SIRT3通过调控CypD乙酰化介导脓毒症相关性脑病的研究
发布时间：2018-07-22 13:03
【摘要】：目的:探讨CypD及其乙酰化在脓毒症相关性脑病小鼠海马组织中的表达水平及其影响。方法3-4月龄雄性C57BL/6J小鼠随机分为假手术组(Sham组,n=21)和盲肠结扎术组(CLP组,n=21)。CLP组实施盲肠结扎术,建造脓毒症相关性脑病模型。造模后第4d至第6d每天行Morris水迷宫测试空间学习记忆能力,术后第7d行空间探索实验,记录小鼠的游泳速度、逃避潜伏期和探索时间。在术后第1d、4d和7d分别使用HE染色切片观察两组小鼠海马组织的损伤;ELISA法检测两组小鼠海马组织中炎症因子IL-6和TNF-α的表达水平,Western Blot法分别测定两组小鼠海马组织中CypD和CypD乙酰化的表达水平。结果:1.与Sham组比较,CLP组小鼠的逃避潜伏期延长(P0.05),探索时间缩短(P0.05),游泳速度比较差异无统计学意义(P0.05)。2.与Sham组比较,CLP组小鼠在术后第1d、4d和7d海马组织损伤明显加重,其中在术后第4d时损伤最显著。3.与Sham组比较,CLP组小鼠在术后第1d、4d和7d海马组织中的IL-6和TNF-α的表达水平均增加(P0.05),其中在术后第4d时炎症因子增加最显著。4.与Sham组比较,CLP组小鼠在术后第1d、4d和7d海马组织中CypD和CypD乙酰化的表水水平均增加(P0.05),其中术后第4d时两者增加最显著。结论:脓毒症相关性脑病小鼠的学习记忆能力下降,其海马组织的损伤明显加重,IL-6、TNF-α以及CypD和CypD乙酰化的表达水平增加,且其表达水平的增加均在术后第4d时最显著。目的:探讨SIRT3是否通过调控CypD乙酰化水平而介导小鼠脓毒症相关性脑病的发生发展。方法:雄性C57BL/6J小鼠,3-4月龄,随机分为六组,每组16只,分别为假手术组(Sham组)、盲肠结扎术组(CLP组)、CypDsiRNA转染组(CypD-si组)、CypD siRNA 对照组(CypD-c 组)、SIRT3 质粒转染组(SIRT3-p 组)和 SIRT3质粒空载组(SIRT3-v组)。除Sham组外,其余五组的小鼠均实施盲肠结扎穿孔术,建立脓毒症相关性脑病模型。CypD-si组和CypD-c组分别用体内转染的方法尾静脉注射siRNA和control siRNA。SIRT3-p组和SIRT3-v组分别尾静脉注射pcDNA3.1和vector pcDNA3.1。造模后第4d每天行Morris水迷宫测试空间学习记忆能力,术后第7d行空间探索实验,记录小鼠的游泳速度、逃避潜伏期和探索时间。在术后第4d用TUNEL法测定六组小鼠海马组织中细胞凋亡水平,测定六组小鼠海马线粒体膜电位的水平以及mPTP的开放程度。用Western Blot法分别测定两组小鼠海马组织中IL-6、TNF-α、caspase-3、CypD、CypD乙酰化和SIRT3的表达水平。结果:1.与Sham组比较,其余五组小鼠的逃避潜伏期明显延长(P0.05),探索时间缩短(P0.05)。与CLP组比较,CypD-si组、SIRT3-P组小鼠的潜伏期缩短(P0.05),探索时间延长(P0.05)。游泳速度的比较差异无统计学意义(P0.05)。2.与Sham组比较,其余五组小鼠海马组织中的细胞凋亡均增加(P0.05)。与CLP组比较,CypD-si组、SIRT3-p组小鼠的细胞凋亡水平减轻(P0.05)。3.与Sham组比较,其余五组小鼠海马组织中mPTP的开放程度增加(P0.05)。与CLP组比较,CypD-si组、SIRT3-p组小鼠海马组织中mPTP的开放程度减少(P0.05)。4.与Sham组比较,其余五组小鼠海马组织中IL-6、TNF-α、CypD和CypD乙酰化的表达水平增加,SIRT3的表达水平降低(P0.05)。与CLP组比较,CypD-si 组、SIRT3-P 组小鼠海马组织中的 IL-6、TNF-α、caspase-3、CypD和CypD乙酰化的表达水平降低,SIRT3的表达水平增加(P0.05)。结论:在脓毒症相关性脑病小鼠的海马组织中,神经细胞坏死和凋亡增加,mPTP的开放增加,IL-6、TNF-α、caspase-3、CypD和CypD乙酰化的表达水平增加,SIRT3的表达水平降低。
[Abstract]:Objective: To investigate the expression level and effect of CypD and its acetylation in the hippocampus of mice with sepsis related encephalopathy. Methods male C57BL/6J mice of 3-4 month old were randomly divided into sham operation group (group Sham, n=21) and cecum ligation group (group CLP, n=21).CLP group to carry out cecal ligation and construct sepsis related encephalopathy model. 6D was used to test space learning and memory ability by Morris water maze every day. After 7d, the swimming speed of mice was recorded and the latency and time of exploration were recorded. The damage of hippocampus tissue in two groups of mice was observed in 1D, 4D and 7d after operation, and ELISA method was used to detect the inflammatory factor IL-6 in the hippocampus of the two groups. The expression level of TNF- alpha and Western Blot method was used to determine the expression level of CypD and CypD acetylation in the hippocampus of two mice. Results: 1. compared with the Sham group, the escape latency of the CLP group was prolonged (P0.05), the exploration time was shortened (P0.05), and the difference of swimming speed was not statistically significant (P0.05).2. compared with the Sham group. After 1D, 4D and 7d, the damage of hippocampal tissue was obviously aggravated, and the most significant injury of.3. was compared with that of the Sham group at the post operation 4D. The expression level of IL-6 and TNF- alpha in the CLP group was increased in 1D, 4D and 7d hippocampus in the CLP group. The level of surface water of acetylation of CypD and CypD in 4D and 7d hippocampus increased (P0.05), and both of them increased most significantly at the time of postoperative 4D. Conclusion: the learning and memory ability of mice associated with sepsis related encephalopathy was decreased, the damage of hippocampus tissue was obviously aggravated, the expression level of IL-6, TNF- a, CypD and CypD increased, and the expression level of the hippocampus was increased. The increase was most significant after operation 4D. Objective: To investigate whether SIRT3 can mediate the development of sepsis related encephalopathy in mice by regulating the level of CypD acetylation. Methods: male