Culture, Identification And Biological Characteristics Of Skeletal Muscle Satellite Cells Of Pig And Preliminary Study About The Porcine MEF2B And MEF2C Gene

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. They become activated when the muscle fiber receives any form of trauma, damage or injury, such as from resistance training overload. The satellite cells then proliferate or multiply, and the daughter cells are drawn to the damaged muscle site. They then fuse to the existing muscle fiber, donating their nuclei to the fiber, which helps to regenerate the muscle fiber. Because of the key role that satellite cells play in skeletal muscle growth, development, and regeneration, many scientists have focused their research on understanding the physiology of this cell. At present, little researches have been carried out about the pig skeletal muscle satellite cells. The myocyte enhancer factor 2(MEF2) that belong to the family of MADS-box regulators plays essential roles in skeletal muscle growth and differentiation during development. MEF2 interacts genetically and physically with different members of the myogenic network and together they cooperate to positively or negatively regulate transcription of downstream muscle specific differentiation genes during development.In this study, a method for culture and identification of the porcine skeletal muscle satellite cells was established and improved. Neonatal porcine skeletal muscle satellite cells were obtained by the two step method of collegenase I and trypsin, and were subject to primary as well as secondary culture in vitro. Morphological observation and growth curve were used to evaluate the proliferative and differentiation ability of skeletal muscle satellite cells. The cells were identified with RT-PCR and cellular immunochemical stain. The results showed that the two step method with collegenase I and trypsin was suitable for collection of skeletal muscle satellite cells. The cells showed strong proliferative ability in the proliferative media and could form myotubes in differentiation media. The porcine MEF2B, MEF2C and CACNG1 genes were cloned using the comparative genomics strategy. The sequence of the porcine MEF2C gene cDNA was 1940-bp which contained the complete coding region, and the porcine CACNG1 gene was 1136bp in length, respectively. In order to obtained the full 5'-untranslated sequence of the porcine MEF2B gene, 5'-RACE experiments has been carried out. The 5' - RACE products and other PCR products were assembled into one 1070-bp sequence of porcine MEF2B gene. The sequences of the porcine MEF2B, MEF2C and CACNG1 genes were submitted to the GenBank of the NCBI database under the GenBank Accession No. DQ343149, DQ366898 and DQ323981, respectively.The CLUSTALW alignment showed the presence of a putative MADS domain in the porcine MEF2B and MEF2C genes. The MADS domain of the MEF2B and MEF2C gene were very conserved with that of human and mouse homologs, suggesting that the MEF2...