| IntroductionTendinopathy,one of the most frequently overuse injuries,is a multifactorial spectrum of tendon disorders which is a clinical syndrome characterized by the combination of activity-related pain,local edema,focal tenderness to palpation,and decreased strength in the affected area.According to statistic,tens of millions of tendon injuries occur worldwide each year.While there are various treatments of tendinopathy,the therapeutic effect is limited.How to restore the normal structures and functions of the injured tendons is still a great challenge to the orthopedics and sports medicine.With the discovery of tendon stem cells,the research of tendinopathy has entered a new stage.As a specific stem cells of tendon,TSCs play an important role in the maintenance of normal physiological function and pathological process.How to regulate the normal differentiation of TSCs to repair the microtear of tendon is of great significance to the prevention and treatment of tendinopathy.At present,it has been proved that TSCs can be regulated by many factors,such as mechanical stretch,inflammatory factor,hormone cortiso and cytokines.Among numerous regulatory factors,mechanical stretch is the initial factor of tendinopathy,so the importance of regulatory effects of TSCs is self-evident.The mechano growth factor,an alternative splice variant of the IGF-1 gene,exits widely in bone,skeletal muscle,tendon,brain and other tissues,which is activated by mechanical stretch,ischaemia,local damage and before stimuli respectively.In recent years,increasing attention has been paid to MGF because of its unique structure of E peptide and specific functions.It has been proved that MGF plays an important role in the proliferation,migration and differentiation of many kinds of cells.In addition,the expression of MGF mRNA in rat gastrocnemius muscle was significantly increased after different trainings.Olesen et al.established animal models by surgical removal of synergistic muscle to the plantaris muscle of the rat,thus increasing the load to plantaris muscle and tendon and found that the expression of MGF in tendon was rapidly up-regulated earlier than IGF-1,which indicated that MGF plays an important role in the process of collagen synthesis and tendon growth induced by mechanical stress.Zhang et al.documented that the expression of MGF was significantly increased in mice after treadmill training,and the effect was intensity-dependent.However,how the expression of MGF changes in TSCs,the "star cell" in the pathogenesis of tendinopathy,after mechanical stretch and whether MGF plays a role in regulating the proliferation,differentiation and migration of TSCs have not been reported ever before.Moreover,does mechanical drafting and MGF regulate differentiation of TSCs alone or in combination? Is it synergistic or antagonistic? This series of problems are to be solved.Therefore,the main aim of this research was to verify the effect of mechanical stretch on the regulation of differentiation of TSCs and the expression of MGF,and discuss the function of MGF on the proliferation,differentiation and migration of TSCs.Finally,the effect of MGF on the regulation of TSCs differentiation under the condition of mechanical stretch and the mechanisms were discussed.MethodPart 1: TSCs in rats were isolated and identificated,then the mRNA and protein levels of osteogenic,adipogenic and tenogenic differentiation related genes before and after mechanical stretch were detected from the transcription and translation levels by Real-time PCR and Western Blot.Part 2: The cell proliferation of TSCs treated with different concentrations of MGF was detected by CCK8 and real-time dynamic cellular analysis.Then the effects of different concentrations of MGF on the migration ability and cytoskeleton of TSCs were detected by Transwell,scratch test and confocal laser scanning microscope.Moreover,the protein levels of tenogenic differentiation related genes were detected by Western Blot in TSCs,which were treated by different concentrations of MGF or PD98059,the MAPK/ERK pathway inhibitor.Part 3: The relative expression levels of osteogenic,adipogenic and tenogenic differentiation related genes were detected by Real-time PCR and Western Blot in TSCs treated with mechanical stretch and MGF.Result Part 1:1.The relative expression levels of TNC and RUNX2 of the group A(4% 1hz)were significantly increased compared with the static culture group at 48h(P<0.05).The relative expression of TNC of the group B(4% 2Hz)were significantly higher than the static culture group at each time point(P<0.05),while the expression of CEBPα was significantly inhibited at 24h(P<0.05),but there was no significant difference in the relative expression of RUNX2 between the stretch and control group(P>0.05).The relative expression of RUNX2 of the group C(8% 1Hz)were significantly increased compared with the static culture group at 24h(P<0.05),while there were no significant differences in the relative expression levels of adipogenic and tenogenic differentiation related genes between the stretch and control group(P>0.05).The relative expression of CEBPα of the group D(8% 2Hz)were significantly higher than the