Study On The Effect And Mechanism Of VEGF In Promoting The Repair Of Aged Tendon

BackgroundTendon diseases and injuries are a serious problem for the aging population,often leading to pain,disability and a significant decline in quality of life.The incidence of tendon injuries,especially rotator cuff tears,increases with the aging of tendons.The aged tendon is not only difficult and slow to repair,but also difficult to restore normal function after recovery.At present,the treatment of tendon injury and tendon disease for the aged population is quite limited and the benefit people can get is not enough,the main reason is that the effect and mechanism of aging on tendon injury repair need to be further understood.Although the view that aging leads to the dysfunction of TSPCs has been widely accepted by researchers at home and abroad,the specific molecular mechanism has not been elucidated yet.The purpose of this study is to analyze the effect of aging on tendon healing and to explain its potential mechanism from the point of view of cell biology,histology and molecular biology,so as to find an effective target to improve the healing of aged tendon.Objectives1.To figure out the effects of aging on histology and biochemistry during tendon healing.2.To clarify the effect of aging on adipogenic differentiation of TSPCs and its mechanism.3.To screen the change of cytokines in TSPCs microenvironment induced by aging.4.To investigate the effect and mechanism of cytokine VEGF on aged tendon healing.Methods1.Effects of aging on histology and biochemistry in the process of tendon healingThe acute injury model of patellar tendon in young and aged rats was established.Tendon tissues at different time points were collected,RNA and protein were extracted and frozen tissue sections were made.QRT-PCR,western-blot,HE staining,Alcian blue staining,oil red O staining and micro-CT were used to evaluate the histological and biochemical effects of aging on tendon healing.2.Effect and mechanism of aging on adipogenic differentiation of TSPCs.The TSPCs extracted from young and aged rat patellar tendon was induced by adipogenic media in vitro.Firstly,the expression of aging marker β-Gal in aged TSPCs was confirmed by β-Gal staining.Then the adipogenic differentiation ability of the young and aged TSPCs was compared by q RT-PCR,western-blot,immunofluorescence and oil red O staining,and m RNA-seq was used to screen the key molecules and signal pathways that affect the adipogenic differentiation ability of aged TSPCs.3.Screening the changes of cytokines in TSPCs microenvironment caused by agingThe acute injury model of patellar tendon in rats was established.The patellar tendons of young and aging rats were collected at 3,7 and 14 days after operation.The difference of cytokines in patellar tendon between the two groups was detected by cytokine microarrays,and then the differential cytokines were used to stimulate TSPCs with adipogenic induction.The cytokines that can affect the adipogenic differentiation of TSPCs were screened by western-blot.Then the best concentration of this cytokine(vascular endothelial growth factor,VEGF)affecting adipogenic differentiation of TSPCs was screened by concentration gradient experiment.Then the key molecules and signaling pathways of VEGF affecting adipogenic differentiation of TSPCs were analyzed by m RNA-seq,and the effect of VEGF on adipogenic differentiation of TSPCs was verified by inhibiting the expression of this molecule.4.Effect of VEGF on aged tendon healing and its mechanismThe acute injury model of patellar tendon was established and exogenous VEGF was injected into the tendons.Samples were collected for frozen tissue sections.HE staining,Alcian blue staining,immunofluorescence and oil red O staining were used to evaluate the histological effect of VEGF on aging tendon repair.At the same time,tendons were collected to assess the effect of VEGF on biomechanical strength after aging tendon healing.ConclusionsThis research comprehensively describes the regulatory effect of VEGF on the differentiation of TSPCs and the role and mechanism of VEGF during aged tendon healing.First of all,we found that the healing effect of aged tendon was worse than that of young tendon,mainly manifested by the decrease of collagen synthesis and the accumulation of adipocytes in the healing tendons.Considering the important role of TSPCs in the repair of tendon injury,we firstly found that aging reduced the adipogenic differentiation ability of TSPCs by inhibiting PPARγ pathway,thus excluding the accumulation of adipocytes during aging tendon healing was due to the enhancement of adipogenic differentiation caused by TSPCs aging.VEGF,a key factor in the stem cell microenvironment,was screened out through cytokine microarray.Through stimulation experiments in vitro and exogenous intervention experiments in vivo,we proved that VEGF which was lacking in the aged tendon microenvironment could inhibit the adipogenic differentiation of TSPCs.At the same time,VEGF can effectively promote the healing of aged tendons,reduce the accumulation of adipocytes in repaired tendons,promote the formation of collagen in aged tendons and improve the biomechanical strength of aged tendons.At the same time,it is also preliminarily clarified that VEGF affects the adipogenic differentiation of TSPCs and the healing of aged tendons by regulating the expression of SPP-1.