Effects Of Exogenetic Trans-10, Cis-12, CLA On Lipids Synthesis In Bovine Mammary Epithelial Cells (BMECs) And Molecular Mechanism

The t10c12 CLA isomer has been causally related to milk fat depression in dairy cows, although no clear molecular mechanism has been established. The objectives of this study were to examine the effects of exogenetic t10c12 CLA on lipids synthesis in cytoplasm of bovine mammary gland epithelial cells (BMGECs) achieved from tissue culture methods. This study was divided into four tests.Test 1. Culturing and certifying of BMGECs. Culturing process is as follows:Sampling, breast parenchyma isolated from udders of mid-lactation Holstein dairy cow was washed 2-3 times in 75% ethanol and 1% bromogeramine, then immediately brought to our laboratory in DMEM/F-12 medium plus 1% antibiotics on ice; Sample handing, mammary gland tissue was cut into fragments like chyle, then tissue fragments were washed with D-Hanks including antibiotic; Seeding, tissue fragments were seed on the flasks using pointed glass pipette; Culturing, tissue fragments were incubated in a humidified incubator at 37℃and 5% CO2; Purifying and passage, impure primary cells (mixed growth of mammary gland epithelial cells and fibroblasts) those migrated from tissue after several days were isolated and purified using trypsin and EDTA.Morphosis of BMGECs was observed under phase-contrast microscope. Pictures showed that:the purified BMGECs formed oval or polygon, and the fibroblasts formed threadline or multiangular. The growth pattern of BMGECs were plotted one-half logarithmically, and the population doubling time was estimated in the logphase of growth between day 2 and 4. Complete confluence is reached after 5 days, and contact inhibition was observed, indicating a typical growth pattern of normal mammalian cells. BMGECs were detected to be Immunohistochemical positive reaction using antibody-CK18. Secretory vesicles and lipid droplets were observed and aggregated in the apical membrane of the cells. The oil red O staining results demonstrated the existence of milk fat droplet in the cells.The mRNA fragment ofαSⅠcasein was amplified, and the derived PCR products were sequenced and 100% homology to the published sequences of bovine as-casein. Western blotting analysis showed that the synthesized protein had similar molecular weight to the predominant proteins αS1 (31kDa) in ruminant's milk protein, and immunoreactions of the proteins with bovine casein antibodies demonstrated that the protein wereαS-casein.Test 2. Study of different levels t10c12 CLA on BMGECs proliferation. Concentration levels of t10c12 CLA included OμM,37.5μM,75μM,112.5μM,150μM,187.5μM,225μM, 262.5μM,300μM. Survival percentage of BMGECs was assayed using MTT. Results showed that 150μM is the upper limit.Test 3. Effects of t10c12 CLA on milk fat synthesis. BMGECs were treated with 0μM, 37.5μM,75μM,112.5μM,150μM t10c12 CLA. TAG concentrate were significantly inhibited by t10c12 CLA (75-150μ.M). Lipogenesis was inhibited by about 80% when cells were incubated with 150μM t10c12 CLA, and this response is of a magnitude similar to the maximum inhibition seen in vivo with the administration of t10c12 CLA to dairy cows. For this reason,150μM does was used in subsequent experiments.MCFA (P<0.0001), UFA (P<0.0001), and MUFA (P<0.0001) were lessened obviously by exogenetic 150μM trans-10, cis-12, CLA with PUFA unchanged (P=0.3471). Adding 150μM t10c12 CLA made relative mRNA abundance of FAS (P=0.017), ACC (P=0.0461) and SCD (P=0.0021) descend. Exogenetic t10c12 CLA had diverse effects on relative mRNA abundance of genes involving in TAG synthesis in BMGECs. The relative expression levels of DGAT2 (P=0.0198) and ACSL (P=0.0319) went up after adding t10c12 CLA to BMECs culture medium for 48 h. However, AGPAT (P=0.1724) and DGAT1 (P=0.1449) had no obvious response to exogenetic t10c12 CLA.Test 4. Exploration about molecular mechanism of tha t110c12 CLA inhibited milk fat synthesis. SREBP-1 is the upstream component regulating milk fat synthesis. The addition of t10c12 CLA had no effect on the expression of SREBP-1 gene and other genes related to post-translation process of SREBP-1 and the protein synthesis of precursor SREBP-1 (pSREBP-1), maybe inhibited the synthesis or promoted the degradation of bio-active SREBP-1 (bSREBP-1).Conclusion:Functional BMGECs were cultured in vitro successfully. 150μM t10c12 CLA can drawn down the TAG content by inhibiting de novo fatty acid synthesis, and up-regulated the genes expression of TAG synthesis, but down-regulated the genes expression of fatty acid synthesis. The molecular mechanism was speculated tha t10c12 CLA maybe have some effects on activation of pSREB-1 and degradation of bSREBP-1.