The Preliminary Study Of Denervated Skeletal Muscle Fibrosis And The Molecular Mechanism Of Myoblast Transdifferentiation

Objectives:Establish the c57mice models of different time loss of accessory nervedisposal.Muscles were cutted into MASSON staining slices.Cross-sectional areas of thedenervation muscle and the fiber were calculated to get to know after the different time ofdenervation how the tissue fibrosis process went. Thus,it will provide a theoretical basisfor us to choose the best treating time for clinical patients who suffered from laryngealnerve denervation. At the same time through the Westernblot, Real-time PCR andimmunohistochemical fluorescence to detect protein levels, mRNA level of fibrosisassociated factors such as changes and expressions. In addition, cultivate C2C12musclestem cells in vitro, with different concentrations of transforming growth factor beta1(TGFbeta1) induction of C2C12cells produce fiber transformation, explore TGF beta1, C2C12cells induced the effect and mechanism of fibrosis transformation, to help finding the bestnerve repair time,providing the experimental basis and theoretical basis for clinical drugintervention.Methods:1. c57mice accessory nerve denervation model were settled in different time30healthy male c57mice were randomly divided into six groups, and according to thepostoperative time divided into normal group, postoperative1week,2weeks,3weeks,4weeks,8weeks group, each group contains only5mice. Intraperitoneal injection of4%chloral hydrate (0.5ml/100g)0.3ml, after the success of the anesthesia, isolated rightsternocleidomastoid and fully exposed the accessory nerve, along the accessory nerveroots cut off5mm long nerve. In1week,2weeks,3weeks,4weeks,8weeks afteroperation we get the specimens. After getting the muscles, we do muscle MASSONstaining slice, and calcute the nerve muscle cross-sectional area and the muscle fiberdiameter, fiber cross-section in different time.2. Real-time quantitative PCR detect different groups in the organization of alpha smoothmuscle actin (a-SMA), connective tissue growth factor (CTGF) and TGF beta1mRNAexpression level resultsFirst using Genebank to find out alpha SMA, TGF beta1, CTGF and internal mRNAsequence, using Primer6software to design primers, primer synthesis. Secondly, througha series of steps to Real-time PCR amplification. Software is used to calculate the value of the mRNA and comparison.3.Western Blot to detect alpha SMA, CTGF and TGF beta1protein expression levels inthe muscle.According to Western Blot instructions by sds-page gel preparation, sds-pageelectrophoresis, wet electrical transmembrane, membrane closed and antibody incubationand color imaging to detect different time denervation tissue fibrosis related factor alphaSMA, CTGF and TGF beta1protein expression levels.4. Immunofluorescence method to detect alpha SMA, CTGF and TGF beta1positivecells in denervation tissues at different timesRemove frozen slices from a refrigerator, melting in room temperature, rupture membrane,incubation primary antibody overnight at room temperature, then add second antibody,fluorescent dye dyeing cell nucleus, collect images through fluorescence microscope.5.In vitro, different concentrations of TGF beta1induced C2C12cell transdifferentiation,detect the protein expression of alpha SMA in cell.Take C2C12cells away from the liquid nitrogen container, cultivating cells in6wellculture plates, according to the different concentrations of TGF beta1(0ng/ml,0.05ng/ml,0.1ng/ml,0.5ng/ml,1ng/ml,2.0ng/ml,5.0ng/ml,10ng/ml), cells wererandomly divided into8groups, each group include three duplication holes, add1.5mlDMEM containing10%FBS per hole, when cell growth density come near70%, addcorresponding concentrations of TGF beta1respectively to each group, induce thetransdifferentiation of C2C12cell, collecting cells when stipulated time reaches.According to the WB steps, detect the protein expression of alpha SMA under thecircumstance of different concentrations of TGF beta1, in order to get the optimalconcentration of TGF beta1that can preferably induce transdifferentiation.6. Observe the changes of fibrosis related factors when using the optimal concentration ofTGF beta1to induce transdifferentiationCultivate C2C12cells, stimulate the cells with the optimal concentration of TGF beta1when the cell density comes near70%except for one group that is equivalently thecontrol goup(0hour group), and yet cultivate the cells continually. Then collect the cellsand stop cultivation after12,24and48hours. Using WB and immunofluorescence todetect the changes of fibrosis related factors.Results:1.Loss of accessory nerve of c57murine models were successfully settled. As the denervation