焦亡在胆红素诱导的大鼠皮层星型胶质细胞神经毒性中的作用研究
发布时间：2018-11-06 09:45
【摘要】：目的在原代培养大鼠皮层星型胶质中明确焦亡是否参与胆红素诱导的神经细胞损伤及炎症反应;调控焦亡是否能抑制胆红素神经毒性,发挥抗炎保护作用。在胆红素脑病大鼠动物模型上初步探究抑制焦亡核心环节半胱氨酸天冬氨酸蛋白酶-1(caspase1)活化,是否能减轻胆红素神经毒性,改善胆红素脑病模型鼠的生活能力。方法原代培养大鼠皮层星型胶质细胞分为胆红素组、VX-765组、对照组:Western blot检测细胞caspase1蛋白和NLRP3蛋白的表达,改良MTT法检测细胞存活率,比色法检测细胞清乳酸脱氢酶(LDH)释放率,EtBr/EthD2染色法检测细胞膜通透性改变,TUNEL法检测染色质DNA断裂,ELISA法检测培养液上清IL-1β和IL-18的水平。建立胆红素脑病动物模型,Western blot检测脑组织caspase1蛋白的表达,ELISA法检测炎症因子IL-1β的水平,免疫荧光染色检测脑组织GFAP蛋白的表达;VX-765干预后动态观察各组新生鼠的神经系统临床表现,记录新生鼠体重变化评估生活能力结果原代培养大鼠皮层星型胶质细胞,胆红素干预6h、12h后,活化型caspase1和NLRP3蛋白表达显著高于对照组(P0.05);与胆红素组相比,VX-765干预可抑制caspase1活化(P0.05),提高细胞存活率(P0.05),降低LDH释放率(P0.05),减少ErBr摄取(P0.05),而不影响EthD2摄取(P0.05),降低细胞TUNEL染色阳性率(P0.01),减少培养液上清IL-1β和IL-18的释放(P0.01)。胆红素脑病动物模型,建模后12h,胆红素组较对照组,脑组织中活化型caspase1表达增加(P0.05),IL-1β水平增高(P0.01),脑组织切片皮层区星型胶质细胞活化(P0.05)。与胆红素组相比,VX-765组新生鼠建模后异常神经系统表现减少(P0.01),生活能力改善(P0.05)。结论在原代培养大鼠皮层星型胶质细胞中证实,焦亡参与了胆红素神经毒性的发生机制;抑制焦亡可减轻胆红素神经毒性,发挥抗炎保护作用。在胆红素脑病动物模型上证实,腹腔注射VX-765抑制caspase1活化,可改善胆红素脑病模型鼠的生活能力。
[Abstract]:Objective to determine whether pyrolysis is involved in bilirubin induced neuronal injury and inflammation in primary cultured rat cortical astrocytes, and to regulate whether pyrolysis can inhibit bilirubin neurotoxicity and play an anti-inflammatory and protective role. To explore whether inhibiting the activation of cysteine aspartate protease 1 (caspase1) in the core link of pyrolysis in rats with bilirubin encephalopathy could reduce the neurotoxicity of bilirubin and improve the living ability of rats with bilirubin encephalopathy. Methods Primary cultured rat cortical astrocytes were divided into bilirubin group and VX-765 group. The expression of caspase1 protein and NLRP3 protein were detected by: Western blot in control group. The survival rate of cells was detected by modified MTT method. The release rate of lactate dehydrogenase (LDH) was detected by colorimetric method, the membrane permeability was detected by EtBr/EthD2 staining, the DNA breakage of chromatin was detected by TUNEL method, and the levels of IL-1 尾 and IL-18 in supernatant of culture medium were detected by ELISA method. The expression of caspase1 protein in brain tissue was detected by, Western blot, the level of inflammatory factor IL-1 尾 was detected by ELISA method, and the expression of GFAP protein in brain tissue was detected by immunofluorescence staining. After the intervention of VX-765, the clinical manifestations of nervous system of newborn rats were observed dynamically, and the changes of body weight of newborn rats were recorded. The results showed that primary cultured rat cortical astrocytes were cultured, and bilirubin was treated for 6 h or 12 h. The expression of activated caspase1 and NLRP3 protein was significantly higher than that of control group (P0.05). Compared with bilirubin group, VX-765 intervention inhibited caspase1 activation (P0.05), increased cell survival rate (P0.05), decreased LDH release rate (P0.05), decreased ErBr uptake (P0.05), but did not affect EthD2 uptake (P0.05). The positive rate of TUNEL staining was decreased (P0.01) and the release of IL-1 尾 and IL-18 in the supernatant was decreased (P0.01). Bilirubin encephalopathy animal model, 12 hours after modeling, bilirubin group than the control group, brain tissue activation caspase1 expression increased (P0.05), IL-1 尾 level increased (P0.01), cerebral slices of astrocytes activation (P0.05). Compared with the bilirubin group, the abnormal nervous system of VX-765 group decreased after modeling (P0.01), the ability of life improved (P0.05). Conclusion in primary cultured astrocytes of rat cortex, pyronacia is involved in the mechanism of bilirubin neurotoxicity, and the inhibition of pyracercosis can reduce the bilirubin neurotoxicity and play an anti-inflammatory and protective role. It was proved in the animal model of bilirubin encephalopathy that intraperitoneal injection of VX-765 inhibited the activation of caspase1 and improved the livability of mice with bilirubin encephalopathy.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R722.1
