Effect On The Gene Expression Profile Of L6 Cells Treated By Co-culture With Schwann Cells

Recently researchers express greater concern on studies of the mechanism of repair, regeneration and reconstruction of damaged skeletal muscle on cell and molecular levels, given the incidence of skeletal-muscle-relative diseases (e.g. skeletal muscle injury, muscular dystrophy, muscular atrophy and hypertrophy) increases. To construct a compound skeletal muscle by applying tissue engineering principles and methods is a creative research work, and would be useful for repair muscle injury.Myoblast, which is differentiated from satellite cell, originates from mesoblastema of embryo. Satellite cells exist between sarcolemma and basement membrane of muscle fiber. Under physiological condition, in a static and undifferentiated state. When being damaged, necrosis of muscle fiber of skeletal muscle occurs, exhibiting macrophages phagocytizing necrotic cells and basement membrane of muscle fiber. Meanwhile, the skeletal satellite cells are activated to proliferate and differentiate intospindly myoblasts, fusing reciprocally into myotube and developing into regenerated muscle fiber. Schwann cells (SC) are glial cells of peripheral nervous system centering on peripheral nerve axons, engendering myelinated or unmyelinated nerve fiber, and they secrete various neurotrophic factors, preventing necrosis of damaged neuronal somas, prompting axon myelinization thereby facilitating regeneration of peripheral and central nervous system. Schwann cells proliferate actively and release mass of neurotrophic substance when peripheral neuromuscular damage occurs, to boost regeneration of nerves and participate in maintaining normal morphology and function of normal skeletal muscle, repairing of damaged muscle and nerves. Rapid proliferation of myoblast cultured in vitro and significant increase of fusion rate of myotube after stimulation by ciliary neurotrophic or nerve extract indicate promotions to proliferation and differentiation of myoblast by neural factors.For a better clinical application, tissue engineered artificial musclewith three-dimensional cultured pure muscle cells should be transferred into nerval-vascular-muscular complex style. To thoroughly understand the molecular mechanism of the process of repairing damaged muscle, Schwann cells, as an influencing factor, were found to participate the course of muscle regeneration . Though effect of Schwann cell on myoblast had been affirmed, it is still unclear about changes of involved gene expression in myoblast. Consequently, studying the changes of gene expression of myoblast co-cultured with Schwann cells will provide theoretical support to complex skeletal muscle reconstruction.This study stands on related work of prophase, which constructs tissue-engineered skeletal muscle with Schwann cell cords while Schwann cells are cultured on tubular scaffold with which skeletal muscle scaffolds are connected. However it is the uncertainty of the molecular mechanism of reciprocal regulation between myoblast and Schwann cells that inhibits further study.In this study researcher firstly obtains primary cultured rat Schwann cells, then co-culture with myoblast cell line L6, cell proliferation and differentiation were observedby cell counting under inverted microscope. Data indicates that at the early stage of proliferation myoblast co-cultured with Schwann cells appear no obvious differences from control groups until obviously advanced emergence of logarithmic growth phase on the 3rd day, while formation rate of myotube of the treated groups is higher than those of control groups. Detections on actins on two observation points, the 3rd and the 5th day, show high expression of cells in treated groups, which is much significant than control groups. All these resultsreflect that the participation of Schwann cells could prompt the process of proliferation and terminal differentiation of myoblast effectively. To look deeper into the morphological changes of myoblast caused by co-culture, this study adopts genome-wide microarray to test the alternations in expression of gene profile when Schwann cells affect the myoblast in progress, in order to clarify alternations in expression of gene involved and metabolic process, in the process of induced differentiation. With qualified RNA obtained from cells from both treated and control groups, hybridize between purified markers and genome-wide microarray. Analysis on hybridized chip sifts 12 significant up-regulated genes and 221 significant down-regulated genes. By adopting software PANTHER to analyze acquired genes of differential expression, myoblast co-cultured with Schwann cells involve various alternation of intracellular metabolism process during proliferation and differentiation. Those differently expressed genes are related to cell proliferation and differentiation, signal transduction, immunity and epidemic prevention, glucose metabolism, nucleic acid metabolism, transport of calcium and iron ions, and suggest that those intracellular metabolism paths mentioned above are pivot routes when Schwann cells affect proliferation and differentiation of myoblast. The differences of expression of those critical genes are likely being the reason of terminal differentiation of myoblast therefore further study might contribute to terminal differentiation theory of myoblast and provide theoretical basis for researches on compound skeletal muscle based on Schwann cells and myoblast.