Method Construction And Effect Study Of Simulating Intramuscular Fat Environment

Intramuscular fat is closely related to meat quality and flavor.Studies have shown that the Wnt signaling pathway affects intramuscular fat formation by inhibiting the differentiation of proadipocytes and the Hedgehog signaling pathway by blocking the differentiation of multipotent mesenchymal cells into adipocytes.Simulation of the formation environment of intramuscular fat cells is the basis of research on the formation of intramuscular fat cells.At present,most of the existing studies are on the analysis of a single cell,but there are both muscle cells and fat cells in the intramuscular fat environment,the use of a single cell can not well simulate the formation environment of intramuscular fat cells.Transwell cell co-culture is a newly developed indirect culture method,which can explore the interaction between two kinds of cells on the basis of physical separation in vitro.Myostatin(MSTN)is a negative regulator of muscle growth secreted by muscle cells.Previous studies using single adipose mesenchymal stem cells have shown that MSTN can affect intramuscular fat formation,but how MSTN plays a role in the presence of both muscle cells and preadipose cells is not clear.Therefore,in this study,C2C12 cells and 3T3-L1 cells were co-cultured in Transwell chamber to simulate the environment of intramuscular fat formation.Changes of 3T3-L1 cells were detected after MSTN treatment,so as to study the effect of MSTN on intramuscular fat formation.The main results are as follows:1.Establishment of Transwell cell co-culture systemThe induced differentiation of C2C12 cells was used to simulate muscle cells in muscle tissue,and 3T3-L1 cells were used to simulate preadipocytes in muscle tissue.A Transwell chamber was used to construct a co-culture environment,with C2C12 cells at the top and 3T3-L1 cells at the bottom.Isolated cultured 3T3-L1 cells were used as control.The results showed that the expression level of PPARγ in 3T3-L1 cells after co-culture was significantly decreased(P<0.01),and the formation of lipid droplets also decreased.These results indicated that the adipogenic ability of 3T3-L1 cells was inhibited after co-culture with C2C12 cells.This phenotype is consistent with the small number of adipocytes in muscle tissue of individual animals.Transwell cells with 3T3-L1 cells were inserted into cell culture plates with C2C12 cells for co-culture,and C2C12 cells cultured alone were used as control.The two groups were treated with adipogenic induction medium DIM.The results showed that the expression of PPARγ in C2C12 cells after co-culture was significantly decreased(P<0.01),and the formation of lipid droplets also decreased.These results indicated that the co-culture with 3T3-L1 cells inhibited the adipogenesis of C2C12 cells.The above indicated that the intercellular factors of the two kinds of cells in the co-culture system constructed by us could influence each other in physical isolation,which was in line with the experiment expectation.It indicates that our co-culture system can be used to simulate the intramuscular environment,and can be used for the subsequent study on the influence of MSTN on intramuscular fat formation in the intramuscular environment.2.MSTN inhibited the expression of PPARγ in 3T3-L1 cells under co-culture conditionTranswell chamber was used to construct a co-culture system with C2C12 cells in the upper compartment and 3T3-L1 cells in the lower compartment.The experimental group was treated with cell medium containing 100 ng/m L MSTN,and the control group was treated with ordinary medium.The two groups were simultaneously treated for 48 h.At the end of the experiment,lipid droplets appeared in 3T3-L1 cells in both groups after oil red O staining.The expression level of PPARγ in 3T3-L1 cells of control group was significantly higher than that of experimental group(P<0.01).These results indicated that MSTN inhibited the expression of PPARγ in 3T3-L1 cells under co-culture condition.3.effect of MSTN on two signal pathways under co culture conditions3T3-L1 cells and C2C12 cells were co-cultured with the Transwell cell co-culture system constructed in this study.3T3-L1 cells were treated with MSTN medium for 48 h,and collected at four time points of 0h,12 h,24h and 48 h for Q-PCR and WB to detect the changes of the two signaling pathways.Q-pcr results showed that the expression levels of c-My C,Cyclin D1 and Wisp2 genes in Wnt signaling pathway were decreased(P<0.05),while Axin2 expression was increased(P<0.05).WB results showed that the phosphorylation levels of Smad2 and Smad3 were significantly decreased(P<0.01),the phosphorylation level of C-My C was significantly decreased(P<0.01),and the protein expression of Wisp2 was also significantly decreased(P<0.01).These results indicated that MSTN treatment in co-culture decreased the activity of Wnt signaling pathway.Meanwhile,Smo and Gli2 genes in the Hedgehog signaling pathway were significantly down-regulated(P<0.05),Gli1 expression at the protein level was extremely significantly decreased(P<0.01),and Smo phosphorylation level was extremely significantly down-regulated(P<0.01).This indicates that the treatment of MSTN in co-culture reduces the activity of Hedgehog signaling pathway.Conclusions:(1)The Transwell chamber co-culture system constructed in this study can well simulate the environment of intramuscular fat formation,and can be used to study the formation of intramuscular fat.(2)In co-culture with C2C12 cells,MSTN treatment weakened the activity of Wnt signaling pathway in 3T3-L1 cells.(3)In co-culture with C2C12 cells,MSTN treatment weakened the activity of Hedgehog signaling pathway in3T3-L1 cells,which was conducive to adipogenesis of 3T3-L1 cells.