水通道蛋白8對膿毒癥大鼠肝細胞線粒體形態(tài)的影響
發(fā)布時間：2018-09-12 08:59
【摘要】：膿毒癥(sepsis)是指感染引起的全身性炎癥反應綜合征(systemic infla-mmatory response syndrome, SIRS),是危重癥患者病死的主要原因之一,進一步發(fā)展可引起重癥膿毒癥(severe sepsis)膿毒癥休克(septic shock)和多器官功能障礙綜合征(multiple organ dysfunc-tion syndrome, MODS)肝臟是膿毒癥狀態(tài)下最易受累的器官之一,肝功能不全是膿毒癥發(fā)展為多器官功能衰竭(multiple organ failure,MOF)的早期表現(xiàn)之一膿毒癥的發(fā)病機制尚不十分明確,可能的發(fā)病機制包括:炎癥因子風暴凝血系統(tǒng)紊亂和微循環(huán)障礙免疫抑制、線粒體功能障礙和機體氧供與氧耗失衡等肝細胞線粒體是機體營養(yǎng)物質(zhì)和能量代謝中心,線粒體功能障礙在膿毒癥狀態(tài)下,尤其是膿毒癥休克患者的病情發(fā)展中起著很重要的作用水通道蛋白8(aquaporin-8,AQP8)是在肝細胞線粒體內(nèi)膜(inner mitochondria membrane,IMM)上表達的一種水分子通道,是具有高度選擇性和高效轉(zhuǎn)運水分子的特異孔道,對維持肝細胞線粒體的結(jié)構(gòu)和功能具有較大的影響本研究使用盲腸結(jié)扎穿孔法制備膿毒癥大鼠模型,旨在探討膿毒癥造成肝細胞線粒體形態(tài)的變化,以及線粒體內(nèi)膜AQP8在膿毒癥介導的肝損傷中的所發(fā)揮的作用 目的 研究膿毒癥狀態(tài)下水通道蛋白8(AQP8)在肝細胞線粒體膜上表達程度對線粒體形態(tài)變化的影響,為進一步探尋改善線粒體膜AQP8蛋白表達的方法,以期能起到對膿毒癥介導肝損傷的保護作用 方法 24只SD大鼠隨機分為2組:對照組和膿毒癥組,每組12只采用盲腸結(jié)扎穿孔術(shù)制備膿毒癥實驗動物模型,對照組進行假手術(shù),完成除盲腸結(jié)扎穿孔外的實驗過程觀察兩組動物的體溫呼吸頻率心率變化和對外界刺激的反應,達到膿毒癥診斷標準后繼續(xù)觀察1h,然后在麻醉后用斷頭法處死實驗大鼠;取出大鼠肝臟,一部分新鮮肝臟用于制作電鏡標本,進行超微結(jié)構(gòu)觀察,另一部分肝臟用于提取線粒體,后采用Western-blotting法測定線粒體AQP8蛋白表達水平,并用RT-PCR法檢測肝細胞線粒體AQP8mRNA水平采用SPSS13.0統(tǒng)計軟件進行分析計量資料采用單因素方差分析(one-wayANOVA) 結(jié)果 1.線粒體超微結(jié)構(gòu)變化 對照組肝細胞膜邊界清楚完整,內(nèi)質(zhì)網(wǎng)及線粒體數(shù)目較多且形態(tài)正常而膿毒癥組肝細胞線粒體顯著腫脹嵴變寬短,并出現(xiàn)線粒體膜破裂,內(nèi)質(zhì)網(wǎng)也出現(xiàn)擴張斷裂及空泡化 2.肝細胞線粒體AQP8蛋白表達 以prohibitin作為內(nèi)參,用AQP8/prohibitin表示AQP8蛋白相對表達水平,將對照組的AQP8蛋白相對表達水平設為1,計算出膿毒癥組(n=12)AQP8蛋白相對表達水平為0.61±0.08,較對照組減少平均約39%,差異有顯著統(tǒng)計學意義(F=62.71,P0.01) 3肝細胞線粒體AQP8mRNA的表達 以β-actin作為內(nèi)參,AQP8/β-actin表示AQP8mRNA相對表達量,將對照組的AQP8mRNA相對表達水平設為1,膿毒癥組AQP8mRNA相對表達量為1.59±0.24;AQP8mRNA表達較對照組增加平均約59%,差異有顯著統(tǒng)計學意義(F=67.85, P0.01) 結(jié)論 1.采用盲腸結(jié)扎穿孔術(shù)可以成功制備大鼠膿毒癥模型; 2.膿毒癥可以介導急性肝損傷,肝細胞線粒體超微結(jié)構(gòu)發(fā)生明顯的變化; 3.膿毒癥狀態(tài)下肝細胞線粒體AQP8蛋白表達下調(diào),可能是導致線粒體腫脹變性的重要因素之一; 4.膿毒癥狀態(tài)下肝細胞線粒體AQP8蛋白表達下調(diào),而AQP8mRNA表達增加,提示可能存在代償機制,但由于存在轉(zhuǎn)錄后抑制AQP8蛋白并未能回升
[Abstract]:Sepsis is a systemic inflammation-mmatory response syndrome (SIRS) caused by infection, which is one of the main causes of death in critically ill patients. Further development of sepsis shock and multiple organ dysfunction syndrome (MODS) can cause severe sepsis. Unc-tion syndrome (MODS) liver is one of the most susceptible organs in sepsis. Liver dysfunction is one of the early manifestations of sepsis developing into multiple organ failure (MOF). The pathogenesis of sepsis is still unclear. Possible pathogenesis includes: disturbance of the inflammatory factor storm coagulation system and microorganisms. Hepatocyte mitochondria, such as circulatory dysfunction, immune suppression, mitochondrial dysfunction, and imbalance of oxygen supply and consumption, are the center of nutrients and energy metabolism. Mitochondrial dysfunction plays an important role in the development of septic symptoms, especially in patients with septic shock. A water molecule channel expressed on the inner mitochondria membrane (IMM) is a highly selective and efficient channel for transporting water molecules. It has a great influence on the maintenance of the structure and function of hepatocyte mitochondria. In this study, we used cecum ligation and perforation method to prepare sepsis rat model. Morphological changes of hepatocyte mitochondria and the role of mitochondrial endometrial AQP8 in sepsis-mediated liver injury
