Isolation And Culture Of Porcine Skeletal Muscle Satellite Cells By Single Muscle Fibers And Their Myogenic Differentiation

Skeletal muscle is composed of terminally differentiated multinuclear, postmitotic myofibers. Regeneration of skeletal muscle therefore depends on an undifferentiated mononuclear muscle progenitor pool, of which satellite cells are the principal component. Muscle satellite cells are quiescent myogenic stem cells situated between the basal lamina and plasmalemma of mature skeletal muscle fibers, It has the proliferation and differentiation potential. Homeostasis, hypertrophy and repair of adult skeletal muscle are carried out by satellite cells. Morphologically, they are characterized by a high nucleus-to-cytoplasm ratio and reduced organelle content. However, the mechanism what controls satellite cell self-renews and differentiates is mostly undefined .Now think that quiescent satellite cells express the paired-box 7 (Pax7) transcription factor , when activated, they express Pax7 together with MyoD. After proliferation, most satellite cells down regulate Pax7 and differentiate. However , Other satellite cell progeny maintain Pax7 expression but lose that of MyoD, withdraw from both cell cycle and immediate myogenic differentiation, and return to a quiescent-like state .Single-fiber muscle cultures have been used to examine satellite cell gene expression, myogenesis on fibers, regenerative capacity and activation processes. This model is very powerful in that it allows for the maintenance of the satellite cell and fibers interaction while eliminating nonmuscle influences and maintaining satellite cell quiescence. Whereas domestic yet see reports .Thus , in this paper was aimed to establish a culture system to isolate satellite cells by single fiber isolating and then culture from porcine skeletal muscle by collagenase I digestion in vitro. Satellite cells on the fibers were identified by immunocytochemistry. Then, satellite cells moved from fibers were analyzed by immunocytochemistry. Satellite cells were passaged and cell growth curve were draw. At this time, cells were induced to myocytes, and the expression of skeletal muscle specific protein was assessed by western blot. Its main research results are as follows:1. Satellite cells adhered to freshly isolated fibers appeared oval and these cells moved from fibers and proliferated gradually. Furthermore, more than 90% of satellite cells positive for Pax7 and MyoD.2. Moreover, MyoD protein was highly expressed in proliferated stage, while the protein expression of myogenin and MyHC was detected only in the differentiating cells.3. After induction of muscle differentiation , Compared with traditional DMâ… differentiation media,DMâ…¡differentiation media is more advantageous to cell convergence, differentiation and multinucleated muscle tube formation. At this time muscle differentiation markers myosin heavy chain (MyHC) and Myogenin displayed positive staining in these cells.To sum up : The study successfully establishes a in vitro culture method for porcine skeletal muscle single fiber, and obtains high purity skeletal muscle satellite cells;Secondly,Optimized of pig skeletal muscle satellite cell proliferation and differentiation training system; Finally, preliminary analysis of the muscle specificity protein expression rules of porcine skeletal muscle satellite cells .Which will provide experimental material for mobility, proliferation and differentiation of skeletal muscle satellite cells.