Experimental Study Of Autologous Adult Stem Cell Transplantation For Hindlimb Skeletal Muscle Injury And The Effect Of Resveratrol On It

Objective:The purpose of the experiment in this article is divided into three parts.First,by establishing a model of skeletal muscle tension failure,using resveratrol(Res)as an adjuvant treatment,to detect the repair effect of Res on skeletal muscle injury,,To explore the effect of stem cell transplantation after skeletal muscle injury,three,the use of autologous bone marrow mesenchymal stem cell transplantation plus resveratrol(BM-MSC+Res)program to intervene in repair of skeletal muscle injury,and explore the promotion of BM-MSC+Res in promoting Role and mechanism in the rehabilitation of damaged skeletal muscle structure and function.Method:1.Resveratrol treatment of skeletal muscle injury:Normal group(N=3),natural recovery group(NR=15),resveratrol group(Res=15).Except for group N,blunt bruise model of skeletal muscle was made by blunt violence method.After injury,NR group was not treated,and Res group was given Res intragastric treatment(15mg/kg/d).The animals were killed 1,3,7,14,21d after injury.Observe the local changes of skeletal muscle after blunt contusion by direct-view method and perimeter measurement method,HE and Masson staining are used to observe the pathological changes of skeletal muscle tissue.IHC and Western blot detection of basic fibroblast growth factor(bFGF)in skeletal muscle,Insulin-like growth factor-1(IGF-1),metastatic growth factor(TGF-β1),type I collagen(COL-I)expression.2.Autologous adult stem cell transplantation for skeletal muscle injury:33 New Zealand rabbits were randomly divided into three groups:normal group(N=3),control group(C=15),and BM-MSC transplantation group(BM-MSC=15).Except group N,the blunt bruise model of skeletal muscle was made by blunt violence method.Group C was injected with cell-free low-glucose DMEM culture medium after injury.BM-MSC group was injected with autologous BM-MSC at the injury site for treatment after skeletal muscle injury.The experiment is the same as Method 1,and the cells are tested.3.Experimental study on autologous stem cell transplantation for hind limb skeletal muscle injury and the effect of resveratrol on it:48 New Zealand rabbits were randomly divided into three groups:normal group(N=3),resveratrol group(Res=15),bone marrow mesenchymal stem cell transplantation(BM-MSC=15)and stem cells Resveratrol adjuvant therapy group after transplantation(BM-MSC+Res=15).Except group N,the blunt bruise model of skeletal muscle was made by blunt violence method.Res group was given intragastric treatment of Res.BM-MSC group was treated with autologous mesenchymal stem cells after injury of skeletal muscle.Res group received autologous BM-MSC transplantation and Res intragastric therapy after injury.Subsequent experiments are the same as Method 1,supplemented by electron microscopy and ultrasound.Result:1.Res treatment of skeletal muscle injury:Direct view method:The swelling peak reached 12 hours after skeletal muscle injury,and the swelling completely disappeared after 72 hours;HE staining:Group N muscle fibers are polygonal,regular in shape,closely arranged,and the nucleus is evenly distributed under the sarcolemma without hyperplasia and deflation.Muscle membrane is intact;blood cells ooze out in the NR group and Res group on the 1st day,inflammatory cells begin to infiltrate on the 3rd day to reach the peak on the 7th day,and muscle fiber morphology basically returns to normal on the 21st day after injury.The repair time is better than the NR group;Masson staining:there are very few collagen fibers in normal muscle cells,which increase gradually after injury and reach a peak around 14d.The content of collagen fibers in the injured area of the Res group is lower than that of the NR group.IHC:bFGF and IGF-1 proteins were diffusely stained in the muscle fiber cytoplasm.The absorbance values of bFGF and IGF-1 proteins showed an upward trend and then a downward trend after injury..Both the NR group and Res group reached a peak on 7d,and were still higher than N on 21d.Group,and the absorbance values of bFGF and IGF-1 protein in Res group were higher than those in NR group 7 days before injury and lower than those in NR group after 14 days of injury,the difference was statistically significant(P<0.05);TGF-β1,COL-I The protein is diffusely stained in the cytoplasm of muscle fibers,and the protein absorbance value of the protein also increases first and then decreases after the injury.Both the NR group and the Res group reached a peak at 7 days after injury.During the entire injury process,TGF-β1,COL-The protein I absorbance values were higher than the Res group,the difference was statistically significant(P<0.05);Western Blot:the expression of related factors was parallel to the results of IHC data.2.Autologous adult stem cell transplantation to treat skeletal muscle injury:Flow cytometric identification showed that rabbit bone marrow mesenchymal stem cells were positive for CD90 and negative for CD45;direct-view method:swelling in the injured area,12h to swelling value,swelling subsided after 72h to the pre-experiment state;HE staining:Group C and BM-In the MSC group,blood cells oozed out on the 1st day after injury.On the 3rd day,the inflammatory cells began to infiltrate to reach the peak on the 7th day.The morphology of the muscle fibers returned to normal on the 21st day after the injury.Masson staining showed that the content of collagen fibers in normal muscle cells was very small.The damage gradually increased with the formation of scar tissue.The collagen fibers gradually increased and reached a peak at 7-14 days.The content of collagen fibers in