Variations Of CD8~+ T Cell Activity And Its Related Molecular Mechanisms In Apolipoprotein C3 Transgenic Mice

Apolipoprotein C-?(Apo C3)is a water-soluble low molecular protein capable of carrying lipids and stabilizing lipoproteins.It mainly inhibits lipoprotein lipase(LPL)activity and inhibits liver uptake of triglyceride-rich lipoproteins(TRL s)and their residues,and plays an important role in plasma lipoprotein metabolism.The results of basic clinical research reveal that Apo C3 is not only a sufficient representative independent risk factor in the cardiovascular disease during clinical pathogenesis,but also closely linked to the risk of other diseases such as type 2 diabetes,hypertension and obesity.At present,it has been shown that Apo C3 acts as a key protein involved in lipid metabolism in vivo and regulates the functions of various immune cell differentiation and inflammation.Our group used Apo C3 transgenic mice and P-407 mice models.to determine the effection of the spleen immune cells frequency in Apo C3 mice and whether it directly regulates CD8 T cell function;then explore the characteristics of energy metabolism of CD8 T cell and its related molecular mechanisms in Apo C3 mice.Part ?.Frequency and activity of spleen immune cells in apolipoprotein C3 transgenic mice and P-407 miceObjective:To analyze the effect of Apo C3 overexpression on the biological activity of spleen immune cells,and to determine whether Apo C3 can directly regulate the phenotype and activity of immune cells.Methods:1.Healthy SPF male ICR mice weighing about 20?25g were divided into two groups:P-407 group and control group.The mice were intraperitoneally injected with P-407 at a dose of 0.5 g/kg body weight for 28 days,and a long-term HTG model was established.The control mice were intraperitoneally injected with the same amount of PBS every other day.2.Using ICR background Apo C3 transgenic mice and P-407 mice as research models,blood was collected from venous plexus in Apo C3 transgenic mice,P-407 mice,and wild-type mice,and the plasma triglyceride(TG)content was measured.Mice with plasma TG levels ? 1000 mg/dL(11.3 mmol/L)were selected for subsequent experiments.3.After prepared a single cell suspension of the spleen,the frequency of the immune cells in the spleen of the transgenic mice,P-407 mice and wild-type mice were respectively detected by flow cytometry.4.To detect the phenotype and activity of CD8 T cells in Apo C3 transgenic mice and P-407 mice.5.After spleen cells of Apo C3 transgenic mice and wild-type mice spleen cells were co-cultured with colon cancer cells MC38 at a concentration of 1:1,1:3,and 3:1,the expression of CD107a in CD8 T cells was examined.Results:1.The plasma triglyceride content of Apo C3 transgenic mice and P-407 mice was significantly higher than wild-type mice,which provided an ideal animal model for subsequent experiments.2.Compared with wild-type mice,the frequency of CD3 CD8+,CD3 CD8+NKG2D+cells in spleen of Apo C3 transgenic mice increased significantly,but the frequency of CD3 CD8+,CD3+CD8 NKG2D cells in spleen of P-407 mice reduced;the frequency of myeloid-derived suppressor cells(MDSC)increased in Apo C3 mice,while the frequency of MDSC decreased in P-407 mice;the frequencies of CD3-NK1.1 and CD3-NK1.1 NKG2D cells in both spleens were significantly reduced;the frequency of CD8 CD62L-CD44 and CD8+CD44+KLGR1 in Apo C3 transgenic mice increased,effector T cells were dominant,and CD8 T cells secreted IFN-y activity highly,while P-407 mice showed an opposite trend.4.The expression of CD 107a in CD8 T lymphocytes was up-regulated.Conclusions:1.The plasma triglyceride levels of Apo C3 transgenic mice and P-407 mice were significantly increased.2.The frequency and activity of CD8 T cells in Apo C3 transgenic mice were up-regulated,but the frequency of CD8 T cells in P-407 mice was down-regulated.3.The frequency of myeloid suppressor cells in Apo C3 transgenic mice increased,while the frequency of MDCS in P-407 mice decreased;both NK cells frequencies showed a downward trend.4.The killing ability of CD8 T lymphocytes to target cells was enhanced in Apo C3 transgenic mice.Part ?.Characteristics of energy metabolism of CD8 T cell and its related molecular mechanisms in Apo C3 transgenic miceObjective:To investigate the energy metabolism characteristics and its related molecules of CD8+ T cell in Apo C3 transgenic miceMethods:1.Using Apo C3 transgenic mice as a research model,blood was drawn from venous plexus of Apo C3 transgenic mice and wild-type mice,and the plasma triglyceride(TG)content was detected.2.After the mice were sacrificed,the spleen of Apo C3 transgenic mice was prepared into single cell suspension,and both in Apo C3 transgenic mice and wild-type mice,the CD8 T lymphocytes of spleen were selected by magnetic bead positive sorting.Detect the expression of lipoprotein lipase LPL was detected by western blot.3.The expression of fatty acid uptake receptor(CD36)on the CD8 T cell was detected by Flow cytometry in physiological state;CD8 T cells were stimulated with IL-2(10 ng/ml)in combination with CD3 antibody and CD28 antibody for 72 hours.under resting and activated state,the expression level of glucose transporter Glutl±hexose kinase HK?(glycolysis key enzyme),intracellular fatty acid oxidation rate-limiting enzyme-carnitine fatty acyltransferase(CPT)and fatty acid synthesis rate-limiting enzyme-acetyl-CoA carboxylase(ACC1)were detected.4.The expression changes of intracellular LKB1,AMPKa,AKT,mTOR and mTOR downstream target proteins HIF-1? and c-Myc were detected.Results:1.Compared with wild-type mice,the expression of Apo C3 target protein LPL was up-regulated in Apo C3 transgenic mice.2.In resting and activated state,the expression of glucose transporter Glutl,hexokinase HK ? and phosphorylated acetyl-CoA carboxylase 1 P-ACC1 were up-regulated in CD8 T cell;after activation,the expression of intracellular fatty acid oxidation rate-limiting enzyme-carnitine fatty acyltransferase CPT1 was decreased;the expression of CD36 on the CD8 T cells in Apo C3 mice was enhanced.3.The expression of P-mTOR was significantly increased in the CD8 positive T cells;there was no significant change in the expression of AMPKa,P-AMPKa,LKB1 and P-LKB1 in CD8 T cells;after activation,the expression of HIF-1? and c-Myc in the downstream target of mTOR was significantly increased.Conclusions:1.The effector CD8+T cells was increased in Apo C3 transgenic mice,and uptaking of glucose and free fatty acids increased;the level of anaerobic glycolysis,fatty acid synthesis were increased,the fatty acid oxidative phosphorylation was decreased.2.In Apo C3 transgenic mice,changes in energy metabolism and activity of CD8 T cells may be related to Akt-mTOR signaling.