本文编号：2313927
[Abstract]:Objective to determine whether pyrolysis is involved in bilirubin induced neuronal injury and inflammation in primary cultured rat cortical astrocytes, and to regulate whether pyrolysis can inhibit bilirubin neurotoxicity and play an anti-inflammatory and protective role. To explore whether inhibiting the activation of cysteine aspartate protease 1 (caspase1) in the core link of pyrolysis in rats with bilirubin encephalopathy could reduce the neurotoxicity of bilirubin and improve the living ability of rats with bilirubin encephalopathy. Methods Primary cultured rat cortical astrocytes were divided into bilirubin group and VX-765 group. The expression of caspase1 protein and NLRP3 protein were detected by: Western blot in control group. The survival rate of cells was detected by modified MTT method. The release rate of lactate dehydrogenase (LDH) was detected by colorimetric method, the membrane permeability was detected by EtBr/EthD2 staining, the DNA breakage of chromatin was detected by TUNEL method, and the levels of IL-1 尾 and IL-18 in supernatant of culture medium were detected by ELISA method. The expression of caspase1 protein in brain tissue was detected by, Western blot, the level of inflammatory factor IL-1 尾 was detected by ELISA method, and the expression of GFAP protein in brain tissue was detected by immunofluorescence staining. After the intervention of VX-765, the clinical manifestations of nervous system of newborn rats were observed dynamically, and the changes of body weight of newborn rats were recorded. The results showed that primary cultured rat cortical astrocytes were cultured, and bilirubin was treated for 6 h or 12 h. The expression of activated caspase1 and NLRP3 protein was significantly higher than that of control group (P0.05). Compared with bilirubin group, VX-765 intervention inhibited caspase1 activation (P0.05), increased cell survival rate (P0.05), decreased LDH release rate (P0.05), decreased ErBr uptake (P0.05), but did not affect EthD2 uptake (P0.05). The positive rate of TUNEL staining was decreased (P0.01) and the release of IL-1 尾 and IL-18 in the supernatant was decreased (P0.01). Bilirubin encephalopathy animal model, 12 hours after modeling, bilirubin group than the control group, brain tissue activation caspase1 expression increased (P0.05), IL-1 尾 level increased (P0.01), cerebral slices of astrocytes activation (P0.05). Compared with the bilirubin group, the abnormal nervous system of VX-765 group decreased after modeling (P0.01), the ability of life improved (P0.05). Conclusion in primary cultured astrocytes of rat cortex, pyronacia is involved in the mechanism of bilirubin neurotoxicity, and the inhibition of pyracercosis can reduce the bilirubin neurotoxicity and play an anti-inflammatory and protective role. It was proved in the animal model of bilirubin encephalopathy that intraperitoneal injection of VX-765 inhibited the activation of caspase1 and improved the livability of mice with bilirubin encephalopathy.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R722.1
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相关期刊论文 前2条
1 陈艳霞;王家勤;许建文;;新生儿高胆红素血症行为神经测定及婴幼儿期智能发育随访[J];实用儿科临床杂志;2007年14期
2 毛健;富建华;陈丽英;王晓明;薛辛东;;重度高胆红素血症新生儿苍白球磁共振成像特征及其临床意义[J];中华儿科杂志;2007年01期
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