The effects of myostatin gene inhibition byan antisense oligodeoxynucleotide and its immunocytochemical localization ın c2c12 myoblasts

Büyüme faktörlerinin, kas hücrelerinin proliferasyonu ve diferansiyasyonu üzerine etkilerim araştırmak üzere, satellit hücrelerini temsilen $C_2C1_2$ çizgili kas hücreleri (miyoblast) yaygın bir şekilde kullanılmaktadır. Transforming growth faktör ailesinin son yıllarda izole edilmiş bir üyesi olan myostatin'in çizgili kas büyümesini engelleyen bir faktör olduğu kabul edilmektedir. Bu çalışmanın amacı, antisense Oligodeoxynucleotide (ODN) tarafından ekspresyonu inhibe edilen myostatin geninin, $C_2C1_2$ satellit hücrelerinin proliferasyonu ve diferansiyasyonu üzerine etkisini araştırmak ve myostatin proteininin lokalizasyonunu belirlemektir. $C_2C1_2$ hücreleri antisens ve kontrol gurubu (birer mikrogram ODN) için ayrı ayrı olmak üzere, Lipofectamin aracılığı ile transfekte edildi. Proses edilmemiş (aktif hale gelememiş) myostatin proteininin, $C_2C1_2$ hücrelerinde lokalizasyonunu tespit için standart immunositokimya tekniği (3,3-diaminobenzidine tetrahydrochloride; DAB) kullanıldı. Myostatin İnhibisyonunun miyotüp oluşumunu hızlandırdığı gözlemlendi. Proses edilmemiş myostatin proteini yaygın olarak miyoblast ve miyotüplerin sitoplazmasında bulundu.

Anahtar Kelimeler:

fareler, antisens DNA, hücre kültürü, büyüme engelleyiciler, immünositokimya, kaslar, iskelet kası, dönüştürücü büyüme etkeni

Antisens oligodeoxynucleotide aracılığı ile myostatin geni inhibisyonunun C2C12 miyoblast hücreleri üzerine etkisi ve myostatin'in immunositokimyasal lokalizasyonu

$C_2C1_2$ skeletal muscle cell line as a representative of satellite cells has been used extensively in the effects of muscle growth factors on muscle cell replication and differentiation. Myostatin, a recently identified member of transforming growth factor-b family, has been widely accepted as a negative regulator of .skeletal muscle growth. The purpose of the present study was to determine whether myostatin inhibition by an antisense oligodeoxynecleotide (ODN) alters proliferation and/or differentiation rates and its location in $C_2C1_2$ satellite cells. $C_2C1_2$ cells were transfected with one microgram of ODNs for corresponding antisense and control groups via lipofectin reagent. Standard immunocytochemistry technique with 3,3-diaminobenzidine tetrahydrochloride (DAB) was used to localize unprocessed myostatin protein in $C_2C1_2$ cells. Myostatin inhibition stimulated myoblast differentiation to give rise to myotubes. Positive staining for unprocessed myostatin was mainly detected in the cytoplasm of myotubes and myoblasts.

Keywords:

mice, antisense DNA, cell culture, growth inhibitors, immunocytochemistry, muscles, skeletal muscle, transforming growth factor,

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