外源线粒体对对乙酰氨基酚损伤的肝细胞的保护作用研究
发布时间：2019-01-05 16:44
【摘要】：对乙酰氨基酚(acetaminophen,APAP)是一种广泛使用的解热镇痛药。治疗剂量的APAP疗效明确,用药安全。但过量服用APAP可以引起严重的肝损伤,表现为肝细胞坏死。APAP引起肝细胞损伤的重要靶标是线粒体。APAP所致肝细胞毒性可能是通过干扰细胞线粒体的生物合成破坏线粒体结构和功能,进而破坏肝细胞的功能。尽管乙酰半胱氨酸是临床上治疗APAP中毒的标准药物,但其给药方式、剂量、时间仍存在争议。因此,寻找减轻APAP肝毒性的途径非常重要。本文中我们以构建了APAP致肝损伤的细胞模型,研究外源线粒体对APAP损伤的细胞的影响。首先我们构建了线粒体可以表达绿色荧光蛋白的HepG2细胞,并从此细胞中提取绿色荧光线粒体,同时采用线粒体特异性的红色荧光染料(MitoTracker Red CMXRos)对正常的HepG2细胞中提取的线粒体进行染色,获得红色荧光线粒体。将制备好的荧光线粒体与HepG2细胞共孵育后,实时观察细胞摄取线粒体的情况,结果显示细胞在10 min内可以摄取外源线粒体。然后用巨胞饮抑制剂—盐酸阿米洛利、网格蛋白抑制剂—高渗蔗糖溶液预处理细胞30 min后,测定细胞对外源线粒体的摄取率,结果显示当盐酸阿米洛利预处理细胞后,摄取率显著降低。此外,我们用詹姆斯绿B对小鼠肝脏线粒体染色,并测定其直径、膜通透性及肿胀度。激光粒度仪测定结果显示,我们提取的线粒体的直径主要分布在531.2?955.4 nm之间,线粒体肿胀度检测结果显示,线粒体溶液540 nm的吸光度较稳定,线粒体的膜结构稳定。最后,我们确定APAP合适的作用浓度为10 mM、损伤时间为24h,成功建立APAP致肝损伤的细胞模型,并初步探讨小鼠肝脏线粒体对APAP损伤的细胞的保护作用。将细胞分为3组,分别为正常对照组、模型组和外源线粒体保护组。我们检测了3组细胞的凋亡率、细胞培养液中谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、细胞中谷胱甘肽(glutathione,GSH)、三磷酸腺苷(adenosine triphosphate,ATP)的含量、线粒体形态和膜电位的变化。检测结果显示,与正常对照组相比,模型组细胞的凋亡率增加;细胞培养液中ALT、AST的含量显著增加;细胞中ATP、GSH的含量显著降低;线粒体膜电位显著降低且发生空泡化。与模型组相比,外源线粒体保护组细胞的凋亡率减少;细胞培养液中ALT、AST的含量降低;细胞中ATP、GSH的含量增加;线粒体膜电位有所恢复;线粒体空泡化程度减小。本研究发现,HepG2细胞在10 min内可以摄取外源线粒体,摄取机制可能是巨胞饮。我们提取的线粒体状态正常,可用于功能性检测。外源线粒体对APAP损伤的肝细胞具有保护作用。
[Abstract]:Paracetamol (acetaminophen,APAP) is a widely used antipyretic analgesics. The therapeutic dose of APAP is effective and safe. But overdose of APAP can cause serious liver damage. The main target of hepatocyte damage induced by APAP is mitochondria. The toxicity induced by APAP may destroy the structure and function of mitochondria by interfering with the biosynthesis of mitochondria, and then destroy the function of hepatocytes. Although acetylcysteine is the standard drug for the treatment of APAP poisoning, its administration, dosage and time are still controversial. Therefore, it is very important to find ways to alleviate the hepatotoxicity of APAP. In this paper, we constructed a cell model of liver injury induced by APAP and studied the effects of exogenous mitochondria on APAP cells. First of all, we constructed HepG2 cells which can express green fluorescent protein from mitochondria and extracted green fluorescent mitochondria from the cells. At the same time, mitochondria specific red fluorescent dye (MitoTracker Red CMXRos) was used to stain mitochondria extracted from normal HepG2 cells to obtain red fluorescent mitochondria. After the prepared fluorescent mitochondria were incubated with HepG2 cells, the uptake of mitochondria was observed in real time. The results showed that the cells could absorb exogenous mitochondria within 10 min. Then the cells were pretreated with Giant Cell Inhibitor-Amiolol Hydrochloride and reticulin Inhibitor-hyperosmotic Sucrose solution for 30 min, and the uptake rate of exogenous mitochondria was measured. The results showed that the cells were pretreated with amiloride hydrochloride for 30 min. The uptake rate decreased significantly. In addition, James green B was used to stain mouse liver mitochondria, and its diameter, membrane permeability and swelling degree were measured. The results of laser particle size analyzer showed that the diameter of the extracted mitochondria was between 531.2 and 955.4 nm, and the swelling degree of mitochondria showed that the absorbance of the solution 540 nm was stable and the membrane structure