Effects Of Leptin On Milk Protein Syntheses In Bovine Mammary Epithelial Cells

In this experiment, epithelial cells culture techniques and cell biology and molecular biology techniques were used to study leptin on milk protein syntheses of bovine mammary epithelial cells. The findings include three parts as follOw:1. Mammary epithelial cells were obtained from the lactating Holstein cows by enzyme digestion and cultured successfully in vitro. The cells after purified by trypsin were stored in liquid nitrogen, and detected the survival rate of recovered cells. Immunofluorescence staining skeleton protein-keratin indicated that the cells we cultured really were bovine mammary epithelial cells. Observing from inverted microscope, cells were cobblestone appearance and adherence. The results showed that:the cells we harvested can be used to the following studies.2. On the basic of culture system of mammary epithelial cells in vitro, the treatments of cells included4does of leptin (0,10,100and1000ng/mL) with (1μg/mL) or without (0) prolactin (PRL), and0ng/mL leptin was the control group. The results of detecting cell proliferation by MTT were that leptin promoted cell proliferation as time dependence and at the same time,10and100ng/mL leptin uncommonly promoted cell proliferation, but1000ng/mL leptin significantly inhibited proliferation. The effect without PRL is not notable.3. The experiment design of this part was same to part2. The expression of genes was from rt-PCR experiments and protein secretion was analyzed by Elisa kit. The results showed that:(1) the expression of main milk protein genes was enhanced by leptin in the presence of PRL, and there were different effects with different leptin treatments.(2)1000ng/mL leptin inhibited PRL receptor (PRLR) expression significantly with PRL and PRL obviously induced PRLR expression.(3) the expression of JAK2was same to that of milk protein, but there was no effect of leptin on the expression of STAT5.(4) no effect of leptin on the secretion of α-casien protein,10ng/mL leptin hastened the secretion of β-casien protein and1000ng/mL leptin inhibited that significantly with PRLThe study is a useful exploration on molecule intercom between mammary fat pad and epithelial cells and in order to lay a theoretical foundation for future practical application of animal husbandry.