Effects Of TGF-?1 And MGF On The Proliferation And Differentiation Of Rabbit Skeletal Muscle Satellite Cells In Vitro

Objective: To explore the methods of isolation,extraction and culture of rabbit skeletal muscle satellite cells and observe the effects of different concentrations of TGF-?1 and MGF on the proliferation and differentiation of rabbit skeletal muscle satellite cells in vitro.Methods: 1.The skeletal muscles were taken from the newborn healthy rabbits of1-week old.The skeletal muscle satellite cells(SMSCs)were extracted by the methods of modified mixed enzyme digestion and differential adherence,and the primary SMSCs were identified by immunohistochemistry stain of desmin.2.Well-grown third generation SMSCs were inoculated on the 96-well plate at a density of 8x103/ml,and divided into control group,TGF-?1 groups(0.5?1?2?5?10?20ng/ml)and MGF groups(15?30?45?75?100?125ng/ml).The proliferation and differentiation of SMSCs in different groups were evaluated by CCK-8 and morphological observation at 24 hours intervals,and the relative optimal concentration of TGF-?1and MGF were selected.3.CCK-8 was employed in measuring the OD value of SMSCs in different relative optimal concentrations of TGF-?1 and MGF groups(control group,TGF-?1group,MGF group,TGF-?1 + MGF group)and the levels of Myf5 and Myogenin m RNA were detected by Real time PCR(control group,TGF-?1group,MGF group)at 0,2,4,and 6 days.Results:1.Immunocytochemical staining showed that Desmin was strongly expressed in rabbit SMSCs.2.CCK-8 measured results in different concentrations of TGF-?1 and MGF groups(1)TGF-?1 group: Effect of proliferation appeared from the fourth day,the OD value of 10ng/ml group was significantly higher than that of the control and other groups(P< 0.01).At fifth day,the OD value of all different concentration TGF-?1 groups were higher than that of the control group respectively(P<0.01),and 10ng/ml group was significantly higher than other groups(P<0.01),20ng/ml group was significantly higher than that of 0.5ng/ml,1ng/ml,2ng/ml and 5ng/ml groups(P<0.05).At sixth day,the OD value of all different concentration TGF-?1 groups were higher than that of the control group respectively(P<0.01),10ng/ml group was significantly higher than other groups(P<0.01),0.5ng/ml group was lower than 1ng/ml and 2ng/ml groups(P<0.01),1ng/ml group was no significantly different from the 2ng/ml group(P>0.05).The 10ng/ml TGF-?1 was selected as the best relative effect concentration.(2)MGF group: Effect of proliferation appeared on the second day,the OD value of all different concentration MGF groups were higher than that of the control group respectively(P<0.01),100ng/ml group was significantly higher than the 15ng/ml,30ng/ml and 45ng/ml groups respectively(P<0.05).From the third to fifth day,the change trends of OD value were similar to the second day.At sixth day,the OD values of 45ng/ml,75ng/ml,100ng/ml and 125ng/ml groups were significantly higher than that of the control group respectively(P<0.01),but the 15ng/ml and 30ng/ml groups were no significantly different from the control group respectively(P>0.05).At the same time the OD values of 15ng/ml and 30ng/ml groups were significantly lower than that of other MGF groups respectively(P<0.01).The OD value of 100ng/ml group was significantly higher than that of other MGF groups respectively(P<0.01).The 100ng/ml MGF was selected as the relative best effect concentration.(3)The relative optimal concentration group: On the second day,the OD value of MGF group(100ng/ml)was significantly higher than that of the control group(P<0.01),there was no significant difference between TGF-?1 group(10ng/ml)and TGF-?1+ MGF group(P>0.05).From the third to sixth day,the OD values of TGF-?1and MGF groups were significantly higher than that of the control group respectively(P<0.01),and MGF group was higher than that of TGF-?1 group(P<0.01).The TGF-?1 + MGF group was no different from the control group(P>0.05).3.The m RNA levels of Myf5 and MyogeninThe m RNA level of Myf5 increased first and then decreased gradually in the control,TGF-?1,and MGF groups whereas Myogenin in the three groups showed low-level expressions and then a slowly rising trend.The m RNA level of Myf5 in TGF-?1 group on day 4 was significantly higher than that on day 0(P<0.01)and also higher than the same period of the control group but with no significant difference(P>0.05).The m RNA level of Myogenin in TGF-?1 group was lower than the control group in six days but there was no significant difference(P>0.05).At second day,the m RNA level of Myf5 in MGF group was significantly higher than the control and TGF-?1 groups(P<0.05).At fourth day,the change trends of the m RNA level of Myf5 was similar to second day but there was no significant difference(P>0.05).The m RNA level of Myogenin in MGF group was significantly lower than that in the control and TGF-?1 groups at fourth and sixth days(P<0.05).Conclusion:1.The rabbit SMSCs were successful obtained by modified mixed enzyme digestion and differential adherence,and identified by immunohistochemistry staining of desmin.2.TGF-?1 could induced the proliferation of rabbit SMSCs in the range of 0.5-20ng/ml and the optimal concentration was 10ng/ml.But it had a weak effect on the differentiation of rabbit SMSCs.3.MGF had a strong proliferation effect on rabbit SMSCs in the range of15-125ng/ml and the optimal concentration was 100ng/ml.It also had a strong inhibitory effect on differentiation.4.When the relative optimal concentration of TGF-?1 and MGF acted on the rabbit SMSCs respectively,MGF promotes proliferation stronger than TGF-?1.There was no proliferation effect when combined the relative optimal concentration of TGF-?1 and MGF.