| Objective:Observation was made on bone marrow precursor dendritic cells(pDCs) of mice infected with Ascaris lumbricoids to investigate the immune stimulating activity of the cells in the aspects of stimulating proliferation of lymphocytes and the toxicity of cytotoxic T cells.Methods:1.Establishment of animal models (ascaris-infected C57BL/6 mouse): Each mouse was administered 1000 eggs through lavage. At the 1d, 11d and 34d of post-infection, mice were desected for obtaining bone marrow cells.2. Experiment grouping and lymphocyte proliferation experiments:The experimental groups were divided into 6 groups, i.e., groups A,B, C, D, E and F. The group A stands for the co-culture of DCs with splenocytes both from infected mice; the group B stands for the co-culture of DCs from infected mice with splenocytes from uninfected mice; the group C stands for the co-culture of DCs from uninfected mice with splenocytes from infected mice; the group E stands for the co-culture of DCs from uninfected mice but pulsed with ABF, and splenocytes from the uninfected mice; the group F stands for the- co-culture of DCs from uninfected mice but challenged with B16 tumor antigen and splenocytes from uninfected mice; and control group D, which is the co-culture of DCs and splenocytes both from uninfected mice. The capacity of DCs to induce splenocytes proliferation was detected by MTT test after 1,11,34 d.3. Induction, culture and identification of DCs:Bone marrow cells were isolated from mouse femur and tibia under aseptic conditions .Induction cytokine was added to culture system.Bone cells were observed under inverted phase microscope. DCs were assayed CD80 by flow cytometry .Simultaneously specimens for transmission electron microscopy were prepared.4. Isolation and culture of splenocytes: Splenocytes were prepared under aseptic condition, with cell density regulated to 1×106/ml and harvested to be incubated in 5% CO2 at 37℃.5. Expression of CTL cytokine: Supernatant was obtained from the above mentioned CTL cultured after 72h. The expression of some cytokines like IL-2, TNF-α, IFN-γ,IL-4, IL-10 and IL-5 was measured by enzyme-linked immunosorbent assay kit.6. Making growth curve of B16: Cells in the logarithmic phase were digested, counted routinely and inoculated into 24-well cultivation plates. Cell counts in three wells were made everyday for 7 days to understand the growth tendence of the target cell.7. The influence of CTL to B16: Taking the CTL as responsive cell and B16 as target cell. The influence of CTL on B16 was detected by MTT method.Results:1. The degree of differentiation and morphology varied at various stages of DCs culture . After being cultured for 7 days, distinct dendritic processes could be seen on the cell surface. Under transmission electron microscope, typical DCs morphological characteristics can be seen. Flow cytometry of CD80 proved it to be highly expressed,CD80 result of mice infected was﹙74.53%±6.42%﹚, CD80 result of mice uninfected was(50.12%±3.25%)。2. Lymphocyte proliferation assays,on the 1d of post-infection,OD result of B group was higher than that of D group;on the 11d of post-infection, OD result of B group was slightly lower than that of D group; on the 34d of post-infection,OD result of B group was slightly higher than D group. OD showed no significant difference between B group and D group at the same time.Each group OD showed significant difference statistically at different intervals of time.3. Cytokine excretion assays on the 1d,11d of post-infection, IL-4 excerted by B group was higher than that by D group; on the 34d of post-infection,IL-4 excerted by B group was lower than that by D group. on the 1d of post-infection, IFN-γexcerted by B group was higher than that by D group; on the 11d,34d of post-infection IFN-γexcerted by D group was higher than that by B group, Each group IFN-γand IL-4 excreted by CTL had significant difference statistically at the same time and different times.But excertion of IL-2,IL-5,IL-10,TNF-αwas not assayed.4. B16 cells entered the logarithmic phase on the 2nd day and grew most vigorously on the 5th day, then went to plateau gradually.5. On the 34th day of worm infection, each group suggested no inhibition effects.Conclusions1. pDCs from the infected mice showed immunity stimulating functions.2. pDCs from the infected mice did stimulate the proliferation of lymphocytes in spleen, and the growth rate varied with infection condition.3. Levels of cytokines IFN-γand IL-4 secreted by CTL which was induced by pDC from infected mice varied with infection condition.4. CTL induced by pDC from mice infected for 34d had no inhibition effect found on target cell B16.
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