高压氧预处理对体外大鼠脑小胶质细胞产生细胞因子的影响

背景高压氧可以减轻脑组织的缺血再灌注损伤,这种作用与高压氧对小胶质细胞的调节作用有着密切的联系.目的探讨高压氧对体外大鼠脑小胶质细胞活性、分泌产生白细胞介素1、肿瘤坏死因子α和一氧化氮的影响.设计完全随机分组设计,对照实验.单位上海第二军医大学海医系潜水医学教研室、免疫教研室、病理教研室、实验动物中心.材料实验于1999-05/2000-01在解放军第二军医大学潜水医学实验室及免疫教研室完成.实验选用新生1日龄SD大鼠30只.方法1]用消化法培养新生SD大鼠的脑组织小胶质细胞.非特异性酯酶染色及细胞免疫化学染色鉴定小胶质细胞.2]原代小胶质细胞按2×105/孔浓度接种于48孔培养板,将小胶质细胞随机分以下5组对照组未经高压氧处理;高压氧(0.2 MPa 1 h)预适应3,7,10,14 d组.经各时程高压氧处理后,随机分成2大组,其中一组细胞培养液添加细菌脂多糖(激活小胶质细胞)1 mg/L,另一组则不添加.3]白细胞介素1活性测定采用胸腺细胞增殖法.肿瘤坏死因子α活性测定采用L929细胞的细胞毒性试验.以Griess法测定亚硝酸的含量代表一氧化氮含量.4]两样本比较采用未配对计量资料t检验.主要观察指标不同时程高压氧预处理后大鼠脑小胶质细胞在静息和激活不同状态下白细胞介素1、肿瘤坏死因子α活性和一氧化氮含量.结果SD大鼠30只均进入结果分析.1]静息小胶质细胞细胞外液白细胞介素1、肿瘤坏死因子α活性和一氧化氮含量各组间差异不明显.2]脂多糖激活的小胶质细胞细胞外液白细胞介素1活性和一氧化氮含量高压氧预处理10和14 d组明显低于对照组10 d组0.409±0.014,(5.21±0.77)μmol/L;14 d组0.381±0.004,(4.93±1.02)μmol/L,P＜0.05].3]脂多糖激活的小胶质细胞细胞外液肿瘤坏死因子活性高压氧预处理14 d组明显低于对照组(51.20±1.13)%,(70.10±2.26)%,P＜0.05].结论0.2 MPa的高压氧预处理可抑制活化小胶质细胞分泌产生白细胞介素1、肿瘤坏死因子α和一氧化氮,但对于静息小胶质细胞无明显作用.

Effect of hyperbaric oxygen pretreatment on cytokine production by in vitro cultured rat brain microglias

BACKGROUND: Hyperbaric oxygen can reduce brain ischemic-reperfusion injury, and this effect is closely related to the modulation of hyperbaric oxygen on microglias.OBJECTIVE: To explore the influence of hyperbaric oxygen on the activity of in vitro cultured brain microglias and secretion of interleukin-1, tumor necrosis factor α and nitric oxide (NO).DESIGN: Completely randomized grouping design, control experiment.SETTING: Teaching and Research Section of Diving Medicine, Teaching and Research Section of Immunity, Teaching and Research Section of Pathology, and the Experimental Animal Center, the Second Military Medical University of Chinese PLA.MATERIALS: This experiment was carried out at the laboratory of Diving Medicine as well as Teaching and Research Section of Immunity, the Second Military Medical University of Chinese PLA, between May 1999 and January 2000. Thirty neonatal SD rats of 1-day birth age were selected for the experiment.METHODS: 1] Brain microglias from newborn SD rats were cultured with digestion method, and microglias were identified with non-specific phosphodiesterase staining and cellular immunochemical staining. 2] Primary microglias were inoculated on 48-well culture board by 2×105/well and randomized into 5 groups: control group without hyperbaric oxygen pretreatment, and hyperbaric oxygen (0.2 MPa 1 hour) pretreatment 3, 7, 10,14 days groups. Cells in groups with hyperbaric oxygen pretreatment at the above various time points were then further divided into 2 subgroups, with one added with culture medium containing bacterium lipopolysaccharide of 1 mg/L (for microglia activation), but not in the other group. 3] Interleukin-1 activity was determined using thymocyte proliferation method. The activity of tumor necrosis factor-α was assessed with L929 cell toxicity test.Nitrous acid content detected by Griess method represented NO content.4] t-test was used to compare the differences in non-paired quantitative data between the two groups.MAIN OUTCOME MEASURES: The activity of interleukin-1 and tumor necrosis factor a and NO content in resting and evoked brain microglias in rats at various time points of hyperbaric oxygen pretreatment.RESULTS: Thirty SD rats entered the result analysis. 1] The activity of interleukin-1 and tumor necrosis factor α and NO content in resting brain microglias: The two groups did not differ obviously. 2] Interleukin-1activity and NO content in lipopolysaccharide-evoked brain microglias: They were significantly lower in 10-day and 14-day hyperbaric oxygen pretreatment groups than those in control group 10-day group: 0.409±0.014,(5.21±0.77) μnol/L; 14day group: 0.381±0.004, (4.93±1.02) μmol/L, P ＜ 0.05].3] The activity of tumor necrosis factor α in evoked brain microglias: It was obviously lower in 14day hyperbaric oxygen pretreatment group than in control group (51.20±1.13) %, (70.10±2.26) %, P ＜ 0.05].CONCLUSION: Pretreatment with 0.2 MPa hyperbaric oxygen can suppress the secretion of interleukin-1, tumor necrosis factor α and NO by evoked microglias, but has not obvious effects on resting microglias.