| Objective To observe and analyze the therapeutic effect and significance of 1, 25-dihydroxyvitamin D3 on the body weight, platelet account and other aspects in the successful established immune thrombocytopenia(ITP) model in mice. And to measure the expression level of CD80, CD86 and MHC II that were presented on the surface of the mouse bone marrow-derived DC cell, then, to investigate the mechanism of action of 1,25-dihydroxyvitamin D3 treatment. For the clinical application of 1,25-dihydroxyvitamin D3 treatment of ITP and other autoimmune diseases, it may provides relevant theoretical basis. Methods 1. Anti-mouse platelet serum prepared in guinea pigs: Took the anti-coagulated whole blood of BALB/c mice, isolated the platelet and suspension were made and thoroughly mixed with the equal volume of complete or incomplete Freund’s adjuvant. The mixture were injected subcutaneously in guinea pigs’ back, groin and back paws, at the 0, 1,2,4-week. In the fifth week, the guinea pigs’ non anti-coagulated whole blood was taken. After centrifugation, the supernatant obtained is the GP-APS. Intraperitoneally administered different proportions of GP-APS to BALB/c mice, observed the amplitude and duration of thrombocytopenia, and selected the appropriate dilution. 2. Grouping and establishment of the chronic model: The mice were divided into three groups: saline control group, ITP model group and 1, 25-dihydroxyvitamin D3 treatment group. The model group was given the GP-APS intraperitoneally, while the treatment group, on the basis of the model group, was administered 100 ng / d of 1, 25-dihydroxyvitamin D3 by intravenous injection, and the saline control group was given the same volume of saline through both intraperitoneal and intravenous injection. We have taken the blood sample after 24 hours of each administration, measured platelet account and recorded the weight, bleeding tendencies. After two weeks of the injection, the mice were sacrificed and dissected; smeared with the bone marrow that has taken out; counted the number of megakaryocyte under the microscope. 3. Cell culture: randomly choose each two BALB/c mouse from treatment group and the model group. While in a sterile environment, washed out the bone marrow from the cavity, filtered after collecting, and washed twice to remove red blood cells. Adding sterile complete medium(which contains GM-CSF 20 ng / ml, IL-4 10 ng / ml, fetal calf serum and anti-double), resuspended the cells, and marked and moved, in accordance with the previous groups, to the 6-well plates. 4. The morphological changes: placed the six-well plates under an inverted microscope, and observed such as changes in cell morphology every day. 5.Flow cytometry measure the expression of CD86, CD80 and MHC II molecule: the cells were collected on the seventh day, adding the PE-cy5-CD86, FITC-CD80 and PE-MHCⅡto the tube, incubated at room temperature and dark conditions for 30 minutes. After washing twice, resuspended the cells then analyzed on board. Results After the treatment of 1, 25-dihydroxyvitamin D3, from the 10 th day of the injection, the body weight of treatment group, compared with the model group, was significantly increased(p <0.05); and its platelet account, compared with the model group, was also increased significantly(p<0.01), bleeding tendency evident was lower(x <0.05), the difference was statistically significant. Conclusion 1. 1,25-(OH)2D3 showed a therapeutic effect on the general status(such as the diet and body weight), bleeding tendency, platelet account and production of the ITP mice model. 2. 1,25-(OH)2D3 played a reduced role in expression levels of mice DC cell costimulatory molecules and MHC II, and may inhibit DC maturation, and thereby induced the immune tolerance type of DC cells. 3. 1,25-(OH)2D3 may have achieved the therapeutic effect on ITP mice model, by inducing the immune tolerance type of DC cells. |
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