objective
To study the effect of aquaporin 8 (AQP8) expression on mitochondrial morphology of hepatocytes in septic symptoms, and to explore a method to improve the expression of AQP8 protein on mitochondrial membrane, so as to protect liver from sepsis-induced liver injury.
Method
Twenty-four SD rats were randomly divided into two groups: control group and sepsis group. Twelve rats in each group were subjected to cecum ligation and perforation to establish sepsis experimental animal model. The control group was subjected to sham operation. The experimental process except cecum ligation and perforation was completed. The changes of body temperature, respiratory rate, heart rate and response to external stimuli were observed in the two groups to achieve sepsis diagnosis. After 1 hour, the rats were killed by decapitation after anesthesia. The liver of the rats was taken out, some of the fresh liver was used for making electron microscopic specimens, and the other part of the liver was used for extracting mitochondria. The expression of AQP8 protein in mitochondria was determined by Western blotting, and the hepatocytes were detected by RT-PCR. Mitochondrial AQP8 mRNA levels were analyzed by SPSS13.0 statistical software and measured by one-way ANOVA.
Result
1. mitochondrial ultrastructural changes
In the control group, the boundary of hepatocyte membrane was clear and intact, the number of endoplasmic reticulum and mitochondria was large and the morphology was normal, but in the sepsis group, the mitochondrial swelling cristae became wider and shorter, and the mitochondrial membrane ruptured, the endoplasmic reticulum distended to rupt and vacuole.
2. expression of mitochondrial AQP8 protein in hepatocytes
Using Prohibitin as internal reference, AQP8/prohibitin was used to express the relative expression level of AQP8 protein. The relative expression level of AQP8 protein in control group was set to 1. The relative expression level of AQP8 protein in sepsis group (n=12) was calculated to be 0.61 (+ 0.08), which was about 39% lower than that in control group (F=62.71, P 0.01).
3 expression of mitochondrial AQP8mRNA in hepatocytes
Using beta-actin as internal reference, AQP8/beta-actin expressed the relative expression level of AQP8 mRNA. The relative expression level of AQP8 mRNA in control group was set to 1, AQP8 mRNA in sepsis group was 1.59 [0.24] and AQP8 mRNA expression was increased by 59% on average compared with control group (F = 67.85, P 0.01).
conclusion
1. the sepsis model can be successfully prepared by cecal ligation and puncture.
2. sepsis can induce acute liver injury, and the ultrastructure of mitochondria of liver cells is obviously changed.
3. The down-regulation of AQP8 protein expression in hepatocyte mitochondria under septic symptoms may be one of the important factors leading to mitochondrial swelling and degeneration.
