| Rheb (Ras homolog enriched in brain) is one of the small G protein and is highly conserved in many organisms from bacteria to human. Rheb gene belongs to a family of genes called RAS, and is expressed in a variety of animal organs and tissues. Rheb has a variety of functions, including promoting formation, storage the fat, cell cycle regulation, regulation of myocardial cell growth and so on.Recent studies have found that growth and development of muscles are associated with mTOR signaling pathway. Rheb gene is the upstream gene of mTOR signaling pathway can regulate the function of muscle cells. Since mTOR signaling pathway is so important to the physiological processes of skeletal muscle satellite cells, Rheb as its upstream regulatory factors necessarily related changes in skeletal muscle work. The study of Rheb overexpression or gene interference on Aerbasi cashmere goats’muscle satellite cells in vitro can help us to explore the role and influence of Rheb in the cashmere goat muscle growth.Rheb is mTOR signaling pathway upstream regulation genes, to participate in the regulation of skeletal muscle cell proliferation and growth. This study was purposed to explore the underlying role of Rheb in Aerbasi cashmere goat muscle satellite cells.1. The construction of Rheb overexpression vector and interfering carrier vectors.Sequences of Aerbasi cashmere goats Rheb cDNA is cloned correctly. We designed and constructed Rheb overexpression vector and interfering carrier vectors. The Rheb overexpress vector was composed of CAG promoter, Rheb, IRES, AcGFP. the CAG promoter can be efficiently start the expression of target gene, and downstream gene of the IRES-AcGFP express green fluorescence protein, which can detect the expression of Rheb gene in transgenic cell.According to cashmere goat Rheb gene cDNA sequencing, we designed and constructed four Rheb interference carrier vectors and a control vector. We compared transfection efficiency of these four vectors in the goat skeletal muscle cells, High efficiency vectors for Rheb interference will be useful for studying function of Rheb gene in the goat skeletal muscle cells.2. The effects of the Rheb overexpression on development-related gene expression in skeletal muscle satellite cells.The goat skeletal muscle satellite cell lines overexpressing Rheb were established using liposome-mediated method. The cell transfection efficiency, electronic volume and cell cycle were detect by FACS.The mRNA and protein expressions of development-related gene were determined by Real-Time PCR and Western blot respectively. The results showed that fetal fibroblasts transfection efficiency was higher than muscle satellite cells. Overexpression of Rheb gene had no significant effect on both the size of the muscle satellite cells and cell cycle. Overexpression of Rheb could cause an upregulation of MSTN, but the expression level of S6K had no significant changes. The goat skeletal muscle satellite cell lines overexpressing Rheb show a decreasing correlation of gene expression of DES, MyHC, MyoD, Myf5, MyoG, Myf6, MCK.3. The effects of the Rheb interference on development-related gene expression in skeletal muscle satellite cells.The goat skeletal muscle satellite cell lines transfect with Rheb interference vectors were established using liposome-mediated method.The cell transfection efficiency, electronic volume and cell cycle were detected by FACS. The mRNA and protein expression of development-related genes were determined by Real-Time PCR and Western blot respectively. The results showed that fetal fibroblasts transfection efficiency was higher than muscle satellite cells. Lower expression of Rheb gene had no significant effect on both the size of the muscle satellite cells and cell cycle. The down regulation of Rheb could cause increastion of MSTN, but the expression level of S6K had no significant changes. The goat skeletal muscle satellite cell lines reducing Rheb show a increasing correlation of gene expression of DES, MyHC, MyoD, Myf5, MyoG, Myf6, MCK. |
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