Mechanism And Regulation Of GH And IGF-I On Casein Synthesis In Bovine Mammary Epithelial Cells

This experiment was conducted to study the effects of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on milk casein synthesis, gene expression of key kinase and regulatory factors regulating milk protein synthesis, proliferation and apoptosis of bovine mammary epithelial cells using the methods of isolation and culture of epithelial cells of mammary gland and the related techniques of cellular and molecular biology.The objective of experiment1was to isolate and culture bovine mammary epithelial cells in vitro and identified gene expression of receptors of GH and IGF-I. The bovine mammary tissue obtained from a live lactating cow by biopsy method and the epithelial cells were isolated and cultured by using collagenase, and then they were distinguished through morphology, expressions of specific proteins (cytokeratin-18) and genes (CSN1S1, CSN1S2, CSN2and CSN3) using inverted microscope, immunofluorescence and RT-PCR method, respectively. The expressions of GHR and IGF-IR was also determined by RT-PCR. It was found that most of the cells after isolated and purified with high viability after frozen and thawed presented characteristic cobblestone-like morphology. Cytokeratin-18, four major casein genes, receptor genes (GHR and IGF-IR) were all detected. These findings showed that bovine mammary epithelial cells with high purity were obtained and GH exerts its roles may through acting on its receptor on bovine mammary epithelial cells directly or by IGF-I indirectly.The aim of experiment2was to determine effects of GH and IGF-I on gene and protein expression of κ-casein in bovine mammary epithelial cells. Growth medium without serum was took as the control group, the media of other groups were supplemented with GH (100ng/mL), IGF-I (100ng/mL) or GH (100ng/mL)+IGF-I (100ng/mL). After cultured for24h, the relative expressions of mRNA of κ-casein gene (CSN3) were determined by real-time quantitative PCR (RT-qPCR) and the κ-casein synthesis was measured by enzyme-linked immunosorbent assay (ELISA). The results show that CSN3gene mRNA expression were enhanced significantly by each treatment (P<0.05), the results of protein level had a parallel trend with mRNA level but the difference was not significant (P>0.05), moreover, there was no cumulative effects of GH and IGF-I were observed both at mRNA and protein level. The results indicated that the synthesis of κ-casein may be promoted by addition of100ng/mL GH or IGF-I within24hours.The experiment3was conducted to test the effects of GH and IGF-I on gene mRNA expressions of key kinases and regulatory factors regulating milk protein synthesis. Treatment was the same to that of the experiment2and the gene relative expressions of Janus kinase2(JAK2), signal transducer and activator of transcription5(STAT5), E74-like factor5(ELF5), mammalian target of rapamycin (mTOR), ribosomal protein S6kinase1(rpS6Kl), eukaryotic initiation factor4E binding protein1(4EBP1), eukaryotic initiation factor4E (eIF4E) and eukaryotic elongation factor2(eEF2) were detected by RT-qPCR and mTOR expression level at protein level were determined by the method of Western blot. The results found that adding GH alone had tendency to increase the mRNA expression of ELF5(P<0.1) compared with control group, adding IGF-I alone promoted the expression of rpS6K1mRNA and mTOR significantly (P<0.05) but these effects were not augmented by GH further. The results indicate that GH and IGF-I may modulate κ-casein synthesis independently through affecting gene expressions of key kinases and regulatory factors that regulating κ-casein synthesis.The purpose of experiment4was to determine the effects of GH and IGF-I on proliferation and apoptosis of bovine mammary epithelial cells. Treatment of cells was the same to that of the experiment2. Cell proliferation was evaluated by CCK-8method within4to96hours and cell apoptosis rate was tested by a FITC-Annexin V/PI double staining assay within24hours. The expression of IGFBP-5gene was also determined using RT-qPCR. The results showed that the cell proliferation was not be facilitated adding GH alone (P>0.05), however, it was promoted by IGF-I from4to6h notably (P<0.05) and was boosted by GH+IGF-I significantly within4to96hours (P<0.05) which was not significant compared to supply IGF-I alone (P>0.05). Both GH and IGF-I had inhibitory effects on cell apoptosis (P<0.05) and this effect was further enhanced when GH cooperated with IGF-I. The roles might be concerned with the reduced expression of IGFBP-5mRNA. This study demonstrated that GH and IGF-I can regulate the number and activity of bovine mammary epithelial cell through promoting cell proliferation and inhibiting cell apoptosis to stimulate lactation.