焦亡在膽紅素誘導(dǎo)的大鼠皮層星型膠質(zhì)細(xì)胞神經(jīng)毒性中的作用研究
發(fā)布時(shí)間：2018-11-06 09:45
【摘要】：目的在原代培養(yǎng)大鼠皮層星型膠質(zhì)中明確焦亡是否參與膽紅素誘導(dǎo)的神經(jīng)細(xì)胞損傷及炎癥反應(yīng);調(diào)控焦亡是否能抑制膽紅素神經(jīng)毒性,發(fā)揮抗炎保護(hù)作用。在膽紅素腦病大鼠動物模型上初步探究抑制焦亡核心環(huán)節(jié)半胱氨酸天冬氨酸蛋白酶-1(caspase1)活化,是否能減輕膽紅素神經(jīng)毒性,改善膽紅素腦病模型鼠的生活能力。方法原代培養(yǎng)大鼠皮層星型膠質(zhì)細(xì)胞分為膽紅素組、VX-765組、對照組:Western blot檢測細(xì)胞caspase1蛋白和NLRP3蛋白的表達(dá),改良MTT法檢測細(xì)胞存活率,比色法檢測細(xì)胞清乳酸脫氫酶(LDH)釋放率,EtBr/EthD2染色法檢測細(xì)胞膜通透性改變,TUNEL法檢測染色質(zhì)DNA斷裂,ELISA法檢測培養(yǎng)液上清IL-1β和IL-18的水平。建立膽紅素腦病動物模型,Western blot檢測腦組織caspase1蛋白的表達(dá),ELISA法檢測炎癥因子IL-1β的水平,免疫熒光染色檢測腦組織GFAP蛋白的表達(dá);VX-765干預(yù)后動態(tài)觀察各組新生鼠的神經(jīng)系統(tǒng)臨床表現(xiàn),記錄新生鼠體重變化評估生活能力結(jié)果原代培養(yǎng)大鼠皮層星型膠質(zhì)細(xì)胞,膽紅素干預(yù)6h、12h后,活化型caspase1和NLRP3蛋白表達(dá)顯著高于對照組(P0.05);與膽紅素組相比,VX-765干預(yù)可抑制caspase1活化(P0.05),提高細(xì)胞存活率(P0.05),降低LDH釋放率(P0.05),減少ErBr攝取(P0.05),而不影響EthD2攝取(P0.05),降低細(xì)胞TUNEL染色陽性率(P0.01),減少培養(yǎng)液上清IL-1β和IL-18的釋放(P0.01)。膽紅素腦病動物模型,建模后12h,膽紅素組較對照組,腦組織中活化型caspase1表達(dá)增加(P0.05),IL-1β水平增高(P0.01),腦組織切片皮層區(qū)星型膠質(zhì)細(xì)胞活化(P0.05)。與膽紅素組相比,VX-765組新生鼠建模后異常神經(jīng)系統(tǒng)表現(xiàn)減少(P0.01),生活能力改善(P0.05)。結(jié)論在原代培養(yǎng)大鼠皮層星型膠質(zhì)細(xì)胞中證實(shí),焦亡參與了膽紅素神經(jīng)毒性的發(fā)生機(jī)制;抑制焦亡可減輕膽紅素神經(jīng)毒性,發(fā)揮抗炎保護(hù)作用。在膽紅素腦病動物模型上證實(shí),腹腔注射VX-765抑制caspase1活化,可改善膽紅素腦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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R722.1
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R722.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 陳艷霞;王家勤;許建文;;新生兒高膽紅素血癥行為神經(jīng)測定及嬰幼兒期智能發(fā)育隨訪[J];實(shí)用兒科臨床雜志;2007年14期
2 毛健;富建華;陳麗英;王曉明;薛辛東;;重度高膽紅素血癥新生兒蒼白球磁共振成像特征及其臨床意義[J];中華兒科雜志;2007年01期
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