Regulation Of ITGB6 Gene On The Proliferation Of Porcine Skeletal Muscle Satellite Cells

The mechanism of pig skeletal muscle growth and development has been receiving much attention.Skeletal muscle,which has the highest proportion of body weight in pigs,its growth and development directly affect the performance of the pig.The growth and development of skeletal muscle is a complex biological process,including the formation and proliferation of muscle cells,the formation of myotubes and muscle fibers,and the terminal maturation stage.With the development of life sciences,scientists have a deep understanding of the regulation mechanism of skeletal muscle development and the regulation of related genes,but there are still many genes involved in the regulation of skeletal muscle growth and development that have not yet been studied.It has been reported that the ITGB6 gene is differentially expressed during the development of skeletal muscle in pigs,and regulates the development of skeletal muscle under the effect of epigenetic modification.Therefore,we began to study the regulation of the ITGB6 gene on the development of skeletal muscle in pigs.Firstly,we isolated pig skeletal muscle satellite cells from one-day-old piglets as a model to study the development mechanism of pig skeletal muscle;secondly,we constructed the eukaryotic expression vector pcDNA3.1(+)-ITGB6 and synthesized the interference fragment of the ITGB6 gene,and then we transfected them into pig skeletal muscle satellite cells.Finally,we studied the regulation effect of the ITGB6 gene on the proliferation of pig skeletal muscle satellite cells by CCK-8,EdU,Western Blot and flow cytometry.The main results are as follows:(1)We used the tissue digestion method to isolate primary pig skeletal muscle satellite cells from the muscle tissue of one-day-old piglets and purified them by the differential adhesion method.The results showed that the porcine skeletal muscle satellite cells proliferated quickly and exhibited strong myogenic characteristics,and a large number of myotube formation could be observed under the microscope after differentiation.(2)We used flow cytometry to identify the purity of porcine skeletal muscle satellite cells.The results showed that the PAX7-positive cells in the purified pig skeletal muscle satellite cells reached 98.5%,indicating that the satellite cells were of higher purity and could be used in subsequent experiments.(3)We transfected porcine skeletal muscle satellite cells with pcDNA3.1-EGFP and identified their transfection efficiency.The statistical results of GFP positive cells showed that the transfection efficiency of porcine skeletal muscle satellite cells was 41.2%.(4)We used the CCK-8 and EdU to analyze the effect of up/down-regulation of ITGB6 gene expression on the proliferation of porcine skeletal muscle satellite cells.It was found that up-regulating the expression of the ITGB6 gene inhibited the proliferation activity of porcine skeletal muscle satellite cells,and down-regulating the expression of the ITGB6 gene promoted the proliferation activity of porcine skeletal muscle satellite cells.(5)We used Western Blot technology to analyze the effect of up/down-regulation of ITGB6 gene expression on Ki67 expression in pig skeletal muscle satellite cells at the protein level.The results showed that up-regulating the expression of ITGB6 gene inhibited the expression of Ki67 in porcine skeletal muscle satellite cells,and down-regulating the expression of ITGB6 gene promoted the expression of Ki67 in porcine skeletal muscle satellite cells.(6)We used flow cytometry to analyze the effect of up/down-regulation of ITGB6 gene expression on the satellite cell cycle of porcine skeletal muscle.The results showed that upregulating the expression of ITGB6 gene would reduce the ratio of S-phase cells in porcine skeletal muscle satellite cells,and down-regulating the expression of ITGB6 gene would increase the ratio of S-phase cells in porcine skeletal muscle satellite cells.In summary,the ITGB6 gene inhibits the proliferation of skeletal muscle cells by inhibiting cell synthesis and ultimately inhibits the development of skeletal muscle.