| Muscle satellite cells (SCs) which provided with strong potential aspect ofself-renewal, proliferation and differentiation and could across the germinal layer todifferentiate into certain cell types in vitro were first discovered by Mauro in frog tibialisanterior muscle. Luxi cattle as a world known beef breed, has been enrolled into thecategories for key protected breed of national livestock and poultry genetic resources. Thepurpose of this study is to establish amplification system for Luxi cattle embryonic skeletalmuscle satellite cells in vitro, and to explore its biological characteristics such as cellproliferation, differentiation with low, medium and high three levels. The results are listedas follows:(1) In this study, we isolated and purified bovine SCs in skeletal muscle from cattlelegs using mechanical separation, combined enzyme digestion and differential adhesionmethodologies. Then optimized best culture system for SCs amplification in vitro andsuccessfully passages to the23generation. It was founded that the SCs had higher cellviability after cryopreservation.(2) The results of immunocytochemistry staining for detection of surface markersexpression in SCs showed that P4, P9, P20generations of Bovine SCs were positivelyexpressed with Desmin and MyoD. The results of RT-PCR detection for SCs surfacemarkers expression showed that all of P4, P9, P20generations of Bovine SCs werepositively expressed with Desmin, Pax7, c-Met, Myf5. And the result of Grayscale analysisshowed that the Variation of the same surface markers from different passages were notsignificant and coincidence with expressional situation of SCs surface marker.(3) All kind of growth curves that went through certain stages including latency phase,logarithmic growth phase and stationary phase from P4, P9and P20. Generations of bovineSCs were presented typically “S” shape. Different passages of SCs all have a well colonyformation but the rates of colony formation were decreased gradually with the increasingnumber of cell passages. The results of flow cytometry analysis for SCs cell cycle showedthat the cell proportions were increased in G0/G1period and decreased in S periodaccording with the increased number of cell passages.(4) The number of Bovine SCs chromosome is2n=60, and the morphology ofchromosomes were no distortion. The chromosomes are proximal to the centromerechromosomes except two sex chromosomes. Those evidence indicating that the isolated SCs in this expriment were all originated form normal Luxi cattle embryos.(5) Induced P4, P9, P20generation of bovine SCs to differentiated into myobalsts,adipocyte, osteoblast, neurogenic and epidermal cells under different inducing conditions.Then applying corresponding staining methodology to detected the inducing affects. Theresults showed that the staining methods applied here were all expressed positively. AfterSCs were induced into different cells, we analyzed the expression of corresponding surfacemarkers including fast skeletal myosin in myotubules, PPARγ and LPL in adipocyte,osteopontin and collage type Ⅰin osteoblast, MAP2and nestin in neurogenic, and α6andCK19in epidermal cells by RT-PCR. Afterword, the results showed those markers were allexpressed positively. Grayscale analysis On expressional amount of target genes showedthat the differences of differential aspect in low-passage and middle-passage SCs were nosignificant difference. But the differential aspect of high-passage cells were reducedsignificantly.These results confirmed that the Luxi cattle embryonic SCs were suitable for isolationand culture in vitro. The low, middle and high-passages SCs were still possess strongbiological characteristics such as self-renewal, proliferation and differentiation afterpassage cultured repeatedly. Besides, Cattle SCs had the advantage of easily and massiveobtained characteristics. So SCs would be used as an ideal seed cells type to protect thevaluable genetic resources Luxi cattle. In addition, it will provide experimental basis forthe clinical usage of SCs. |
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