| The HS6ST2 gene is belong to the HS6ST gene family and can encode the 6-O Sulfotransferases 2 which can specific modifying the 6-Osulfation site of the heparan sulfate(HS) chains connected on the heparan sulfate proteoglycans(HSPG). It has been reported that the HS6ST2 involved in Notch, Wnt and TGFβ signal pathway that are the pivotal signal pathys which involved in the regulation about proliferation and differentiation process of satellite cells, and HS6ST2 was a potential candidate gene associated for skeletal muscle growth and development. However, the regulatory mechanism of HS6ST2 gene behind it is still unclear. Therefore, we isolated and cultured the original generation of skeletal satellite cells of Hailan laying hens and Avian broilers at three different age (1week,2 weeks and 6 weeks), and then check the HS6ST2 gene expression level in satellite cells of laying hens and broiler through RT-PCR method. For research the function of HS6ST2 gene in chicken satellite cells proliferation and differentiation regulation and signal transduction of the Wnt/β-catenin、TGFβ/MSTN、Notch signal pathways,we constructed the overexpression vector and shRNA vectors of chicken HS6ST2 gene, and transfected those vectors into chicken original generation satellite cells, then weprobed the effects on chicken satellite cells through RT-PCR method, CCK8 cell proliferation test method and cell morphology observation method,etc. The main findings are as follows:(1) The primary skeletal muscle satellite cells of laying hens and broilers were isolated and cultured, and the purity of all cells identified by immunofluorescence was more than 90%, after the isolation, the satellite cells number of the same week old laying hens were significantly more than that of broilers, and this difference would reduce with the day-old increasing; the differentiation of primary satellite cells of laying hens were lower than that of broilers, in the cultural process, myotubes appeared in the satellite cells of laying hens were approximately 12 hours in advance than that of broilers.(2) In RT-PCR detection, we found that the HS6ST2 gene mRNA expression level in vitro culture stages were higher than those of broilers in primary skeletal muscle satellite cells of 1W,2W,6W old laying hens that were isolated and cultured, and when in vitro adherent Oh, the HS6ST2 mRNA expression amount among the satellite cells were significantly different (P< 0.05) in different week old laying hens and broilers, the HS6ST2 mRNA expression quantity in satellite cells of both laying hens and broilers would reduce with the day-old increasing; this showed that the HS6ST2 gene mRNA expression in satellite cells of both laying hens and broilers satellite existed significant differences.(3) After the construction of HS6ST2 gene over expression vectors and interference vectors and the transfection of chicken skeletal muscle satellite cells, compared with the positive control group, after the interference of HS6ST2 gene, the cell number significantly reduced, but the myotubes appear time and yield in cells were earlier and higher than that of the control group, but after the over expression of HS6ST2 gene, the situation was in contrast with the interference group. Each cell proliferation curve after the transfection in each group by the construction of CCK8 assay the showed that compared with the positive control group, the value-added rate of interference group was significantly lower, and the value-added rate of super table group increased significantly; in addition MyoG, MyoD, MyHC mRNA expression in satellite cells of interference group was significantly higher than that of the positive control group (P< 0.01), Pax7 gene expression levels were significantly lower than those in the positive control group (P< 0.01), the situation of over expression group was in contrast with interference group. These results suggested that HS6ST2 gene have the functions of promoting the proliferation and inhibiting of differentiation of satellite cells.(4) After the expression on Wnt/β-catenin, TGF β/MSTN, Notch pathway key protein gene in satellite cells by detecting RT-PCR the interference and over expression of HS6ST2 gene showed that, compared with the positive control group, GSK3βgene mRNA expression level decreased significantly (P< 0.05 or P< 0.01) in Wnt/(3-catenin pathway of satellite cells in interference group, but β-catenin expression increased significantly (P< 0.01); the ACVR1, ACVR2 gene mRNA expression level decreased significantly (P< 0.05 or P< 0.01) in TGFβ/MSTN pathway; the Notchl, Hairyl, Hey2 gene mRNA expression level decreased significantly (P< 0.05 or P< 0.01) in Notch pathway, but Numb gene mRNA expression level significantly increased (P< 0.01); the situations of over expression group and interference group were on the contrary. These results suggested that the HS6ST2 gene had a positive regulatory role for the Notch and TGFβ/MSTN pathway in satellite cells but had negative regulatory role for Wnt/β-catenin signal passway. |
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