4. The expression of AQP8 protein in hepatocyte mitochondria was down-regulated and the expression of AQP8 mRNA was up-regulated in septic symptoms, suggesting that there may be compensatory mechanism, but the inhibition of AQP8 protein after transcription did not recover.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R459.7
[Abstract]:Sepsis is a systemic inflammation-mmatory response syndrome (SIRS) caused by infection, which is one of the main causes of death in critically ill patients. Further development of sepsis shock and multiple organ dysfunction syndrome (MODS) can cause severe sepsis. Unc-tion syndrome (MODS) liver is one of the most susceptible organs in sepsis. Liver dysfunction is one of the early manifestations of sepsis developing into multiple organ failure (MOF). The pathogenesis of sepsis is still unclear. Possible pathogenesis includes: disturbance of the inflammatory factor storm coagulation system and microorganisms. Hepatocyte mitochondria, such as circulatory dysfunction, immune suppression, mitochondrial dysfunction, and imbalance of oxygen supply and consumption, are the center of nutrients and energy metabolism. Mitochondrial dysfunction plays an important role in the development of septic symptoms, especially in patients with septic shock. A water molecule channel expressed on the inner mitochondria membrane (IMM) is a highly selective and efficient channel for transporting water molecules. It has a great influence on the maintenance of the structure and function of hepatocyte mitochondria. In this study, we used cecum ligation and perforation method to prepare sepsis rat model. Morphological changes of hepatocyte mitochondria and the role of mitochondrial endometrial AQP8 in sepsis-mediated liver injury
objective
To study the effect of aquaporin 8 (AQP8) expression on mitochondrial morphology of hepatocytes in septic symptoms, and to explore a method to improve the expression of AQP8 protein on mitochondrial membrane, so as to protect liver from sepsis-induced liver injury.
Method
Twenty-four SD rats were randomly divided into two groups: control group and sepsis group. Twelve rats in each group were subjected to cecum ligation and perforation to establish sepsis experimental animal model. The control group was subjected to sham operation. The experimental process except cecum ligation and perforation was completed. The changes of body temperature, respiratory rate, heart rate and response to external stimuli were observed in the two groups to achieve sepsis diagnosis. After 1 hour, the rats were killed by decapitation after anesthesia. The liver of the rats was taken out, some of the fresh liver was used for making electron microscopic specimens, and the other part of the liver was used for extracting mitochondria. The expression of AQP8 protein in mitochondria was determined by Western blotting, and the hepatocytes were detected by RT-PCR. Mitochondrial AQP8 mRNA levels were analyzed by SPSS13.0 statistical software and measured by one-way ANOVA.
Result
1. mitochondrial ultrastructural changes
In the control group, the boundary of hepatocyte membrane was clear and intact, the number of endoplasmic reticulum and mitochondria was large and the morphology was normal, but in the sepsis group, the mitochondrial swelling cristae became wider and shorter, and the mitochondrial membrane ruptured, the endoplasmic reticulum distended to rupt and vacuole.
2. expression of mitochondrial AQP8 protein in hepatocytes
Using Prohibitin as internal reference, AQP8/prohibitin was used to express the relative expression level of AQP8 protein. The relative expression level of AQP8 protein in control group was set to 1. The relative expression level of AQP8 protein in sepsis group (n=12) was calculated to be 0.61 (+ 0.08), which was about 39% lower than that in control group (F=62.71, P 0.01).
3 expression of mitochondrial AQP8mRNA in hepatocytes
Using beta-actin as internal reference, AQP8/beta-actin expressed the relative expression level of AQP8 mRNA. The relative expression level of AQP8 mRNA in control group was set to 1, AQP8 mRNA in sepsis group was 1.59 [0.24] and AQP8 mRNA expression was increased by 59% on average compared with control group (F = 67.85, P 0.01).
conclusion
1. the sepsis model can be successfully prepared by cecal ligation and puncture.
2. sepsis can induce acute liver injury, and the ultrastructure of mitochondria of liver cells is obviously changed.
3. The down-regulation of AQP8 protein expression in hepatocyte mitochondria under septic symptoms may be one of the important factors leading to mitochondrial swelling and degeneration.
4. The expression of AQP8 protein in hepatocyte mitochondria was down-regulated and the expression of AQP8 mRNA was up-regulated in septic symptoms, suggesting that there may be compensatory mechanism, but the inhibition of AQP8 protein after transcription did not recover.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R459.7
【共引文獻】
相關(guān)期刊論文 前10條
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