The Effect Of Mechanical Loading On Expression Of RhoA/ROCK Molecules In Tendon Stem Cells And These Preliminary Observations Of Osteogenetic Differentiation

Tendinopathy is one of the most common sports injury diseases. It is seriously affectthe athlete’s sports career. In people’s normal physiological conditions， the movementprocess of tendon injury and tendon repair maintained a certain balance. When this balanceis broken, it will result in pathological，and tendon micro damage is aggravating，eventually become tendinopathys.In our previous found in clinical work，in the tendinopathys patients, tendon tissuecontains heterotopic ossification, and heterotopic ossification is one of the importantpathological manifestations of tendinopathy. The causes for tendon caused by excessive useof tendon minor damage， but the tendons of ossification of origin are not very clear. Wefound that under the condition of certain mechanical loading， TSCs can direclyosteogenetic differentiation. It is show that mechanical loading can lead TSCs to directiondifferentiation，and aggravate tendon injury. Envently lead to the failure of tendon repairitself. How to control the TSCs osteogenetic differentiation is one of the key scientificproblems to avoid Tendinopathy pathogenesis. So the regulation for TSCs osteogeneticdifferentiation and the mechanism function of tendon micro damage repair are an importantscientific problem and significance clinical value.In the mesenchymal stem cells (MSCs) related research, it is found that RhoA is animportant member of the family of Rho，and it is mechanical sensitivity. Mechanicalloading can make its activation and produce biological effection. It may participate in theprocess of TSCs osteogenesis. ROCK is a major downstream receptor family of RhoA.Inrecent years， the reseaches found that RhoA/ROCK affect stem cell fate ofdifferentiation. Similarly, in terms of regulate the osteogenetic differentiation of TSCs，RhoA/ROCK signaling molecules may play a major role. Whether mechanical loading isregulate TSCs osteogenetic differentiation through the RhoA/ROCK signaling molecules isstill to be further discussed. Based on previous reports， we put forward the following hypothesis: mechanicalloading can cause expression of RhoA/ROCK in TSCs， thereby to accelerate theosteogenetic differentiation in terms of TSCs，blocking the expression RhoA/ROCKsignaling molecules in TSCs， the expression of osteogenesis differentiation is downregulated after mechanical loading inTSCs.Our research is divided into three parts1. The effect of mechanical loading on expression of osteogenesis molecules intendon stem cells.1.1Respectively from two aspects of gene expression and protein expression，reserchthe osteogenesis differentiation index Runx2and BMP2expression level and change trendafter TSCs under different loading conditions.1.2The result of the experiment1.2.1Different intensity of loading on BMP2and Runx2gene expressionUnder the load1day,4%and8%loading strength, the mRNA expression of BMP2had no significant change. In the load2days and3days，4%load, the mRNA expressionof BMP2express no significant change in intensity， loading strength under8%BMP2mRNA expression upregulated (P <0.05).At different time points (1day，2days，3days)， Runx2mRNA expression in theloading of4%and8%under the intensity compared with the control group with significantdifference (P <0.05).1.2.2Different intensity of loading on BMP2and Runx2protein expressionBMP2under different time point4%intensity control group had no significantdifference (P>0.05) compared， in the load1day，2days， Loading of8%strength underBMP-2expression compared with the control group had no significant increase (P>0.05)，load three days significantly increased compared with control group.(P＜0.05).In1day，2days，3days, loading of Runx2，4%intensity compared with the controlgroup had no significant increase (P>0.05). In1days，8%the load loading strength underRunx2expression compared with the control group had no significant increase (P>0.05)，load2days，3days significantly increased compared with control group.(P＜0.05).1.3Summary1.3.1In certain mechanical loading can make the TSCs Runx2and BMP2expression 1.3.2Runx2and BMP2expression levels increase with the increase of the intensity ofload2The effect of mechanical loading on expression of RhoA/ROCK in tendon stemcells.2.1Respectively from two aspects of gene expression and protein expression，reserchthe RhoA and ROCK expression level and change trend after TSCs under different loadingconditions.2.2The result of the experiment2.2.1Mechanical Loading of TSCs RhoA，the influence of the ROCK gene expressionlevels:In the Loading of different time (1day，2days，3days) of each group of RhoA，ROCK gene expression with the increase of the intensity of loading increasing trend;Statistically difference (P <0.05).2.2.2Mechanical Loading of TSCs RhoA，the influence of ROCK protein expressionin:In load after1days，RhoA，ROCK protein expression level under different variablesshould be no significant change (P>0.05)， the load in2days，3days， compared withcontrol group，4%，8%， load should be variable RhoA， ROCK increasing trend ofprotein expression (P <0.05).2.2.3Mechanical Loading effects on cell proliferationIn the Loading of different time (1day，2days，3days) of each group， the loadshould be variable was4%， no significant effect on cell proliferation. Load should bevariable was8%， reduced cell proliferation， compared with the control group withstatistical difference (P <0.05).2.3Summary2.3.1In mechanical loading can make the TSCs RhoA and ROCK signal moleculeexpression;2.3.2RhoA and ROCK expression levels rise with the increase of intensity of load.3The effect of mechanical loading on expression of osteogenesis molecules intendon stem cells after blocking the expression of RhoA/ROCK.3.1In search for the optimal conditions of the experiment，from two aspects of gene expression and protein expression， study RhoA， ROCK molecules of TSCs osteogenesisRunx2expression of differentiation index.3.1.1Experimental conditions and groupingLoad should be8%,1Hz,4h/d and load3d. Experimental group: TSCs load; TSCs+load; Loading of TSCs+C3toxin; TSCs+Y27632+load3.1.2Cells were collected for Real-timePCR detection.3.2The result of the experiment3.2.1In8%loading strength，4h/d， under the condition of load for3consecutivedays of TSCs osteogenetic differentiation markers Runx2gene expression3.2.2Join RhoA inhibitors C3toxin, TSCs after Loading the condition of the Runx2gene expression is not added C3toxin group cut;3.2.3Join ROCK inhibitors TSCs Y27632in these conditions after the load Runx2gene expression is not added Y27632toxin group cut;3.3Summary3.3.1C3toxin inhibits RhoA， can make the TSCs osteogenesis of mechanical loadinginduced differentiation markers levels drop;3.3.2Rainfall distribution on Y27632inhibition of ROCK， can make the TSCsosteogenesis of mechanical Loading induced differentiation markers levels drop;3.3.3RhoA/ROCK molecules play a key role in mechanical loading induced TSCsosteogenetic differentiation.