.(4)Both the cell and animal tests confirmed that the HMW adiponectin model have basic biological activity.By verifying with detections of NF-?B,AMPK and other indicators in ob/ob mice and the evidence of cell experiments,two polymer models with biological activities of HMW adiponectin have been proved successfully constructed.(5)The relationship between HMW adiponectin and AKT has been investigated.By analyzing the indicators such as AMPK,PI3 K,T-cadherin and AKT,the effects of adiponectin monomer and HMW adiponectin on AKT activation were determined.Adiponectin monomer can activate AKT by stimulating both of AMPK(primary pathway)and PI3 K,whereas HMW adiponectin can significantly activate AKT by stimulating PI3 K.(6)The relationship between HMW adiponectin and T-cadherin expression has been explored.In skeletal muscle and endothelial cells of ob / ob mice,both adiponectin and HMW adiponectin can promote the expression of T-cadherin mildly.The effect of adiponectin is weak and HMW adiponectin is slightly stronger.The study confirmed that the HMW adiponectin model obtained by combining adiponectin fusion protein and nano-microspheres has corresponding biological functions,and the model has been successfully constructed,which can provide support for the subsequent research of HMW adiponectin.