BM-MSC group was lower than that in group C;IHC showed:bFGF,IGF-1 protein content showed a trend of increasing first and then decreasing after injury.Both groups reached a peak at 7d.The protein content of BM-MSC was higher than that of group C at 7d before injury.BM-MSC histones were at 14d and after injury The content was lower than that in group C,but at 21 days after injury,the BM-MSC group and group C were still higher than normal,the difference was statistically significant(P<0.05);TGF-(31,COL-I protein content appeared first after injury The trend of rising and falling is that the C group and the BM-MSC group both reached the peak at 7d,and the C group was higher than the BM-MSC group in the whole process of injury,the difference was statistically significant(P<0.05);Western Blot showed:bFGF,The changes of IGF-1,TGF-β1 and COL-Ⅰ protein content were parallel to the IHC experimental data.3.Experimental study on autologous stem cell transplantation to treat hind limb skeletal muscle injury and the effect of resveratrol on it:Direct view method:swelling in the injured area,12 hours to the peak of swelling,and the swelling subsided after 72 hours to the state before the experiment.Ultrasound showed that the elasticity of skeletal muscles decreased after injury.Res and BM-MSC treatment showed a significant improvement in elasticity,but it was weaker than the performance of the BM-MSC+Res group.Electron microscopy results showed that blunt contusion of skeletal muscle resulted in Z-line water streak or rupture,sarcoplasmic reticulum swelling or vacuolation,mitochondrial accumulation under the sarcolemma and different sizes,swollen mitochondria in muscle fibers,and scarce ridges.After blunt contusion of skeletal muscle,it was treated with resveratrol intervention,bone marrow mesenchymal stem cell transplantation,and bone marrow mesenchymal stem cell transplantation supplemented with resveratrol treatment.The results showed that the skeletal muscle Z line and mitochondria The degree of abnormal changes in ultrastructure and other ultrastructures were significantly alleviated;HE showed that the normal group of rabbit muscle fibers were polygonal,regular in shape,closely arranged,and the nucleus was evenly distributed under the sarcolemma without hyperplasia and deflation,and the sarcolemma was intact.In each model group,blood cells exuded 1 day after injury,inflammatory cells infiltrated 3 days after injury,7 days after injury,inflammatory cells gradually decreased,with scar tissue formation,and 21 days after injury,muscle fiber morphology basically returned to normal;Masson staining showed:collagen in normal muscle cells The fiber content was relatively small,and it increased gradually after injury,reaching a peak at 7-14 days.During the whole injury process,the collagen fiber content of BM-MSC+Res group was lower than that of BM-MSC group and Res group,the difference was statistically significant(P<0.05).IHC showed that the bFGF and IGF-1 proteins were all stored in the cytoplasm,and the bFGF and IGF-1 protein contents showed an upward trend and then a downward trend after injury.Except for the normal group,they reached a peak at 7 days.The content of bFGF protein in the BM-MSC+Res group was higher than that in the BM-MSC group and Res group 7 days before injury,but the difference was not statistically significant(P>0.05).3-7 days after injury,IGF-in the BM-MSC+Res group 1 The protein content was higher than that of BM-MSC group and Res group,the difference was statistically significant(P<0.01);14 days after injury,the protein content of bFGF in BM-MSC+Res group was lower than that of BM-MSC group and Res group,the difference was statistically significant Significantly(P<0.01),the model group returned to normal at 21 days after injury;the IGF-1 protein content of the BM-MSC group and Res group was still higher than the normal group at 21 days after injury,the difference was statistically significant(P<0.05),the difference between the BM-MSC+Res group and the normal group was not statistically significant.The protein content of TGF-β1 and COL-I showed a trend of rising first and then decreasing after injury.Each model group reached a peak at 7 days after injury,and the protein content of BM-MSC+Res group was lower than that of BM when TGF-β1 protein and 3 and 7 days.-The difference between the MSC group and the Res group was statistically significant(P<0.01).During the entire injury,the COL-I protein content of the BM-MSC+Res group was lower than that of the BM-MSC group and the Res group,and the difference was statistically significant(P<0.05);Western Blot showed:bFGF,IGF-1,TGF-β1 and COL-I protein content changes parallel to the IHC experimental data.Conclusion:1.Res treatment of skeletal muscle injury:Resveratrol can promote the repair of skeletal muscle by up-regulating and down-regulating the expression of bFGF and IGF-1 proteins and inhibiting the overexpression of TGF-β1 and COL-I proteins.2.Autologous adult stem cell transplantation to treat skeletal muscle injury:Autologous bone marrow mesenchymal stem cell transplantation can inhibit the overexpression of TGF-β1 and COL-I proteins by up-regulating and down-regulating the expression of bFGF and IGF-1 proteins,thereby promoting the repair after skeletal muscle injury.3.Experimental study on autologous stem cell transplantation for the treatment of hind limb skeletal muscle injury and the effect of resveratrol:Adjuvant therapy with resveratrol after autologous bone marrow mesenchymal stem cell transplantation can up-regulate an d down-regulate the expression of bFGF and IGF-1 proteins,inhibit the overexpression of TGF-β1 and COL-I proteins,and then promote the repair of skeletal muscle injury.