Date of Award
Master of Science
Vermont P. Dia
Doris H. D'Souza, Tao Wu
Exosomes are extracellular vesicles involved in intercellular communication. Bovine milk exosomes stability and their biological effects on macrophages have not been extensively investigated. Hence, the objectives of this study were to characterize bovine milk exosomes and investigate their effects on RAW 264.7 macrophages under normoxia and hypoxia. Bovine milk exosomes were isolated using differential centrifugation and identified by the presence of exosomal markers (ALIX, TSG101, and CD81) and particle size. The effect of in vitro digestion and pH on stability of milk exosomes was investigated. The biological activity of milk exosomes in RAW 264.7 under normoxia was determined by measuring proliferation, expression of proliferation markers p53, p21, cyclin D1, CDK2 and β-catenin, cell cycle, and the protective effect of exosomes on cisplatin-induced cytotoxicity. The effect of exosomes on RAW 264.7 under hypoxia was assessed by measuring proliferation, ROS production, cytokines, cell cycle, and the expression of hypoxia marker HIF-1α and proliferation markers p53 and p21. Exosomes were positive for exosomal markers and their size was 106.8 nm ± 3.4. Exosomal marker TSG101 was detected after digestion and exposure to different pH values. The treatment of exosomes significantly increased proliferation in cells under normoxia and had a protective effect against cisplatin-induced apoptosis. Proliferation markers were significantly affected by exosome treatment under normoxia. Cell cycle analysis in normoxia showed that exosome treatment reduced the percentage of pre-apoptotic cells while arresting the cells in G2/M phase. Hypoxic conditions reduced the viability of cells by 27% while exosome treatment significantly increased the cell viability. Generation of ROS under hypoxia was significantly reduced in the cells treated with exosomes. Hypoxia alone did not affect TNF-α, but exosome treatment significantly increased TNF-α in a dose-dependent manner under hypoxia. Cell cycle analysis showed that hypoxia alone arrested cells in the G0/G1 phase whereas exosome treatment reduced the percentage of cells in this phase. The results demonstrated that bovine milk exosomes isolated from commercially available milk are stable and affect macrophage proliferation under normoxia and hypoxia indicating the potential role of exosomes in the immune system. Further research is needed to understand milk exosomes’ role in human health.
Matic, Svjetlana, "STABILITY OF BOVINE MILK EXOSOMES AND THEIR EFFECT ON PROLIFERATION OF RAW 264.7 CELLS. " Master's Thesis, University of Tennessee, 2019.