Study On The Lipid Metabolism Of Jurkat Cells And HepG2 Cells Infeted By HTLV-1 Virus

BackgroundIntracellular lipid droplets serve as a form of fat storage, and also as a multi-functional complicated organelle involved in many biological processes. A variety of complicated metabolic diseases, such as obesity and high cholesterol, have been reported to be associated with the abnormalities of intracellular lipid droplets in number and function, indicating that lipid droplets are directly or indirectly involved in the related diseases. It has been demonstrated that lipid droplets participate in regulation of immune response in tissues and cells, particularly in regulating lymphocyte function. The occurrence of related diseases may be resulted from immunoregulatory dysfunction of lymphocytes induced by abnormal lipid droplets, and the mechanism remains unclear.Adult T-cell leukemia (ATL) is a disease directly related to a human T-cell leukemia virus type-Ⅰ (HTLV-1) infection which could last for a decade before the onset of ATL. It still remains elusive that whether the intracellular lipid metabolism altered or not in such a long-term latent period, particularly what is the influence of HTLV-1 infection on lipid metabolism in T cells, and whether these changes will cause a cellular immune response disorders that induce ATL. Therefore, further study of the impact of the HTLV-1 on intracellular lipid metabolism has been shown of great significance.Objectives1. Observe the changes of lipid droplets after co-culture with fat cells, and explore the lipid metabolism in Jurkat cells and HepG2 cells after HTLV-1 virus infection.2. Analyze the influence of HTLV-1 infection on the apoptosis and survival of Jurkat cells and HepG2 cells.3. Detect the expressions of lipid-metabolism-related genes after HTLV-1 infection. Explore mechanisms of viral infection affect on cellular lipid metabolism.Method1. Cell culture:(1) MT2 cells (treated with colchicine and carrying HTLV-1 virus) were co-cultureed with human peripheral blood lymphocyte leukemia line Jurkat T cells, 24 h later, these cells were stained with oil red O and the changes of lipid droplets compared with the untreated control Jurkat cells were observed.(2) The same method was used to observe changes of lipid droplets in co-cultured human hepatoma cell line HepG2 cells compared to untreated HepG2 cells.2. Collect cells of control groups and co-cultured groups were collected and. the RNA and protein was extracted respectivily. Successful infection of the two types of cells was proved by detection of characteristic expression of HTLV-1 Tax protein using PCR, Real-timePCR and Western Blot.3.Cells of control groups and co-cultured groups were extracted. After Nile Red (Nile red) staining, analysis cell lipid droplets changes and apoptosis by using flow cytometric.4. Pcmv-Tax and PcDNA6-HIF1a recombinant plasmids were constructed and into Jurkat cells by electroporation. Gene expression was detected by Western Blot.5. Statistical analysis:the experimental datas were analyzed using SPSS 19.0 software, and the results were represented as means±standard deviation (x±s).P<0.05 means statistically significant, and P<0.01 was considered significant.Result1. microscopic observation of cell morphology Jurkat cells were co-cultured with the MT2 cells.24h later, observed under an inverted microscope, MT2 cells were tightly wrapped in Jurkat cells, similar to the "Flower Mission" shape. MT2 cells and HepG2 cells were co-cultured for 24h.Observed under an inverted microscope,the two type of cells just scattered.2. Microscopic observation of lipid droplets after Oil Red O staining The lipid droplets in HTLV-1 infected Jurkat cells, were significantly increased compared with the control group, while the lipid droplets in HTLV-1 infected HepG2 cells, had no significant difference compared to the control group. Semi-quantitative determination indicated that the number of extracted lipid droplets, in HTLV-1 infected Jurkat cells were higher than that in the control group, while that in HepG2 cells just a little more than the control group.3. Flow cytometry results of Nile red staining Lipid droplets of virus-infected Jurkat cells werer more than the control group. There wese no significant changes of lipid droplets between virus-infected HepG2 cells and control group. Compared with the control group, the number of apoptotic cells were increased in HTLV-1-infected Jurkart cells.4. PCR and Western Bloting results In HTLV-1-infected Jurkart cells, the expression of Tax, HIF1a, ADRP, CAV1, BCL-2 and P65 were up-regulated.5. The expression of recombinant plasmids Pcmv-Tax, PcDNA6-HIFla transfected Western Blot results showed that Tax and HIF1a were successfully expressed in the Pcmv-Tax and PcDNA6-HIF1a transfected Jurkart cells.Conclusion1.HTLV-1 can increase the accumulation of lipid droplets in Jurkat cells, accompanied by lipid metabolism-related gene expression changes, especially the dramatically higher expression level of HIF1a, HIF1a may be an important factor in HTLV-1 induced accumulation of fat in Jurkat cells.2. The accumulation of lipid droplets did not change significantly in HTLV-1 infected non-lymphoid HepG2 cells.