static culture group at 48h(P<0.05),while the expression of TNC was significantly inhibitedat 24h(P<0.05),but there was no significant difference in the relative expression of RUNX2 between the stretch and control group(P>0.05).2.The relative expression levels of MGF mRNA of all the stretch groups were significantly increased compared with the static culture group at each time point.The expression level of MGF mRNA was significantly increased in group B(4% 2Hz and group C(8% 1Hz)at 12h(P<0.05),and the highest peak of MGF mRNA expression emerged at 24h(P<0.05).Part 2:1.CCK-8 assays demonstrated that the cell proliferation of TSCs was significantly enhanced after treated by 5ng/ml MGF for 24 h or more(P<0.05),while there were no significant differences between other groups in OD(P>0.05).The RTCA showed that TSCs cell index was slightly higher than other groups after being treated with 1ng/ml and 5ng/ml MGF,with no differences between them.When treated with 10,25,50ng/ml MGF or 5ng/ml MGF+10μg/ml PQ401,the cell index of TSCs were significantly lower than that of the non-treatment group(P<0.05).The cell index of TSCs treated with 5ng/mlMGF+50μM PD98059 was significantly lower than all other groups(P<0.05).2.The results of scratch-healing experiments indicated that the migration abilities of TSCs with 1,5 and 10ng/ml MGF treated were significantly increased than that of the control group,and The TSCs migrate most actively when treated with 5ng/ml MGF(P<0.05).The result of transwell system showed that MGF stimulated TSCs migration in all of the concentrations we detected,with cell migration at concentrations of 5.0 and 7.5 ng/mL being particularly significant(P<0.05).Moreover treatment with 5ng/ml MGF and 10μg/ml PQ401 significantly reduced the number of TSCs migration and OD value compared with the treatment with 5ng/ml MGF alone,and the differences were statistically significant(P<0.05).After exposure to 5ng/ml MGF and 10μg/ml PQ401,compared with other groups,the cell morphology changed significantly with the minimum number of cell migration and the lowest OD value.Confocal laser scanning microscopy showed that after treatment with 0,2.5,5.0,7.5,10.0 ng/ml MGF treatment,the cytoskeleton of TSCs had good continuity and strong fluorescence intensity,the cells presented a typical long spindle shape,and F-actins were arranged in order.After treatment with 25 and 50ng/ml MGF,the morphology of TSCs turned irregular,and the cytoskeleton was obviously discontinuity disorganization with weak fluorescence.When the TSCs were treated with 10μg/ml PQ401,the morphology was significantly changed,and the cytoskeleton was broken and the fluorescence was extremely weak.3.The results of Western Blot indicated that the relative expression levels of TNC and SCX protein in 5ng/ml and 10ng/ml MGF group were significantly higher than those in control group(P<0.05).There were no significantly differences between TSCs treated with 1.0 and 25ng/ml MGF and control group in relative expression levels of TNC and SCX protein(P>0.05).Part 3:1.The results of real-time PCR showed that when TSCs treated by 0,2.5 and 5.0 ng/ml MGF were stretched(4% 2Hz)for 24 h,the relative expression of SCX and TNC mRNA was significantly increased compared with the static culture group(P<0.05).Furthermor,the relative expression of RUNX2 and DLX5 as well as AP2 and PPARγ mRNA was significantly lower than that of static culture group(P<0.05).When compared the TSCs treated by 50μM PD98059 and 5ng/ml MGF with the TSCs treated by 5ng/ml MGF alone,the relative expression of all gene mRNA was significantly decreased after being stretched(4% 2Hz)for 24h(P<0.05).2.The results of Western blot showed that when TSCs treated by 0ng/ml,2.5ng/ml and 5.0ng/ml MGF were stretched(4% 2Hz)for 24 h,the relative expression of TNC protein was significantly increased compared with the static culture group(P<0.05).Furthermor,the relative expression of RUNX2 and CEBPα protein was significantly lower than that of static culture group(P<0.05).When compared the TSCs treated by 50μM PD98059 and 5ng/ml MGF with the TSCs treated by 5ng/ml MGF alone,the relative expression of all gene protein was significantly decreased after being stretched(4% 2Hz)for 24h(P<0.05).ConclusionPart 1: Different conditions of mechanical stretch inducing TSCs to differentiate into different cells.4% 2Hz 24 h is the optimal stretch condition for tenogenic differentiation,8% 1Hz 24 h is the optimum stretch condition for osteogenic differentiation,and 8% 2Hz 48 h is the optimal stretch condition for adipogenic differentiation.Mechanical stretch can increase the expression level of MGF in TSCs,and MGF is related to the tenogenic differentiation.Part 2: MGF can enhance the migration and proliferation of TSCs via activation of MAPK/ERK signaling pathway,and can induce the differentiation of TSCs into tendon cells.Part 3: Mechanical stretch and MGF can jointly promote the differentiation of TSCs into the tendon and inhibit the adipogenic and osteogenic differentiation.Furthermore,MGF is capable of inducing TSCs differentiate under mechanical stretch by activating the MAPK/ERK pathway. |
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