C57BL/6J mice were divided into six groups at 3-4 months of age, 16 rats in each group, respectively, the sham operation group (group Sham), the cecum ligation group (group CLP), and the CypDsiRNA transfection group (CypD-si). Group (group CypD-c), CypD siRNA control group (group CypD-c), SIRT3 plasmid transfection group (SIRT3-p group) and SIRT3 plasmid unloaded group (group SIRT3-v). Except for Sham group, the other five groups of mice were performed cecum ligation and perforation, and a sepsis related encephalopathy model was set up in the.CypD-si group and CypD-c components in vivo. Group A.SIRT3-p and group SIRT3-v were injected with pcDNA3.1 and vector pcDNA3.1., respectively, to test space learning and memory ability by Morris water maze every day after 4D and Morris water maze was tested every day. The swimming speed of mice was recorded, the incubation period and the time of exploration were avoided. The cells in the hippocampus of six groups of mice were determined by TUNEL method at the end of the operation. The level of apoptosis, the level of mitochondrial membrane potential and the opening degree of mPTP in the hippocampus of six groups of mice were measured. The expression level of IL-6, TNF- alpha, Caspase-3, CypD, CypD acetylation and SIRT3 in the hippocampus of two mice was measured by Western Blot method. Results: 1. and Sham group compared with the Sham group, the escape latency of the remaining five groups of mice was obviously prolonged (P0.05). Time shortened (P0.05). Compared with group CLP, the latency of mice in group CypD-si and SIRT3-P was shortened (P0.05), and the time of exploration was prolonged (P0.05). The difference of swimming speed was not statistically significant (P0.05) compared with group Sham (P0.05), the apoptosis in the hippocampus of the other five groups increased (P0.05). The decrease of apoptosis level (P0.05).3. was compared with that in group Sham, and the degree of opening of mPTP in the hippocampus of the other five groups increased (P0.05). Compared with the CLP group, the opening degree of mPTP in the hippocampal tissues of the CypD-si group and the SIRT3-p group was reduced (P0.05).4. compared with the Sham group, and the other five groups of mice hippocampus The level of expression of SIRT3 decreased (P0.05). Compared with group CLP, IL-6, IL-6, TNF- a, Caspase-3, CypD and CypD acetylation in group CypD-si and SIRT3-P group decreased, and SIRT3 expression level increased (P0.05). Conclusion: in the hippocampus of mice with sepsis related encephalopathy, the necrosis and apoptosis of nerve cells in the hippocampus of mice with sepsis related encephalopathy. The expression of mPTP, IL-6, TNF-, Caspase-3, CypD and CypD increased and the expression level of SIRT3 decreased.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7;R747.9
本文编号：2137550
[Abstract]:Objective: To investigate the expression level and effect of CypD and its acetylation in the hippocampus of mice with sepsis related encephalopathy. Methods male C57BL/6J mice of 3-4 month old were randomly divided into sham operation group (group Sham, n=21) and cecum ligation group (group CLP, n=21).CLP group to carry out cecal ligation and construct sepsis related encephalopathy model. 6D was used to test space learning and memory ability by Morris water maze every day. After 7d, the swimming speed of mice was recorded and the latency and time of exploration were recorded. The damage of hippocampus tissue in two groups of mice was observed in 1D, 4D and 7d after operation, and ELISA method was used to detect the inflammatory factor IL-6 in the hippocampus of the two groups. The expression level of TNF- alpha and Western Blot method was used to determine the expression level of CypD and CypD acetylation in the hippocampus of two mice. Results: 1. compared with the Sham group, the escape latency of the CLP group was prolonged (P0.05), the exploration time was shortened (P0.05), and the difference of swimming speed was not statistically significant (P0.05).2. compared with the Sham group. After 