A Culture System For Dendritic Cells Induced From Murine Bone Marrow In Vitro

The current study suggests that the occurrence,development and outcome of atherosclerosis is associated with a chronic inflammation reaction, it often related to a large number of immune cells and inflammatory mediators.Dendritic cells were found to be the most powerful professional antigen presenting cell,which can directly activate the naive T cells in vivo,and they may play an important role in the activate of lymphocytes,the regulation of the naive T lymphocytes proliferation and the mediate of immune tolerance. However,the mostly problem is that they were present in smaller quantities,only about 1% of peripheral mononuclear,which brought great difficulty to research work in immunological function.So we establish a highly efficient culture system for dendritic cells induced from murine bone marrow in vitro, for the following research in AS mouse model provide experimental basis.Objective:1.We have designed this experiment focus on using the method of inducing cells from murine bone marrow in vitro by rm GM-CSF and rm IL-4 to obtain DCs for the following research in AS mouse model provide experimental basis.2.DCs were confirmed by morphological observation,the cell surface molecular detection and stimulate the T cells appreciation ability.Methods:1.The induction and cultivation of DCs from murine bone marrowExecuted C57BL/6 mice by pulling the neck,removed the femur and the tibia in sterile conditions.pricked both ends of the femoral medullary cavity with 1ml syringe needles,extracted RPMI-1640 cultures to rush out the bone marrow,filtered by strainerand added the red blood cells cracking liquid to dissolve red blood cells.Adjusted cells concentration to 5x105/ml by RPMI-1640 completely cultures.vaccinated into culture flasks,and put them into the constant temperature incubator(condition:37 ℃ 、5%CO2).The suspended cells were collected and centrifuged after 48 and 96 hours of culture.The cells were split into two groups in the fifth day.One group added LPS(1μg/ml) to stimulate immature DCs,the other did not add anything.Two suspended cell groups were continue to cultivate to the seventh day.2. Identification of the DCs from murine bone marrow2.1 Cell morphology observationDCs growth and adhesion were observed under inverted microscopy2.2 The detection of DCs surface molecules by flow cytometricCollected suspended cells in culture flasks,after centrifugal and counting,added the antibody and the isotype control antibody to the cells according to the specification.In this condition,the time of incubated avoid light in 4℃was 30 minutes.After centrifugal and washed,we detected the molecular expression of CD11 c,CD80,CD86,MHC-Ⅱ in DCs surface by flow cytometry.2.3 Detected the ability of proliferation that DCs stimulated the allograft T cells in one-way mixed lymphocyte responses with the method of MTTExecuted BALB/c mice by pulling the neck,removed the spleen in sterile conditions.After treated by the lymphocyte separation medium,the monocytes were separated successfully in the spleen cells grinding fluid of mice. T cells were harvested by using nylon column,adjusted cells concentration to 5x105/ml by RPMI-1640 completely cultures as the reaction cells.Took immature DCs and mature DCs from C57BL/6 mice bone marrow as the stimulation cells.According to 1:5,1:10,1:20,1:40 mixed DCs and T cells and vaccinated into 96-well plates,and put them into the constant temperature incubator(condition:37℃、5%CO2) 72 hours. Added 200μl MTT to each hole before the end of the culture. 4 hours later,added 150μl DMSO to dissolve T cellsunder the conditions of normal temperature and vibration.Inspected and recorded the optical density in each hole by enzyme league detector(wave length 570nm).Results:1. The morphology of DCs observationObserved the morphology of DCs with inverted microscopy.After 48 hours induced by rm IL-4 and rm GM-CSF,the morphology of mice bone marrow cells showed different size cell colony.The semi-suspension cells were increased significantly and the surface of cells emergence of several protuberances. In the 5th day, the colony phenomenon of suspension cells were more obvious than before,and the size of cell increased.DCs were collected and divided into two groups,one group added LPS(1μg/ml) to stimulate cells,the other did not add anything.48 hours later,a large number of cells released from the cell colony in LPS group,and the other group was still a colony growth.2.The detection of DCs surface molecules by flow cytometricThe culture supernatant and cells were collected in the 7th day,and detected of DCs surface molecules by flow cytometric.CD11 c was highly expressed in these cell groups,purity was more than 90%.Without LPS stimulation group cells showed low expression of CD80,CD86,MHC- Ⅱ molecules,consistent with immature state.LPS stimulation group cells showed high expression of CD80,CD86,MHC- Ⅱ molecules,consistent with mature state.3.Detected the ability of proliferation that DCs stimulated the allograft T cells in one-way mixed lymphocyte responses with the method of MTTSingle factorial analysis of variance results showed that the ability of proliferation has increase tendency along with the increment of the proportion of DCs.The result in the contrast of two group cells under the same conditions, immature DCs has lower the ability of proliferation than mature DCs,and the difference was statistically significant(p<0.05).Conclusion:This experiment focus on using the method of inducing cells from mice bone marrow invitro,and these cells were confirmed by morphological observation,the cell surface molecular detection and stimulate the T cells appreciation ability.The experiment proves the feasibility of inducing cells from mice bone marrow in vitro by rm GM-CSF and rm IL-4 to obtain DCs,which establish the foundation for the following research in AS mouse model.