Comparison Of Proliferation And Differentiation Capacity Of Skeletal Muscle Satellite Cells In Nanjiang Yellow Goat And Boer Goat

Acting as stem cells in muscle, skeletal muscle satellite cells (SMSCs) play a crucial role in postnatal muscle growth and repair after injury.In this study, we obtained the high-purified SMSCs from longissimus dor si of newborn Nanjiang yellow goat and Boer goat using 0.2% Pronase and Percoll density gradient centrifugation, and identified by using morphological analysis and immunofluorescence of PAX-7. Then, the proliferative capacity of SMSCs from 0 d to 8 d were analyzed by using hemocytometer and CCK-8 kit, and the fusion rate of SMSCs from 3 d to 8 d were evaluated through statistical nuclei of fusion.In addition, to explore the mechanism on proliferation and differentiation of SMSCs, the expression levels of PAX-7, MYOD, MYOG and six p38MAPK signaling pathway genes (TAK1, MKK3, MKK6,p38MAPK, MEF2A and MEF2C) were detected in SMSCs of both breeds at different stages (1 d,3 d,4d and 7 d). It is essential that screening the reference genes of stable expression in SMSCs during the different stages to normalize qRT-PCR data. The expression of nine common internal control genes including β-ACTIN, GAPDH, TOP20, YWHAZ, SDHA, PGK1, RP2, RPL4 and HPRT1 were investigated in SMSCs at 1 d,3 d,4 d and 7 d by employing qRT-PCR, and the data were analyzed using the geNorm software.(1) Immunocytochemistry analysis showed that more than 95% isolated goat SMSCs were positive for monoclonal antibody PAX-7, suggesting that the Percoll density gradient centrifugation was suitable to obtain high-purity SMSCs.(2) Significant higher SMSCs number and OD value of SMSCs at 3 d,4 d and 5 d were found in Nanjiang yellow goat (P< 0.05) compared with Boer goat. Additionally, the exponential growth period emerged earlier in Nanjiang yellow goat than Boer goat. These results imply that the Nanjiang yellow goat SMSCs have greater proliferative capability and growth speed compared with Boer goat.(3) The fusion speed of SMSCs was faster after 4 d, and the fusion rate reached 80% at 8 d. Moreover, the fusion rate of SMSCs was higher (P< 0.05) at 6 d in Nanjiang yellow goat than that in Boer goat.(4) The stability rank of nine reference genes from high to low was PGK1, SDHA, β-ACTIN, RPL4, GAPDH, TOP2β, YWHAZ, RP2, and HPRT1 in SMSCs at different stages, and three genes (PGK1, SDHA and β-ACTIN) were recommended to normalize the qRT-PCR data.(5) The expression level of PAX-7 mRNA was relatively high at proliferative stages and reached the highest levels at 3 d, but was down-regulated in SMSCs during the differential stages in both breeds, and at 1 d and 3 d of proliferative stages of SMSCs in Nanjiang yellow goat were significantly higher (P< 0.05) than those in Boer goat. A relatively high level of MYOD mRNA was found in SMSCs at proliferative and differential stages, with the highest level at 1 d of differential stages. Additionally, the markedly higher levels (P< 0.01) of MYOD were detected at 3 d of proliferative stages and at 1 d of differential stages of SMSCs in Nanjiang yellow goat. MYOG was not expressed in proliferative stage while a high level exhibited at differential stages, and peaked at 1 d of differential stages. These results suggest that PAX-7 and MYOG play roles in proliferation and differentiation, respectively, while MYOD works on both proliferation and differentiation in SMSCs from two breeds.(6) Among the p38MAPK signaling pathway genes, TAK1 and MKK6 were rarely expressed at all stages. The expression levels of MKK3, p38MAPK, MEF2A and MEF2C were relatively high, and their expressions increased when the SMSCs began to differentiate, suggesting these four genes possibly involve in the regulation of SMSCs differentiation. Compared with Boer goat, the expression levels of MKK3 at 4 d of differentiation, MKK6 at 1 d and 4 d of differentiation, p38MAPK at 1 d of differentiation, and MEF2C at 1 d and 4 d of differentiation in SMSCs from Nanjiang yellow goat were significant higher (P< 0.05). No significant differences of MEF2A expression were detected between two breeds, but the abundance of MEF2A was relative high. These results imply that the MKK3, p38MAPK, MEF2A and MEF2C to promote the differentiation of SMSCs combine with the feature of fusion rate in two breeds.