| Objective:We isolated satellite cells from the injured skeletal muscle tissues,and then detected the nucleic acid and protein levels of these intracellular effector molecules,Pax7 and MRFs(MyoD,Myf5,myogenin,MRF4)to explore whether these changes were related to the time of injury or not.Methods:1.Establishment of rat skeletal muscle contusion models: 258 healthy adult male SD rats(200 g ± 5 g)were purchased and randomly selected 6 rats to detect the purity of the extracted satellite cells;the rest rats were randomly divided into experimental group and the control group.The experimental group was further divided into 13 subgroups according to different injury time(0 h,4 h,8 h,12 h,24 h,36 h,48 h,60 h,3 d,4 d,5 d,6 d,7 d),18 rats in each group.The skeletal muscle contusion model of right hind limb was prepared by an improved free falling method.The rats were sacrificed by excessive injection of 2% pentobarbital sodium(300 mg/kg)at stated time points.After that,we extracted the contusion skeletal muscle tissues from the injured area.The rats in the control group were sacrificed followed the same method.2.Morphological observation of contusion skeletal muscle tissues of rats: Tissues were fixed in 4% paraformaldehyde solution for 48 hours and then made into paraffin sections for H&E staining to observe the histopathological characteristics of the injured area.3.Isolation,extraction,purification and identification of quiescent satellite cells: The quiescent satellite cells were isolated,extracted and purified from the skeletal muscle of the right hind limb in rats by using double-enzyme digestion combined with differential attachment method.Then the Pax7 immunofluorescence staining was performed to calculate the extraction efficiency of satellite cells.4.Muscle satellite cells were extracted from skeletal muscle of rats in experimental group and control group by the same method.Six cell samples were inoculated into 24-well plate for immunofluorescence staining.The other six cell samples were stored at-80 C by cell precipitation for PCR test.5.Immunofluorescence staining: Pax7,MyoD and myogenin antibodies were used to detect the content of target proteins in satellite cells,and then FL-StrataQuest software was used to analyze fluorescent images.6.Real-time quantitative PCR experiment: The relative quantification of Pax7,MyoD,Myf5,myogenin and MRF4 mRNA in satellite cells of each group was detected by real-time quantitative PCR with double reference genes.Results:1.H&E staining showed that within 7 days after skeletal muscle injury,rats underwent hemorrhage,inflammation,necrosis of muscle fibers,karyolysis,disappearance of nuclei and formation of a small number of new muscle fibers,which confirmed that the skeletal muscle contusion model was successfully prepared and could be used for subsequent experiments.2.By extracting and identifying the quiescent satellite cells,we found that the sorted cells were round and highly refractive.Data analysis showed that the purity of satellite cells was 89.29%.3.The morphology of sorted satellite cells from different groups was different.The cells extracted from 12 hours after injury group began to appear triangle,polygon and spindle,and the number of spindle cells was higher in 7 days after injury group.The results of immunofluorescence staining showed that the mean Pax7 and MyoD fluorescence intensity per cell began to increase at 12 hours after injury and lasted until 7 days after injury,while the mean myogenin fluorescence intensity per cell increased significantly at 36 hours after injury,with a slight delay in time.We also found that there were many cells with 2 nuclei at the day 7 after injury group.The nuclei of these cells were oval with strong myogenin fluorescence signals allowing us to indirectly speculate that these sorted cells were fused myotubes,which were not yet completely mature.4.Analysis of the results of PCR experiments showed that the level of Pax7 mRNA began to increase 24 hours after injury,and then maintained a high level.MyoD mRNA and Myf5 mRNA levels began to increase 48 hours after injury,and the changes of MyoD mRNA levels were more significant.The changes of myogenin mRNA levels were similar.Compared with MyoD,the changes of myogenin mRNA levels were slightly delayed in time,which was consistent with the changes of proteins.There was no obvious change in MRF4 mRNA level.Conclusion:We can successfully extract skeletal muscle satellite cells with high purity(89.29%).Skeletal muscle satellite cells play an indispensable role in the process of adult skeletal muscle regeneration and repair after injury.The expression levels of Pax7 and MRFs genes and proteins represented injury time-related regularity. |
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