| ObjectiveCD38has various roles in the regulation of cardiovascular disease, cancer andmetabolic diseases, but its role in immune system is rarely reported. Our previousstudy show that B cell development of TLR4mutmice are obstacled, and theexpression of TLR4gene in CD38-/-mice were significantly decreased in the spleen.Our study aims at illustrate the effects of CD38deficiency on the distribution andexpression of TLR4gene, reveal the influences of CD38deficiency to B celldevelopment and its key points, explore the potential role of CD38inanti-inflammatory response and its mechanism. The results of our study will make upthe defection of our knowledge to the roles of CD38in B cell development, and willprovide new targets for medical intervention.Methods1. Identify whether CD38gene of mice were successfully knockout by extractingDNA from tail of mice, and then identifing the expression of CD38and Neogene by PCR;2. The mRNA expression levels of TLR4gene in organs (heart, liver, spleen, lung,kidney, brain, thymus and lymph nodes) of WT and CD38-/-mice were evaluatedby RT-PCR;3. The mRNA and protein expression levels of TLR4gene in spleen of WT andCD38-/-mice were detected by Real-time PCR and Western-blot individually,technology, and the protein expression levels of NF-κB and Sirt1genes weredetect by Western-blot;4. Detect the expression levels of inflammatory factors (including TNF-α, MCP-1,IL-1β and IL-6) in the spleen of WT and CD38-/-mice by Real-time PCR;5. Compare the histological differences between the spleen of WT and CD38-/-miceby both HE staining and immunohistochemistry staining; 6. Sorting and Purifing B cells from spleens of WT and CD38-/-mice with magneticbeads sorting method, and then detect the mRNA and protein expression levelsof TLR4gene in B cells by Real-time PCR and Western-blot, Protein expressionlevels of NF-κB and Sirt1gene were detected by Western-blot;7. Detect the expression level of inflammatory factors (including TNF-α, MCP-1,IL-1β and IL-6) in B cells of WT and CD38-/-mice by Real-time PCR;8. Compare the total number of spleen cells and B cells between WT and CD38-/-mice by cell counting;9. Analyze B cell development, cell cycle and apoptosis by staining B cells withcorresponding surface antibodies and analyze with flow cytometry technology.Results1. All CD38knockout mice we used for experiments are homozygous, whichensured the reliability of the experimental results;2. The expression level of TLR4gene in spleen is the specifically decreased amongmajor organs, and expression of TLR4gene was found nearly2fold decreasingin CD38-/-mice;3. Development of spleen in CD38-/-mice were obstacled, the expression ofinflammatory cytokines such as TNF-α, MCP-1and IL-6are significantlydecreased, however expression of IL-1β is significantly increased;4. Protein expression levels of NF-κB and Sirt1gene in spleen are significantlyincreased in CD38-/-mice;5. The cell number of macrophages in spleen are reduced in CD38knockout mice;6. The number of B cells in CD38-/-mice show a two fold decreasing, andconsistent with the trends in the spleen, expression of MCP-1is significantlydecreased, however expression of IL-1βis significantly increased, but there is noobvious difference in the expression of TNF-α and IL-6;7. The expressing of TLR4gene was decreased in B cells of CD38-/-mice, while theexpression of NF-κB and the Sirt1gene were increased significantly;8. The B cell development in CD38knockout mice were obstacled, which showsthe number of mature B cells were decreased and the transition of T1to T2was inhibited, but the cell cycle and apoptosis of B cell are not affected.ConclusionsOn one hand, CD38gene knockout can cause the decreasing expression ofMCP-1by reducing the expression of TLR4gene in B cells; on the other hand, CD38gene knockout can enhancing expression of Sirt1gene and deacetylate NF-κB,which can inhibit the expression of MCP-1; both of those pathways contribute tosuppressing transition of T1to T2, and inhibiting maturation of B cells. |
PDF Full Text Request