| Adipose Derived Stem Cells（ADSCs）are adult stem cells present in the body′s adipose tissue.Compared with bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells,ADSCs are easy to obtain and little damage is brought to the individual.At the same time,cultured with the appropriate condition,ADSCs could differentiate into multi-lineages cell types,such as adipocyte,osteoblast and chondrocytes,which has great potential in regenerative medicine,medical cosmetology and research on fat metabolism;At the same time,it is of great significance to find out the mechanism of fat differention to improve quality and flavor of the meat and enhance the cold resistance of livestocks.Studies have shown that estrogen can regulate lipid synthesis and accumulation of ADSCs by activating Estrogen receptorα（ERα）and Estrogen receptorβ（ERβ）.G-protein coupled receptor 30（GPR30）,a membrane estrogen receptor discovered in recent years,was found to be expressed in fat,breast,ovary and other organs and tissues,in which mediated the rapid non-genomic effects of estrogen on the stimulation of downstream cascade reaction,and played a certain role in breast proliferation and fat accumulation.However,whether estrogen could participate in the regulation of adipogenic differentiation of ADSCs through GPR30 signaling pathway remains to be further investgated.In this experiment,Guanzhong dairy goats were used as material to isolate and culture ADSCs.Then the expression and distribution patterns of GPR30 and the effects and mechanisms of estrogen on adipogenic differentiation in goat ADSCs were reserched.The experimental method was as follows:（1）The experiments of inducing adipogenic,osteogenic and chondrogenic differentiation were carried out to prove that the isolated and cultured cells could differentiate into adipocytes,osteoblasts and chondroblasts,and it is preliminarily determined that the cultured cells had a multi-lineages differentiation ability of adipogenesis,osteogenesis and chondrogenesis,with stem cell characteristics;The isolated cells were further identified by measuring mesenchymal and hematopoietic stem cells surface markers.Fixed cells were incubated with anti-CD29,CD34,CD44,CD45,CD73,CD90,CD105 antibodies,and analyzed by flow cytometry.The isolated cells were positive for CD29,CD44,CD90,CD105,the mesenchymal stem cell surface markers,and negative for the hematopoietic stem cell markers such as CD34,CD45,CD73.The isolated cells were characterized by surface markers of ADSCs and proved to be goat adipose stem cells.（2）The expression and distribution patterns of GPR30 in ADSCs were studied by immunofluorescence staining,PCR and Western blotting.The results showed that ADSCs cells could transcribe GPR30 m RNA and express GPR30 protein stably.Immunofluorescence staining confirmed that GPR30 expression is localized in the cell membrane.（3）ORO staining,absorbance and Real-time fluorescence quantitative PCR（q RT-PCR）were used to study the effect of estrogen/GPR30 signaling pathway on adipogenic differentiation of ADSCs.The results showed that estrogen and GPR30-specific G1 could effectively promote lipid synthesis and increase the relative levels of adipogenic m RNA such as ACC,ADIPOQ,FABP4,FASN,LPL and PPARγduring adipogenic differention of ADSCs.However,G15,the GPR30 inhibitor,could block the estrogen-promoting effects on adipogenic differentiation of ADSCs.Which indicated that estrogen regulated the adipogenic differentiation of goat ADSCs through GPR30 signaling pathway.（4）ORO staining and q RT-PCR were used to detect effects of estrogen,G1 and G15on adipogenic differentiation of ADSCs in the presence and absence of U0126,the antagonist of extracellular regulated protein kinases 1/2（ERK1/2）.The lipid accumulation and relative levels of adipogenic m RNA were detected in each treatment groups.Treatment with U0126 attenuated the promoting effects of estrogen and G1 on adipogenic differentiation of ADSCs;Lipid accumulation and relative levels of adipogenic m RNA in ADSCs decreased significantly.This indicated that ERK1/2 was involved in the adipogenic differentiation regulation of ADSCs by estrogen/GPR30 signaling pathway.（5）The effects of estrogen/GPR30 signaling pathway on the expression of ERK 1/2were examined by Western blotting.It was found that E2 and G1 could promote ERK1/2phosphorylation;while G15 blocked the effect of 17βE2 on ERK1/2.Combined with above experiments,estrogen/GPR30 signaling pathway could mediate the adipogenic differentiation of ADSCs by activating ERK1/2. |
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