| This paper aimed to study the function of MSTN in the process of multipotential stem cells of muscle (Mesenchymal multipotential stem cells, Adipose-derived stem cell and Muscle satellite cell) differentiating into adiposities and in the growth of adipose tissues. Firstly, we treated different differentiated C3H10T1/2 cells using recombinant human MSTN protein, and then the cells were differentiated into adiposities by DIM treatment. Using this method, we analyzed the role of MSTN on the adipogenesis of C3H10T1/2 cells, and we also analyzed the receptor and the phosphorylated protein of MSTN signaling in this process. Secondly, we analyzed the role of MSTN on the adipogenesis of porcine Adipose-derived stem cell (ADSC) and Muscle satellite cell. Thirdly, we isolated the promoter of MSTN gene. And then, we decomposed transcriptional activity of MSTN promoter which was regulated by adipogenesis related factors. Fourthly, the expression patterns of MSTN in longissimus muscle, back fat and abdominal fat was detected at the stages of 35-day,2-month,3-month,4-month and 6-month Meishan pig respectively, using quantitative real-time PCR methods. The results could provide available information to recognizing the probable influence of MSTN in the growth of adipocyte tissue. The main results are as follows:1. The role of MSTN on the adipogenesis of C3H10T1/2 cellsWhen MSTN treated C3H10T1/2 cells in the process of adipogenesis, there was no difference in the adiposities among the different groups, however, the adiposities was significantly higher in 50 ng/ml of MSTN than control (P< 0.05) when rosiglitazone was added into the medium. When C3H10T1/2 cells were differentiated into adiposities after treated by MSTN, the adiposities was significantly higher in low concentration (5 ng/ml) of MSTN than control (P< 0.05). C3H10T1/2 cells could have the potential of differentiated into fat cells and myoblasts after azacytidine treatment. The adiposities was significantly in 50 ng/ml MSTN group higher than in control, when C3H10T1/2 cells was treated by azacytidine and MSTN. However, there was no difference in the adiposities among the different groups when C3H10T1/2 cells was treated by azacytidine and then induced into adiposities by DIM plus MSTN. Those results indicated that MSTN might inhibited the myogenesis potential and promote the adipogenesis of C3H10T1/2 cells. In the regulation, MSTN may firstly bind to ActRⅡB, and then through ALK4 regulating the expression of downstream genes, ALK5 may have subsidiary role in this process. In the signaling pathway, Smad2/3 and p38-MAPK could be phosphorylated by MSTN. In the myogenesis of C3H10T1/2 cells, the inhibited role could be played through Smad2 and p38-MAPK by MSTN.2. The role of MSTN on the adipogenesis of adipose-derived stem cell (ADSC) and muscle satellite cellWe isolated ADSC cells and MSC cells from porcine fat tissues and semitendinosus muscle respectively. Using immunocytochemistry methods, we found that the vast majority of ADSC cells were positive for CD 44 but not for Desmin. However, most of MSC cells were positive for CD44 and Desmin. When we treated ADSC cells and MSC cells with different concentration of MSTN during DIM (differentiation inducer) treatment, the adipogenesis of ADSC cells and MSC cells were inhibited as the increased concentration of MSTN added into DIM, the inhibited results also shown in MSC cell which was treated with MSTN before DIM treatment, however, there was no influence when ADSC cells was treated with MSTN before DIM treatment. The similar results were confirmed by our real-time PCR data of PPARy and aP2 when we treat cells with MSTN (50 ng/ml) which was similar to the treatment above. Thus we conjecture that MSTN had differently inhibited roles on the adipogenesis of ADSC and MSC cells. Because MSTN is secreted from muscle cell, then we presume that the adiposities in the muscle may mainly come from ADSC cell.3. The transcriptional activity of MSTN promoterBased on the sequence of Sus scrofa MSTN gene (accession number:AY208121), we amplified a 1709 bp (base pair) sequence containing 60 bp of coding sequences (CDS) and 1649 bp of flanking 5’-region of MSTN gene of pig using designed primers. Six progressive deletion constructions of the pig MSTN promoter were analyzed in myoblast, myotube and C3H10T1/2 cells though Dual-luciferase reporter assay. The longest construct pGL3-MSTNp1 was treated with rosiglitazone, DIM or the constitution of DIM. We found that porcine MSTN could be up-regulated by IBMX and rosiglitazone, but not by other constitutions of DIM, Contransfection experiments revealed that MyoD and PPARy could up-regulated the expression of MSTN, but C/EBPa and C/EBPβdown-regulated the expression of MSTN. Based on the result, we conjecture that the expression of MSTN will has a transient increase to exert some function until C/EBPa and C/EBPβare expressed when the cells were induced to differentiate into adipocytes.4. The expression pattern analysis in adipose tissuesRNA was extracted from the longissimus muscle, back fat and abdominal fat of Meishan pig at the stage of respectively 35-day,2-month,3-month,4-month and 6-month. This study examined the expression of MSTN, Activin receptor typeⅡB (ACVR2B), peroxisome proliferator-activated receptor (PPAR) y and adipocyte specific fatty acid binding protein (aP2) in longissimus muscle, back fat and abdominal fat at the stages of 35-day,2-month,3-month,4-month and 6-month Meishan pig respectively, using quantitative real-time PCR methods. The results indicated that the expression of MSTN and ACVR2B in longissimus muscle decreased gradually from the day 35 to the third month point, and then increased. Both MSTN and ACVR2B had a weak positive correlation (r< 0.3) with aP2 and PPARy in longissimus muscle which indicated that MSTN may not influence the adipogenesis in this tissue. Therefore, we conjecture that the decrease may be related to longissimus muscle growth before the third month point, and the increase may be related to the slowing down of longissimus muscle growth after the third month stage. MSTN shown an increased expression pattern in the back fat, and ACVR2Balso increased gradually except the highest point at the stage of 2-month. The expression of aP2 also showed an increased expression pattern in the back fat, indicating that the obesity is increased with the growth of back fat. In addition, MSTN had a positive correlation with aP2 and PPARy in this tissue (P< 0.05), and ACVR2B had a positive correlation with aP2 (P= 0.181) and PPARy (P= 0.187). Thus, we could conjecture that the increased expression patterns of MSTN and ACVR2B in the back fat may be due to MSTN response to obesity in the growth period. Our study found that the expression of PPARy at 2-month and 3-month old was higher than that at the stage of 35-day, and aP2 was increased with the growth of abdominal fat tissue. In addition, MSTN had a negative correlation with aP2 and PPARy, and ACVR2B had a negative correlation with PPARy. However, the expressions of MSTN and ACVR2B at the stage of 35-day were higher than that at other stages (P< 0.05). Taken all these into consideration, adiposities of abdominal fat tissue may has better growth at other stages than that at 35-day. |
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