| Objective:To observe the establishment of immune tolerance induced by injecting the specific antigen through mesenteric vein after surgical brain injury and to explore the mechanism of immune tolerance with non-parenchymal cells in the liver.Methods:1.32New Zealand rabbits were divided randomly into four groups called A, B, C, D. Group A was the control group, group B injecting1ml normal saline group, group C injecting lml MBP(2mg/ml), group D injecting lml single cell suspension group(106/ml). The first day brain injury model of rabbits was made.0.8*0.4*0.5cm brain tissue were prepared for single cell suspension; Within30mins, group B, C, D was received laparotomy looking for mesenteric vein to inject antigens. Seven days later all were killed for Fas/FasL Real-time PCR.2.20SD rats were randomly divided into three groups. Five for control group, five for sham group and10for trauma group. According to different time points, trauma group was divided into2,4,8,16h and24h. Primary cultured rat microglia was cultured with serum centrifugated from venous blood for stimulating24h. MHC-II expression is measured by flow cytometry.3. Another24New Zealand rabbits were divided randomly into three groups named group E, F, G. Modeling as the first part, all rabbits were drained blood on1stday,3rdday,7thday,14thday and21stday to measure concentration of IL-2, IL-10and TGF-β by ELISA method.21days later all were killed, and obtained liver tissues to observe the expression of Foxp+3by immunohistochemical method.Results:1.Real-time PCR demonstrated:after seven days, Fas are highly expressed in all groups, with no significant difference between the groups (p＞0.05); group A and group B hardly expressed FasL, group C FasL expression was significantly increased, higher than group A and group B (p<0.05), group D FasL expression was slightly increased, with no significant difference contrasting group A and group B(p＞0.05).2. Flow cytometry results show:MHC-Ⅱ in the the control group is hardly expressed; in sham it is lower expressed; in the trauma group it was expressed higher with time passing. Comparing with control group and sham group, the difference was significant (P＜0.05). 3. The ELISA demonstrated:Compared with the control group, TGF-beta, IL-10in group F were significantly increased and reached a peak around14days (p＜0.05); while IL-2correspondingly reduced, and the difference between group F and other group was statistically significant (p＜0.05); TGF-beta, IL-10in group G were slightly higher than control group in the14th days, and the difference among them was not statistically significant (p＞0.05). The difference between group G and control group was not statistically significant (p＞0.05).4. Immunofluorescence:in the group E and G, we can hardly see T lymphocytes with orange-red excitation light in the liver paraffin sections, while it can be found in the sinus and perivascular T lymphocytes in group F(p＜0.05).Conclusion:1. Mesenteric vein injection of the specific antigen did not reduce the expression of Fas. The mesenteric vein injection of MBP can induce immune tolerance; single cell suspension can not fully induce immune tolerance.2. Expression of MHC-Ⅱ in microglial cells can increase after brain injury in rats.3. Injection of MBP can induce immune tolerance, while the single cell suspension antigen-specific poor.4. Injection of MBP can increase the postoperative expression level of IL-10and TGF-beta.5. Injection of MBP can induce the expression of Foxp+3. Brain cells were still not able to induce the expression of Foxp+3. |
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