1D, 4D and 7d, the damage of hippocampal tissue was obviously aggravated, and the most significant injury of.3. was compared with that of the Sham group at the post operation 4D. The expression level of IL-6 and TNF- alpha in the CLP group was increased in 1D, 4D and 7d hippocampus in the CLP group. The level of surface water of acetylation of CypD and CypD in 4D and 7d hippocampus increased (P0.05), and both of them increased most significantly at the time of postoperative 4D. Conclusion: the learning and memory ability of mice associated with sepsis related encephalopathy was decreased, the damage of hippocampus tissue was obviously aggravated, the expression level of IL-6, TNF- a, CypD and CypD increased, and the expression level of the hippocampus was increased. The increase was most significant after operation 4D. Objective: To investigate whether SIRT3 can mediate the development of sepsis related encephalopathy in mice by regulating the level of CypD acetylation. Methods: male C57BL/6J mice were divided into six groups at 3-4 months of age, 16 rats in each group, respectively, the sham operation group (group Sham), the cecum ligation group (group CLP), and the CypDsiRNA transfection group (CypD-si). Group (group CypD-c), CypD siRNA control group (group CypD-c), SIRT3 plasmid transfection group (SIRT3-p group) and SIRT3 plasmid unloaded group (group SIRT3-v). Except for Sham group, the other five groups of mice were performed cecum ligation and perforation, and a sepsis related encephalopathy model was set up in the.CypD-si group and CypD-c components in vivo. Group A.SIRT3-p and group SIRT3-v were injected with pcDNA3.1 and vector pcDNA3.1., respectively, to test space learning and memory ability by Morris water maze every day after 4D and Morris water maze was tested every day. The swimming speed of mice was recorded, the incubation period and the time of exploration were avoided. The cells in the hippocampus of six groups of mice were determined by TUNEL method at the end of the operation. The level of apoptosis, the level of mitochondrial membrane potential and the opening degree of mPTP in the hippocampus of six groups of mice were measured. The expression level of IL-6, TNF- alpha, Caspase-3, CypD, CypD acetylation and SIRT3 in the hippocampus of two mice was measured by Western Blot method. Results: 1. and Sham group compared with the Sham group, the escape latency of the remaining five groups of mice was obviously prolonged (P0.05). Time shortened (P0.05). Compared with group CLP, the latency of mice in group CypD-si and SIRT3-P was shortened (P0.05), and the time of exploration was prolonged (P0.05). The difference of swimming speed was not statistically significant (P0.05) compared with group Sham (P0.05), the apoptosis in the hippocampus of the other five groups increased (P0.05). The decrease of apoptosis level (P0.05).3. was compared with that in group Sham, and the degree of opening of mPTP in the hippocampus of the other five groups increased (P0.05). Compared with the CLP group, the opening degree of mPTP in the hippocampal tissues of the CypD-si group and the SIRT3-p group was reduced (P0.05).4. compared with the Sham group, and the other five groups of mice hippocampus The level of expression of SIRT3 decreased (P0.05). Compared with group CLP, IL-6, IL-6, TNF- a, Caspase-3, CypD and CypD acetylation in group CypD-si and SIRT3-P group decreased, and SIRT3 expression level increased (P0.05). Conclusion: in the hippocampus of mice with sepsis related encephalopathy, the necrosis and apoptosis of nerve cells in the hippocampus of mice with sepsis related encephalopathy. The expression of mPTP, IL-6, TNF-, Caspase-3, CypD and CypD increased and the expression level of SIRT3 decreased.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7;R747.9
【参考文献】
相关期刊论文 前1条
1 Qing-Hong Zhang;Zhi-Yong Sheng;Yong-Ming Yao;;Septic encephalopathy: When cytokines interact with acetylcholine in the brain[J];Military Medical Research;2014年02期
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