PAS-Na对锰致大鼠原代小胶质细胞炎症损伤干预作用的研究
发布时间：2018-08-24 11:59
【摘要】：【目的】研究锰对大鼠原代小胶质细胞氧化损伤和炎症因子白细胞介素(IL-1β、IL-6)、肿瘤坏死因子(TNF-α)、前列腺素2(PGE2)表达的影响,探讨对氨基水杨酸钠(PAS-Na)对锰导致的小胶质细胞损伤的干预作用。【方法】(1)原代培养的小胶质细胞经过分离提纯和鉴定后,(1)锰毒性试验:种于96孔板中的细胞分别给予0、200、300、400、500μmol/L浓度MnCl2处理24h。(2)PAS-Na无毒筛选试验:孔接种于96孔板中的细胞分别给予0、50、150、450μmol/L浓度PAS-Na处理24h。(3)PAS-Na干预试验:小胶质细胞被随机分为对照组、染锰组、PAS对照组、50、150、450-PAS干预组,其中对照组、染锰组、PAS对照组给予正常DMEM完全培养基,50、150、450-PAS干预组给予400μmol/L浓度MnCl2处理24h后,对照组给予正常DMEM完全培养基换液,染锰组给予400μmol/L浓度MnCl2处理24h,PAS对照组给予450μmol/L浓度PAS-Na处理24h,50、150、450-PAS干预组分别加入50、150、450μmol/L浓度PAS-Na处理24h。(2)用MTT试剂测定小胶质细胞存活率,DCFH-DA探针标记小胶质细胞氧化损伤情况,酶联免疫吸附法测定细胞上清液IL-1β、IL-6、TNF-α、PGE2炎症因子含量,荧光定量PCR检测小胶质细胞IL-1β、IL-6、TNF-αmRNA表达。【结果】细胞存活率检测:低浓度的MnCl2能刺激原代小胶质细胞的增殖存活率增加,高浓度MnCl2对小胶质细胞有一定的细胞毒性,MnCl2处理24h后小胶质细胞存活率在400μmol/L和500μmol/L组比对照组低(P0.05)。PAS-Na处理24h后,与对照组比较,各剂量处理组细胞形态和存活率没有明显变化(P0.05)。染Mn 24h,50、150、450μmol/L浓度PAS干预24h后,染Mn组细胞存活率比对照组低(P0.05)。与染Mn组比较,50、150、450-PAS干预组小胶质细胞存活率升高,差异有统计学差异(P0.05)。细胞内活性氧DCFH-DA标记检验:发现Mn处理可以显著增加细胞活性氧的生成,50、150、450-PAS干预组细胞活性氧生成比染Mn组少。细胞上清炎症因子:与对照组相比,MnCl2使小胶质细胞上清液中IL-1、TNFα分泌增加,差异有统计学意义(P0.05)。50、150、450-PAS干预组细胞TNFα表达低于染Mn组(P0.05)。小胶质细胞IL-1β、TNF-αmRNA表达:与对照组相比,染Mn组IL-1β、TNFαmRNA表达量增加,差异有统计学意义(P0.05)。50、150、450-PAS干预组小胶质细胞IL-1βmRNA表达低于染Mn组(P0.05)。150、450-PAS干预组小胶质细胞TNF-αmRNA表达低于染Mn组(P0.05)。【结论】(1)过量锰暴露会对原代小胶质细胞造成损伤,导致细胞内活性氧生成,IL-1β、TNF-αmRNA表达量升高,其对应的炎症因子IL-1β、TNF-α分泌增加。(2)PAS-Na对锰导致的小胶质细胞损伤具有一定的干预作用,这可能与PAS-Na的抗氧化和抗炎作用有关。
[Abstract]:[objective] to study the effects of manganese on oxidative injury of rat microglia and the expression of IL-1 尾 -IL-6, TNF- 伪 and PGE2. To investigate the effect of sodium p-aminosalicylate (PAS-Na) on the damage of microglia induced by manganese. [methods] (1) the primary cultured microglia were purified and identified. (1) Manganese toxicity test: cell division in 96-well plate. Don't be treated with 0 200300400500 渭 mol/L MnCl2 for 24 h. (2) PAS-Na nontoxic screening test: cells inoculated in 96-well plate were treated with 0 50150450 渭 mol/L PAS-Na for 24 h. (3) PAS-Na intervention test: microglial cells were randomly divided into control group. The rats in the control group were treated with normal DMEM complete medium 50150450-pas for 24 h, and the control group was treated with MnCl2 at the concentration of 400 渭 mol/L for 24 hours. The control group was treated with the normal DMEM complete medium for 24 hours, and the control group was treated with the normal DMEM complete medium for 24 hours, and the control group was treated with the normal DMEM complete medium for 24 hours. The manganese exposed group was treated with 400 渭 mol/L MnCl2 for 24 h, the control group was treated with 450 渭 mol/L PAS-Na for 24 h, the control group was treated with 50150450 渭 mol/L PAS-Na for 24 h. (2) the survival rate of microglia was measured by MTT reagent and the oxidative damage of microglia was labeled with DCFH-DA probe. Enzyme linked immunosorbent assay (Elisa) was used to detect the content of IL-1 尾 -TNF- 伪 PGE2 in supernatant, and fluorescence quantitative PCR was used to detect the expression of IL-1 尾 -IL-6TNF- 伪 mRNA in microglia. [results] Cell survival rate: low concentration of MnCl2 could stimulate the proliferation and survival rate of primary microglia. The survival rate of microglia in the 400 渭 mol/L and 500 渭 mol/L groups was lower than that in the control group (P0.05). PAS-Na had no significant changes in cell morphology and survival rate compared with the control group (P0.05). The cell survival rate of the Mn group was lower than that of the control group (P0.05) after 24 h PAS treatment with Mn 50150450 渭 mol/L for 24 h. Compared with the Mn group, the survival rate of microglia in the 50 150450-pas group was higher than that in the control group (P0.05). DCFH-DA labeling test of intracellular reactive oxygen species: it was found that Mn treatment could significantly increase the production of reactive oxygen species in cells treated with 50150450-pas, which was less than that in Mn infected group. The expression of IL-1,TNF 伪 in the supernatant of microglia was significantly higher than that in the control group (P0.05). The expression of TNF 伪 in the cells of the intervention group was lower than that in the Mn group (P0.05). The expression of IL-1 尾 -TNF- 伪 mRNA in microglial cells: compared with the control group, the expression of IL-1 尾 -TNF- 伪 mRNA in Mn group was higher than that in control group. The difference was statistically significant (P0.05) .50150450-pas intervention group was lower than Mn group (P0.05) .150450-pas intervention group microglial cell TNF- 伪 mRNA expression was lower than that of Mn group (P0.05). [conclusion] (1) excessive manganese exposure will cause damage to primary microglia cells. The expression of IL-1 尾 -TNF- 伪 mRNA increased, and the corresponding inflammatory factor IL-1 尾 -TNF- 伪 secretion increased. (2) PAS-Na may interfere with the damage of microglia induced by manganese, which may be related to the antioxidant and anti-inflammatory effects of PAS-Na.