| Long-termed severe infection is a very common clinical disease. Systemic inflammatory response syndrome (SIRS), caused by trauma, bum or shock et al, is generally accompanied by explosive inflammatory responses and sustained serious septicemia. It will cause ischemia and even multiple organs failure (MOF). On conditions of no timely rescuing, it is lethal. Patients of sepsis or SIRS are characterized by sustained existence of endotoxemia with impaired adaptive immunity. However, the relationship between endotoxemia and unpaired adaptive immunity is still in the dark.Lipopolysaccharide (LPS) is a toxic part of endotoxin which is a component of Gram negative bacterial cell wall. LPS can stimulate antigen-presenting cells (APCs) such as dendritic cells (DC), monocytes and macrophages, inducing them to synthesize and release some chemokines and proinflammatory mediators which are harmful to the bodies or will even result in septic shock.DCs are currently known to be the most potent professional antigen-presenting cells, which have the unique function of stimulating naive T cells. According to their origins, DCs can be divided into myeloid DCs and lymphoid DCs which are differentiated from CD34+ progenitors or monocytes and lymphocytes respectively. Current study of DCs is mainly focused on myeloid DCs as they are easy to obtain and proliferate. Murine DCs coming from bone marrow progenitors were the subsets employed in our project. Mature DCs express high level of major histocompatibility complex class II (MHC-H), co-stimulatory molecules (CD80, CD86) and some of the adhesion molecules. They can deliver antigens to T cells and startup specific immune responses. Due to the important functions and pivotal roll in the adaptive immunity of DCs, we suppose that sustained LPS stimulation will impair the processes of differentiation and maturation of DCs, and will affect specific immune response.Conventional method was employed to culture murine bone marrow derived dendritic cells (BMDCs) induced by GM-CSF. In the process the group with sustained LPS stimulation, which mimic the sustained infection state in vivo, was aimed to study the effect of long-termed LPS existence on the maturation of DCs, Meanwhile, the group without LPS was used as negative control and the groupstimulated by LPS in the last two days was called 'single LPS stimulation control'. After 7 days of culturing, DCs were harvested to perform the experiments listed as follows: (1)comparing the morphological difference; (2)flow cytometry analysis to measure the phenotype and endocytosis ablity; (3)detecting cytokines secreted in the supernatant by ELISA; (4)examining the capability of antigen presentation by MLR; (5)detecting cell death using Hoechst33258 staining; (6)studying expression level of some signal transduction molecules by Western-blot. The results are showed as follows:Part I. Effect of sustained LPS on the phenotypes and functions of DCs.Researches have confirmed that primed by LPS, DCs undergo a process of maturation. Mature DCs are known as upregulated expression of MHC-II, CD86, CD80, CD11c etc., and increase in the capability of stimulating allogenic T cell proliferation. However, our work indicated that the maturation and function of BMDCs were remarkably slacked when co-culturing BMDCs with 1μg/mg LPS from the early stage of differentiation from bone marrow progenitors until day 7. Under optical microscope, the rumples and protuberances on the membrane were less and blunt. FACS analysis showed that the expression of MHC-II, CD86, CD80 and CD11c on sustained LPS stimulated BMDCs is notably downregulated but the uptake of antigen (FITC-OVA) was enhanced compared to those of single LPS stimulation. The ability of stimulating proliferation of allogenic and homogenic T cells was also impaired. Besides, cytokines released in the supernatant were examined and the results showed that mature BMDC secreting more TNF-α and IL-12 (p70) than that of BMDCs under sustained LPS stimulation. All these results made us believe that BMD... |
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