白介素-37对四氯化碳诱导的小鼠肝纤维化的作用研究

发布时间：2018-08-07 16:13

【摘要】：目的:构建四氯化碳诱导的小鼠肝纤维化模型,探讨白介素-37对肝脏纤维化的作用。方法:60只雄性昆明小鼠随机均分为6组,即正常对照组(对照组),四氯化碳造模组(模型组),空质粒A组,空质粒B组,IL-37质粒干预组A组(实验组A组),IL-37质粒干预组B组(实验组B组),每组又按照处死时间点(8周,12周)随机分成2小组。对照组:腹腔注射生理盐水1ml/kg,2次/周,共8周;模型组:腹腔注射40%CCL4溶液1ml/kg,2次/周,共8周;空质粒组A组:按模型组造模8周,造模开始时即给予空质粒20μg尾静脉高压注射,2次/周,共8周;空质粒B组:按模型组造模8周后停止注射CCL4,然后每周2次给予空质粒20μg尾静脉高压注射,共4周;实验组A组:按模型组造模8周,造模开始时即给予IL-37重组质粒20μg尾静脉高压注射,2次/周,共8周;实验组B组:按模型组造模8周后停止注射CCL4,然后每周2次给予IL-37重组质粒20μg尾静脉高压注射,共4周。8周时从各组中随机选取一半小鼠于注射后禁食24h后处死,取血及肝脏。12周时同法处死剩余小鼠。行以下检测:(1)采用BCA法检测小鼠肝脏组织总蛋白浓度;(2)ELISA法检测小鼠肝组织中的IL-1,IL-6,TNF-α、TGF-β1、Col-Ⅳ、PCⅢ表达水平;(3)电子天平称量小鼠肝湿重及体重,计算肝指数;(4)通过HE染色观察小鼠肝脏病理改变。结果:(1)对肝组织总蛋白的影响:与对照组对比,8周及12周时模型组肝组织总蛋白水平均明显减低(P0.05);而实验组A组肝组织总蛋白水平较模型组高(P0.05)。12周时实验组B组肝组织总蛋白水平比模型组高(P0.05)。空质粒A组、空质粒B组无论8周还是12周肝组织蛋白浓度同模型组比较无显著差异(P0.05)。(2)对肝组织细胞因子il-1、il-6、tnf-α、tgf-β1的影响:与对照组相比,8周及12周时模型组的il-1、il-6、tnf-α、tgf-β1水平明显升高(p0.05)。实验组a组与对照组相比,il-1、il-6、tnf-α、tgf-β1水平明显升高(p0.05);与模型组相比il-1、il-6、tnf-α、tgf-β1水平明显减低(p0.05)。8周及12周时实验组b组与对照组比以上因子水平明显升高(p0.05),12周时实验组b组较模型组il-1、il-6、tnf-α、tgf-β1水平明显减低(p0.05)。空质粒a组、空质粒b组在8周及12周时上述因子水平同模型组无显著差异(p0.05)。(3)对肝纤指标col-Ⅳ、pcⅢ的影响:与对照组比较,8周及12周时模型组的col-Ⅳ、pcⅢ水平明显升高(p0.05)。实验组a组(8周及12周)与模型组相比col-Ⅳ、pcⅢ水平明显减低(p0.05)。8周及12周时实验组b组的col-Ⅳ、pcⅢ水平较对照组明显升高(p0.05),12周时实验组b组的col-Ⅳ、pcⅢ水平较模型组明显减低(p0.05)。8周及12周空质粒a组、空质粒b组同模型组结果无显著差异(p0.05)。(4)对肝指数的影响:与对照组相比,8周时及12周时模型组小鼠肝指数明显升高(p0.05);实验组a组肝指数较模型组明显减低(p0.05)。实验组b组8周时与对照组相比,肝指数升高明显(p0.05),而与模型组比较肝指数无明显差异(p0.05);12周时实验组b组与模型组相比肝指数明显减低(p0.05)。(5)肝组织he染色结果:ishaki肝脏坏死炎症活动评分示:模型组及空质粒组小鼠得分多在(10-14)分,实验组多为(7-9)分,对照组均为(0-3)分。模型组与实验组比较肝脏坏死炎症活动度得分差异具有统计学意义(p0.05)。ishaki肝纤维化评分示模型组得分多在4-5分,实验组多在2-3分,对照组均为0分。模型组与实验组肝纤维化评分比较有显著差异(p0.05)。结论:IL-37能够减轻CCL4诱导的小鼠肝纤维化,可以有效减轻肝组织炎症;其抗肝纤维化机制可能是通过下调炎症因子表达减轻肝脏炎症和减少细胞外基质的表达从而减轻肝纤维化。
[Abstract]:Objective: to construct a model of hepatic fibrosis induced by carbon tetrachloride in mice and explore the effect of interleukin -37 on liver fibrosis. Methods: 60 male Kunming mice were randomly divided into 6 groups, that is, normal control group (control group), carbon tetrachloride model group (model group), empty plasmid A group, empty plasmid B group, A group of IL-37 plasmid intervention group (group A), IL-37 plasmid. Group B of the intervention group (group B of the experimental group), each group was randomly divided into 2 groups according to the time point of death (8 weeks, 12 weeks). The control group was intraperitoneally injected with saline 1ml/kg, 2 times per week, for a total of 8 weeks; the model group was intraperitoneally injected with 40%CCL4 solution 1ml/kg, 2 times per week for 8 weeks; the empty plasmid group A group was given the model group for 8 weeks and the empty plasmid 20 u g tail vein was given at the beginning of the model. Pressure injection, 2 times per week, 8 weeks, empty plasmid B group: after modeling group for 8 weeks to stop injection of CCL4, and then 2 times a week to give empty plasmid 20 mu g tail vein high pressure injection, for a total of 4 weeks, group A: model group for 8 weeks, IL-37 recombinant plasmid 20 mu tail vein high pressure injection, 2 times per week, a total of 8 weeks; experimental group B group: group B group: model group: group B group: group group according to model group: group group: experimental group according to model group After 8 weeks, the injection of CCL4 was stopped, and then 2 times a week, the recombinant plasmid of IL-37 recombinant plasmid was injected with 20 mu g tail vein, and a total of half of the mice were randomly selected from each group for 4 weeks.8 weeks after 24h. The blood and liver.12 weeks were executed with the same method. (1) the total protein concentration of the liver tissue was detected by BCA method (1). 