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2200760
[Abstract]:[objective] to study the effects of manganese on oxidative injury of rat microglia and the expression of IL-1 尾 -IL-6, TNF- 伪 and PGE2. To investigate the effect of sodium p-aminosalicylate (PAS-Na) on the damage of microglia induced by manganese. [methods] (1) the primary cultured microglia were purified and identified. (1) Manganese toxicity test: cell division in 96-well plate. Don't be treated with 0 200300400500 渭 mol/L MnCl2 for 24 h. (2) PAS-Na nontoxic screening test: cells inoculated in 96-well plate were treated with 0 50150450 渭 mol/L PAS-Na for 24 h. (3) PAS-Na intervention test: microglial cells were randomly divided into control group. The rats in the control group were treated with normal DMEM complete medium 50150450-pas for 24 h, and the control group was treated with MnCl2 at the concentration of 400 渭 mol/L for 24 hours. The control group was treated with the normal DMEM complete medium for 24 hours, and the control group was treated with the normal DMEM complete medium for 24 hours, and the control group was treated with the normal DMEM complete medium for 24 hours. The manganese exposed group was treated with 400 渭 mol/L MnCl2 for 24 h, the control group was treated with 450 渭 mol/L PAS-Na for 24 h, the control group was treated with 50150450 渭 mol/L PAS-Na for 24 h. (2) the survival rate of microglia was measured by MTT reagent and the oxidative damage of microglia was labeled with DCFH-DA probe. Enzyme linked immunosorbent assay (Elisa) was used to detect the content of IL-1 尾 -TNF- 伪 PGE2 in supernatant, and fluorescence quantitative PCR was used to detect the expression of IL-1 尾 -IL-6TNF- 伪 mRNA in microglia. [results] Cell survival rate: low concentration of MnCl2 could stimulate the proliferation and survival rate of primary microglia. The survival rate of microglia in the 400 渭 mol/L and 500 渭 mol/L groups was lower than that in the control group (P0.05). PAS-Na had no significant changes in cell morphology and survival rate compared with the control group (P0.05). The cell survival rate of the Mn group was lower than that of the control group (P0.05) after 24 h PAS treatment with Mn 50150450 渭 mol/L for 24 h. Compared with the Mn group, the survival rate of microglia in the 50 150450-pas group was higher than that in the control group (P0.05). DCFH-DA labeling test of intracellular reactive oxygen species: it was found that Mn treatment could significantly increase the production of reactive oxygen species in cells treated with 50150450-pas, which was less than that in Mn infected group. The expression of IL-1,TNF 伪 in the supernatant of microglia was significantly higher than that in the control group (P0.05). The expression of TNF 伪 in the cells of the intervention group was lower than that in the Mn group (P0.05). The expression of IL-1 尾 -TNF- 伪 mRNA in microglial cells: compared with the control group, the expression of IL-1 尾 -TNF- 伪 mRNA in Mn group was higher than that in control group. The difference was statistically significant (P0.05) .50150450-pas intervention group was lower than Mn group (P0.05) .150450-pas intervention group microglial cell TNF- 伪 mRNA expression was lower than that of Mn group (P0.05). [conclusion] (1) excessive manganese exposure will cause damage to primary microglia cells. The expression of IL-1 尾 -TNF- 伪 mRNA increased, and the corresponding inflammatory factor IL-1 尾 -TNF- 伪 secretion increased. (2) PAS-Na may interfere with the damage of microglia induced by manganese, which may be related to the antioxidant and anti-inflammatory effects of PAS-Na.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
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