Extraction And Identification Of Skeletal Muscle Satellite Cells From A Rat Model Of Radiation Muscle Fibrosis

The preferred clinical treatment for nasopharyngeal carcinoma is radiotherapy.However,about 24.34% of patients with nasopharyngeal carcinoma have undergone radical radiation therapy,and have symptoms of radiation induced muscle fibrosis(RIF)such as difficulty in opening mouth,difficulty swallowing,stiffness and pain in the neck,and shoulder dysfunction.RIF can cause coexistence of skeletal muscle damage and fibrosis,which will seriously affect the quality of life and even endanger patients' lives.Muscle regeneration after skeletal muscle injury is usually mediated by skeletal muscle satellite cells(SCs).Reactivation of SCs can deal with muscle damage,proliferate and differentiate into new muscle cells,migrate to the injured site,repair damaged muscles,or maintain the steady state of the stem cell bank.Existing treatments for radiation muscle fibrosis include corticosteroids,vitamin E,pentoxifylline,superoxide dismutase,hyperbaric oxygen therapy,etc.,but the efficacy is not ideal.Current clinical treatments cannot effectively or completely prevent the occurrence of muscle fibrosis after radiation,so it remains an important clinical problem.The exact pathophysiology of RIF is not completely clear,and animal models are needed to simulate clinical relevance and explore good treatments.The purpose of this study: to construct a model of rat radioactive muscle fibrosis through 1.90 Gy radiotherapy;2.to isolate and identify rat skeletal muscle satellite cells;3.to reveal that rat skeletal muscle satellite cells participate in 90 Gy radiotherapy to form radioactive muscle fibrosis in rats The formation of radioactive muscle fibrosis was explored,and new strategies for the prevention and treatment of radioactive muscle fibrosis were proposed.[Method] Seventy-two SD rats,aged about 7 weeks,weighed about 220 g to 250 g.Randomly divided into six groups(12 rats in each group,half of them were male and half were female).5% sodium pentobarbital(0.1 mL / 100g)was intraperitoneally injected into the rat anesthesia,and the surface of the medial rectus femoris muscle of the left femur was marked with a marker.Radiotherapy was given by the Nucletron Micro selectron-HDRIr-192 post-installation system(Nucletron / Netherlands),and the radiation tube was fixed at the mark on the left thigh.The dose normalization point is 0.5 cm below the source center,and the radiotherapy area has a 0.5 cm radius around the marked point.Control group(n = 12)did not receive radiotherapy;5 groups of experimental groups(90Gy-1w group,n = 12;90Gy-2w group,n = 12,90Gy-4w group,n = 12,90Gy-12 w group,n = 12(90Gy-24 w group,n = 12)90Gy single-dose radiotherapy was given.Tissues and cells were extracted from the radiotherapy site at 1st week,2nd week,4th week,12 th week,and 24 th week after radiotherapy,and the results were detected.[Results]1.In the rat model of radioactive muscle fibrosis,obvious fibrosis can be seen in Masson staining,and muscle fiber regenerated by fusion of skeletal muscle satellite cells can be seen in HE staining.2.In the rat model of radioactive muscle fibrosis,TUNEL staining showed that significantly more apoptotic cells were found 4 weeks after radiotherapy.3.In the rat model of radioactive muscle fibrosis,the electron microscope showed that the muscle filaments in the control group of rats were neatly arranged,there was no dissolved muscle filaments,the nucleus was normal and regular,mitochondria were visible,and the structure of the mitochondria was intact.Four weeks after radiotherapy,the myofilaments were neatly arranged and the nucleus was normal,but localized myofilaments were found to be dissolved and destroyed,mitochondria were damaged and vacuolar degeneration occurred.In addition,compared with the control group,more autophagic vesicles were formed.4.In the rat model of radioactive muscle fibrosis,tissue immunofluorescence staining showed that only a few satellite cells were activated in the muscles of the control group,but the muscles of the rats in the radiotherapy group activated many satellite cells after 4 weeks.5.In the rat model of radioactive muscle fibrosis,cellular immunofluorescence shows that the fluorescence intensity of Pax7 + skeletal muscle satellite cells in the proliferation state is lower than that of the proliferating rat L6 myoblasts,and the MyHC + skeletal muscle satellite cells in the differentiation state.the same.[Conclusion] 1.Radiotherapy activates regeneration of rat muscle cells.2.Radiotherapy leads to muscle apoptosis in rats.3.Radiotherapy activates rat autophagy and damages mitochondria.4.Radiotherapy activated the activation of skeletal muscle satellite cells in rat muscle cells at the tissue and cellular levels.We successfully isolated,extracted and identified rat skeletal muscle satellite cells.