Effects Of Glycine On Proliferation, Apoptosis And Gene Expression Of Immune Factors Of Mammary Epithelial Cells In Dairy Cow

The mammary epithelial cells of dairy cow (CCMEC) were used for test purpose to explore effects of glycine (Gly) on proliferation, apoptosis and immune response of CCMEC. Proliferative activity, cycle, apoptosis rate of cells and gene expression of four immune factors (IL-1β、IL-6, IL-8and TNF-a) were detected after cultured with Gly in order to illustrate Gly whether to participate in the process of cell growth and immune regulation, as well as the effects on immune response of CCMEC. The content and results of this paper showed as follows:1. CCMEC were isolated with collagenase digestion and cultured in vitro. After cultured with0,1,2,5,10and20mmol/L Gly for24,48and72h, MTT assay was used to detect cell proliferative activity. The results showed that:low concentration (1mmol/L) of Gly promoted cell proliferation, while high concentration (10-20mmol/L) of Gly inhibited cell proliferation significantly (P<0.05).2. Flow cytometry was used to detect cell cycle of CCMEC with Annexin V-FITC/PI double staining, after CCMEC were cultured with0,1,2,5,10and20mmol/L of Gly. It was found that Gly significantly promoted cell cycle of CCMEC converted to S and G2/M phase (P<0.05), as well as promoted cell division.3. Flow cytometry was used to detect apoptosis rate of CCMEC with Annexin V-FITC/PI double staining, after CCMEC were cultured with0,1,2,5,10and20mmol/L of Gly. The results showed that:Gly significantly promoted apoptosis of CCMEC(P<0.05). In this test, apoptosis rate of CCMEC supplemented with20mmol/L of Gly was the highest.4. Total RNA was extracted, after CCMEC were cultured with0,1,2,5,10and20mmol/L of Gly. Total RNA was transcribed into cDNA. The gene expression of IL-1β, IL-6, IL-8and TNF-α was detected with Real-Time PCR. PCR products were used for agarose gel electrophoresis. The results indicated:CCMEC cultured in vitro had the ability to secrete IL-1β, IL-6, IL-8and TNF-α. Gly could significantly improve gene expression levels of these four immune factors (P<0.05), and the gene expression levels of these four immune factors were the highest when cultured with5mmol/Lof Gly.