2) ELISA method was used to detect IL-1, IL-6, TNF- alpha, TGF- beta 1, Col- IV, PC III expression in mice liver tissue; (3) electronic balance weighed the wet weight and weight of liver in mice and calculated liver index. (4) the pathological changes of liver in mice were observed by HE staining. (1) the effect of (1) on the total protein of liver tissue: compared with the control group, the total egg of the model group was 8 and 12 weeks at the 8 and 12 weeks. The total protein level of the liver tissue in group A of the experimental group was higher than that in the model group (P0.05). The total protein level of liver tissue in the B group was higher than that in the model group (P0.05). The empty plasmid A group and the empty plasmid B group had no significant difference between the 8 weeks and the 12 weeks of the liver tissue protein concentration in the same model group (P0.05). (2) the cause of liver tissue cell causes was (2). The effects of sub IL-1, IL-6, tnf- a, tgf- beta 1: compared with the control group, the level of IL-1, IL-6, tnf- a, and tgf- beta 1 in the model group increased significantly at the 8 and 12 weeks (P0.05). The a group of the experimental group was significantly higher than the control group, IL-1, IL-6, alpha, and beta 1, compared with the model group. The level of B in the experimental group and the control group was significantly higher than that in the control group (P0.05). At 12 weeks, the level of IL-1, IL-6, tnf- a, tgf- beta 1 in the group B of the experimental group was significantly lower (P0.05). The level of the empty plasmid a group and the empty plasmid B group at 8 and 12 weeks were not significantly different from the model group (P0.05). (3) the effect of the liver fibrin index col- IV and the control group: and the control Group comparison, the level of col- IV and PC III in the model group increased significantly at 8 and 12 weeks (P0.05). The a group (8 and 12 weeks) in the experimental group compared with the model group, col- IV, PC III level decreased significantly (P0.05) in the B group of the experimental group at the.8 weeks and 12 weeks, and the PC III level was significantly higher than the control group (P0.05). At the 12 week, the level III level of the experimental group was compared with the model group. Obviously decreased (P0.05).8 week and 12 week empty plasmid a group, empty plasmid B group had no significant difference in the same model group (P0.05). (4) the liver index in the model group was significantly higher than the control group at 8 weeks and 12 weeks (P0.05); the liver index of the a group in the experimental group was significantly lower than that in the model group (P0.05). The B group in the experimental group was compared with the control group at 8 weeks, The liver index was significantly higher (P0.05), but there was no significant difference in liver index between the model group and the model group (P0.05). The liver index of the B group in the experimental group was significantly lower than that in the model group (P0.05) at 12 weeks. (5) the liver tissue he staining results showed that the scores of the ishaki liver necrosis and inflammation were (10-14) scores in the model group and the empty plasmid group, and the experimental group was (7-9), and the experimental group was more than that of the model group. Compared with the experimental group, the scores of liver necrosis and inflammation were statistically significant (P0.05) in the model group and the experimental group (P0.05) the score of the.Ishaki liver fibrosis score in the model group was more than 4-5 points, the experimental group was 2-3 and the control group was 0. The difference between the model group and the experimental group was significantly different (P0.05). Conclusion: IL-37 can be found. Alleviating liver fibrosis in mice induced by CCL4 can effectively reduce inflammation of liver tissue. The mechanism of anti hepatic fibrosis may be to reduce liver inflammation and reduce the expression of extracellular matrix by down-regulation of inflammatory factors and reduce liver fibrosis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.2

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