time goes on, we found that the right sternocleidomastoid atrophysignificantly in mice through naked eye observation, and muscular atrophy made thesternocleidomastoid muscle smaller than the normal ones. By masson staining slices，weobserved that as denervation time goes on, sternocleidomastoid muscle fiber shrink andthe average diameter of muscle fiber decreases, muscle cross-sectional area shrink, andintramuscular interstitial collagen connective tissue increased, to finally have a lot ofmuscle fiber in space. The comparison between normal control group and denervationgroup1week,2weeks,3weeks,4weeks,8weeks show that the difference had statisticalsignificance (P <0.05). Longer duration of denervation, sternocleidomastoid musclefibers appear obvious atrophy and fibrosis, and denervation muscle fibrosis trend slowedafter4weeks, denervation muscle cross-sectional area and collagen cross-sectional areabetween group4weeks and group8weeks is no obvious difference, which indicates thatdenervation4weeks may probably be the most significant period that muscle atrophy andfibrosis happens.2.Real-time quantitative PCR detect alpha SMA, CTGF and TGF beta1mrna expressionin each group. We found that alpha SMA and CTGF transcription level raised to the peakin2weeks after denervation. The TGF beta1transcription raised in1week afterdenervation. Since then the transcription factor declined slightly, but there are still acontinuous expression of a higher level for a long time.3. The protein expression of fibrosis related factors were not found in normalsternocleidomastoid muscle. Alpha SMA and CTGF can be detected positive expressionin1week after nerve loss, and reaches to the peak in2weeks after denervation, then theexpression declined, but higher level positive strip can still be detected. The proteinexpression of TGF beta1in1week after denervation is obviously higher,2,3,4weekshave fallen a bit, but there are still a constant expression. The result is consistent with thePCR results.4. Immunofluorescence method to detect the expression of alpha SMA, CTGF and TGFbeta1.These factors were not found in normal sternocleidomastoid muscle. Alpha SMAand CTGF positive cells emerged in1week after nerve injury. In two weeks after thedenervation the amount of the positive cells reaches to the peak and they mainly locatedin cell cytoplasm and cell membrane. TGF beta1positive cells reaches to the peak in1week after denervation, then the amount of positive cells will decline, but still remain at ahigher level over time. 5. TGF beta1can induce C2C12cell transdifferentiation.The increasing concentration ofTGF beta1will make the expression of alpha SMA increase gradually. When the TGFbeta1concentration is5ng/ml, the alpha SMA amount is the most. While the TGF beta1concentration reaches to10ng/ml, alpha SMA began to decline. Thus we can concludethat5ng/ml is the appropriate concentration of TGF beta1to induce thetransdifferentiation of C2C12cell. After TGF beta1stimulating C2C12cells12hours,the protein expression of collagen-, CTGF and alpha SMA began to rise and continue toexpress. Their expression levels are much higher than the control group.6.Immunofluorescence test indicates that5.0ng/ml TGF beta1stimulate C2C12cells for12hours, collagen-I, CTGF and alpha SMA the three factors express enhancedfluorescent protein, which located in cell cytoplasm and cell membrane. Until48hours,no significant decrease can be seen in the enhanced fluorescent protein.Conclusion:The accessory nerve denervated murine model is settled successfully. As the denervationtime lengthened, the sternocleidomastoid mucle will atrophy and develop fibrosis. Themuscle fiber atrophy and fibrosis gradually aggravate. The most obvious morphologicalchanges happens in4weeks after denervation, and there comes a stable platform periodwhile the muscle fibrosis progress developed slowly after4weeksFibrosis related factors show high expression when muscle denervation happens. TGFbeta1is the first one to reach the expression peak. The expression peaks of CTGF andalpha SMA are showing later than TGF beta1’s. It indicates that denervation will firststimulate the expression of TGF beta1, then through a series of cascade reactions,CTGF and alpha SMA will start to express, all these fibrosis related factors will finallymake the muscle atrophy and develop fibrosis.5.0ng/ml is the optimal concentration of TGF beta1to induce C2C12cellstransdifferentiation. Observing the protein expression and quantitative expression ofcollagen-,CTGF and alpha–SMA by WB and immunofluorescence. Both showed thatafter5.0ng/mlTGF beta1stimulate C2C12, the above three indicators are higher in12hours, and continuously expressed in24hours and48hours.