| Objective: To observe the vitro culture condition of IMDM medium , and establish the model of Blastocystis hominis infecting BABL/C mice;to discuss preliminarily about the characteristic of immune response of infected Blastocystis hominis by observation of the expression level of interleukin 4 ( IL-4 ), interleukin 10 (IL-10) and interferonγ(IFN-γ) in peripheral blood at different time intervals after the mouse were infected by B.h., as well as the Dynamic changing rule related to the number of CD4~+CD25~+Foxp3~+T Cell in BABL/C mice's spleen.Methods: The growth, reproduction and relevant factors of B.h under different culture conditions including PH value, concentration of serum and inoculation-dose were compared, by separation of Blastocystis hominis from the feces of infectors, which were continuously cultivated in vitro using the IMDM culture medium. B.h were cultivated in vitro by IMDM culture medium. For the testing group, the method of intragastric infusion was adopted to infect BABL/c Mice 4×107 per mouse; for the control group, BABL/c Mice were fed with equal culture solution and was fed routinely. Peripheral Blood and spleen were taken out in batches after being infected for 2d, 4d, 2w, 3w and 4w, the changes in number of interleukin-4 (IL-4), interleukin -10 (IL-10) and Interferon–γ(IFN-γ) in each group of Peripheral blood were detected by ELISA. Spleens were simultaneously taken out in preparation for single cell liquid. After coloration and Washing of the surface of the cell were conducted using the McAb of both FITC-CD4 and APC-25, with drilling liquid and stationary liquid used to treat it, then, intra-nuclear staining needed to be executed using PE-FJK-Foxp3 antibody. Changes in the number of CD4~+CD25~+Foxp3~+T Cell Cell were detected through the adoption of FCM.Results: The optimum culture condition for B.h in IMDM medium were as follows: PH value ranged 7.0～8.0, concentrations of new bron bovine serum were more than 10%, the number of inoculation were no less than 1x105 cells per tube, penicillin and streptomycin (104 units/mL) should also be added and anaerobically cultured at 37℃. Subculturing could be conducted for one time in the interval of 3-6 days, the long- term cultivation could then be realized.There was a slightly increase in the number of CD4~+T Cell in the Spleen of BABL/c mice infected with B.h. However, no statistic meaning needed to be taken into account.The number of CD4~+CD25~+Foxp3~+T Cell in the spleen of BABL/c mice infected with Blastocystis homini would obviously rise after declining. It would drop sharply in the fourth week. In comparison with the CD4+CD25+Foxp3+T Cell of BABL/c mice's spleen in Blank group, there should be a statistical significance in terms of the discrepancies among 2d, 4d, 2w, 3w and 4w.The concentration of IL-4 and IL-10 would first decline slowly within one week after being infected, then rising again. Later on, dropped slowly after reaching the peak value in the second week. There should be a statistical significance when it comes to the comparison between testing group and Blank group in the second week. There was a strong downward trend in the concentration of IFN-y in the second week after it went up rapidly within a week, after that, it was up slowly. There was a statistical significance while comparing the difference between testing group and control group after being infected for 6 days and 2 weeks.Conclusion:IMDM culture medium is sutiable for the growth and reproduction of B.h. IMDM can be used in diagnosis and long-term Vitro culture. Immune response should be very complicated after being infected with B.h. While, the CD4+CD25+ Foxp3+T regulatory cell was one of the key components of Immune response, which might have taken part in the early immunosuppression. Th1 immune response will take place 1 week after BABL/c mice are infected with B.h. Immune response will later be shifted to Th2.
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