Functional Study Of SREBP1 In Buffalo Mammary Epithelial Cell

Compared with cow milk, there is more plentiful nutrients in buffalo milk, such as protein, fat, vitamin, and buffalo milk is idea food for human livelihood. Sterol regulatory element-banding protein 1 (SREBP1), an important transcription factor, regulates the expression and activity of enzyme and protein involved in milk fat synthesis to influence on the synthesis and secretion of triglyceride in mammary epithelial cells. In the present study, we identified that milk fat globule (MFG) could be used to study the expression of functional genes in mammary epithelial cells in vivo using qRT-PCR, and we proved that SREBP1 is involved in regulating various enzymes and proteins related with milk fat synthesis and SREBP1 gene may act on ERK1/ERK2 signaling pathway to regulate the expression of PPARy gene using RNAi technology and method of fatty acid treatment. This study provides theoretical foundation for further study the mechanism of milk fat synthesis in buffalo. The main research results are as follows:1. The study identified that the total RNA isolated from MFG mainly derived from mammary epithelial cells and could be used to study the expression of functional genes in mammary epithelial cells. Among the transcript of MFG, milk somatic cells (MSC), mammary gland (MG), there was no expression of adipocyte-specific gene(AdipoQ), leukocyte-specific genes (CSF1, IL1α, CD43) in MFG; the expression of the epithelial cell marker genes (Keratin 8, Keratin 18) in MFG was higher than in MSC and lower than in MG; the expression of fibroblast marker gene (vimentin) in MFG was lower than in MSC and MG; the expression of milk protein genes (LALBA, BLG, CSN2) and milk fat synthesis related genes (ACACA, BTN1A1, FABP3, FAS) in MFG was higher than in MSC and MG.2. The coding sequence of SREBP1 and PPARy was amplified by using milk fat globule as experimental materials, the length of the two genes was respectively 3438bp and 1428bp. 3. Buffalo mammary epithelial cells (BMECs) were successfully isolated and passaged. The BMECs converged as typical cobblestone morphology, formed dome-like structures and papillate structures, could secrete milk fat droplet and its growth curve was S-shaped. The results of immunocytochemistry showed that there were the expressions of Keratin 18 and vimentin in BMECs. 4. SREBP1 gene was involved in the regulation of various enzyme and transported protein during milk fat synthesis. After infecting buffalo mammary epithelial cells with shRNA-SREBP1 lentiviral particle, the mRNA expression of SREBP1 decreased by 45% and the triglyceride concentration of SREBP1 inhibition group in growth medium was extremely significantly higher than that of negative control group(P<0.01). After treating with fatty acid, the triglyceride concentration of oleic acid group and steric acid group in growth medium was extremely signigicantly higher than that of control group(P<0.01), the triglyceride concentration of palmitic acid group in growth medium was extremely signigicantly lower than that of control group(P<0.01); the results of oil red O staining showed that oleic acid and steric acid can inhibit the generation of lipid droplet, and palmitic acid can promote the generation of lipid droplet in mammary epithelial; the results of qRT-PCR showed that, the expression of SREBP1 gene in oleic acid group and steric acid group was downregulated compared with control group, and the expression of SREBP1 gene in palmitic acid group was upregulated. After infected with shRNA-SREBP1 lentiviral particle and treated with fatty acid, the expression trend of ACACA, FABP3, FAS, SCD, ERK1, ERK2, PPARy and Insigl genes was consistent with the expression trend of SREBP1 gene. These results suggested that SREBP1 gene is a central transcription factor in regulating milk fat synthesis and SREBP1 gene may act on ERK1/ERK2 signaling pathway to regulate the expression of PPARy gene.