The Research On The Changes Of Cytokine Levels In The Culture Of Immature And Mature Dendritic Cells Derived From Mouse Bone Marrow

Objective: Dendritic cells(DCs) are not only key controllers of innate and adaptive immunity, but also play a vital role in the immune tolerance. They have long been known to be the most efficient antigen-presenting cells whose functions are different according to their location and phenotype. Multiple sclerosis is a chronic demyelinating and an autoimmune disease of the central nervous system. It is well known that abnormal T lymphocyte activation is closely linked to incidence of MS. DCs are biological monitor between Th1 and Th2 cells, as same as Th17 and Treg. The scientists focus on the study related to the mechanism of immune tolerance of MS recent years. The experiment aims at making a deep discussion on immune response and immune tolerance through detecting the content of IL-23 and IL-35, which are extracted from supernatant of imDCs and DCs that are cultured by improved methods of Son, and seeking a new direction for the treatment of MS.Methods: 1 Separate and cultivate imDCs and mDC from mice bone marrow in vitroFirst, we executed the C57BL/6 mice by pull the neck, soaked them into iodophors and 75% ethanol for 5minutes and 2 minutes, removed muscles from legs with sterile forceps and scissors which were soaked in sterile phosphate buffered saline for a while.Then, the experimenter wiped the legs with sterile gauzes and immersed them into sterile RPMI-1640 for 2minutes. Next, we punched the femur, rushed the bone marrow out, filtered impurities with cell strainer, splitted red cells using lysis buffer and washed cells twice. Finally, cells were resuspended with cell culture media(contain 10% fetal bovine serum, rmGM-CSF(10 ng/ml), rmIL-4(10ng/ml)) and suspended into six pore plates. Add all or half of the total volume of fresh media supplemented enough cytokines(rmGM-CSF(10ng/ml), rmIL-4(10ng/ml)) to refresh the media in 48 hours later and 96 hours later. We could harvest of immature dendritic cells on the fifth day. On the sixth day, we should supplement a litter fresh media(rmTNF alpha(15ng/ml)). Collect mature dendritic cells on the seventh day. 2 Sort imDCs and DCs by flow cytometry: Mark DCs which were harvested on the fifth and seventh day with CD11 c antibody. The CD11c+DCs were DCs what we needed.3 Identificate the imDCs and mDCs 3.1 Observe the morphology of imDCs and mDCs 3.1.1 Observe the morphology of imDCs and mDCs by converted fluorescence microscopy 3.1.2 Observe the morphology of imDCs and mDCs by scanning electron microscopy 3.1.3 Observe the morphology of imDCs and mDCs by transmission electron microscopy3.2 Detect the expression of cell surface molecules by flow cytometric: Test the expression of CD11c?MHCII?CD80?CD86 on the surface of imDCs and DCs.4 Test the content of IL-23 and IL-35 by enzyme linked immunosorbent assay 5 Statistic analysis: The quantitative data was expressed in mean±standard deviation. We could utilize independent sample t test and One-way ANOVA test to compare two or several groups, and all the significant level was set at P<0.05.Results: 1 The morphology of imDCs and mDC 1.1 Observe the morphology of imDCs and mDCs by converted fluorescence microscopy: The number appeared more and the volume became lager of cells with time flying. It was observed that most cells which were cultured for 48 hours were adherent, while more cells became suspended after 96 hours. On the fifth day, some cells gathered into a mass. We could see typical morphology of DCs, whose enation was increasing in parallel with mass. 1.2 Observe the morphology of imDCs and mDCs by scanning electron microscopy: The enation was lager, longer and thicker on the surface of mDCs in contrast with imDCs. Many rugae formed and m DCs shaped like branches. 1.3 Observe the ultrastructure of imDCs and mDCs by transmission electron microscope: It was obviously that the nucleuses were irregular and hemilateral, and there were less organelles and more lysosomes for imDCs. Although the nucleuses of mDCs were alike to imDCs, there were more organelles such as mitochondria, golgi complex, endoplasmic reticulum and less lysosomes. 2 Detect the expression of cell surface molecules by flow cytometric: Flow cytometry was employed to sort suspended cells and trace the expression of costimulatory molecules of im DCs and DCs. A mean of 82.922%(SEM±0.637) and 91.143%(SEM±0.751) of cultured imDCs and DCs stained positively for CD11 c. An average of 46.075%(SEM±5.742) and 68.040%(SEM±2.527) of cultured imDCs and DCs stained positively for both CD11 c and MHCII, respectively. The percentage of both CD11 c and CD80 staining positively in imDCs and DCs are 8.057%(SEM±2.307) and 46.397%(SEM±2.982).On the average,5.943%(SEM±0.308) and 47.330%(SEM±0.430) of the cultured imDCs and DCs stained positively for both CD11 c and CD86. In addition, the average number of imDCs and DCs expressing CD11 c, MHCII and CD80 were 8.810%(SEM±0.642) and 15.810%(SEM±1.773), moreover the percentage of the simultaneously positive cells of CD11 c, MHCII and CD80 in imDCs and DCs reveals to be 5.747%(SEM±0.255) and 10.290%(SEM±0.393). In a word, all the datum were considered significant statistically between imDCs and DCs(P<0.05). 3 Test the content of IL-23 and IL-35 by enzyme linked immunosorbent assay: There existed obvious significance in the expression of IL-23 and IL-35 between imDCs and mDC respectively. As a whole, the level of IL-23 was higher, but the content of IL-35 was lower in DCs than imDCs.Conclusions:1 The experiment not only succeeds in culturing imDCs and DCs in vitro with IL-4?GM-CSF and TNF-?,but also identifys their morphology by various microscopes and the expression of costimulatory molecules on their surface by flow cytometry.2 There is a markedly difference in the expression of IL-23 from imDCs and mDCs, as same as IL-35. The paper not only makes a deep discussion on their significance respectively, what's more, it lays the foundation for later gene transfection.