of mitochondria was stable. Finally, we determined that the appropriate concentration of APAP was 10 mM, for 24 h, and successfully established the cell model of APAP induced liver injury, and preliminarily studied the protective effect of mouse liver mitochondria on the cells damaged by APAP. The cells were divided into 3 groups: normal control group, model group and exogenous mitochondrial protection group. The apoptosis rate, the contents of glutamate-pyruvic transaminase (alanine aminotransferase,ALT), glutamic oxalacetic transaminase (aspartate aminotransferase,AST), glutathione (glutathione,GSH), adenosine triphosphate (adenosine triphosphate,ATP) in three groups were measured. Changes of mitochondrial morphology and membrane potential. The results showed that compared with the normal control group, the apoptosis rate of the model group was increased, the content of ALT,AST in the cell culture medium was significantly increased, the content of ATP,GSH in the cell was significantly decreased, and the mitochondrial membrane potential was significantly decreased and vacuolated. Compared with the model group, the apoptosis rate of exogenous mitochondrial protection group decreased, the content of ALT,AST in cell culture medium decreased, the content of ATP,GSH in cell increased, the mitochondrial membrane potential recovered and the degree of mitochondrial vacuolation decreased. In this study, we found that HepG2 cells could absorb exogenous mitochondria within 10 min, and the mechanism of uptake might be giant cell drink. Our extracted mitochondria are in normal state and can be used for functional detection. Exogenous mitochondria can protect hepatocytes from APAP damage.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2402036
[Abstract]:Paracetamol (acetaminophen,APAP) is a widely used antipyretic analgesics. The therapeutic dose of APAP is effective and safe. But overdose of APAP can cause serious liver damage. The main target of hepatocyte damage induced by APAP is mitochondria. The toxicity induced by APAP may destroy the structure and function of mitochondria by interfering with the biosynthesis of mitochondria, and then destroy the function of hepatocytes. Although acetylcysteine is the standard drug for the treatment of APAP poisoning, its administration, dosage and time are still controversial. Therefore, it is very important to find ways to alleviate the hepatotoxicity of APAP. In this paper, we constructed a cell model of liver injury induced by APAP and studied the effects of exogenous mitochondria on APAP cells. First of all, we constructed HepG2 cells which can express green fluorescent protein from mitochondria and extracted green fluorescent mitochondria from the cells. At the same time, mitochondria specific red fluorescent dye (MitoTracker Red CMXRos) was used to stain mitochondria extracted from normal HepG2 cells to obtain red fluorescent mitochondria. After the prepared fluorescent mitochondria were incubated with HepG2 cells, the uptake of mitochondria was observed in real time. The results showed that the cells could absorb exogenous mitochondria within 10 min. Then the cells were pretreated with Giant Cell Inhibitor-Amiolol Hydrochloride and reticulin Inhibitor-hyperosmotic Sucrose solution for 30 min, and the uptake rate of exogenous mitochondria was measured. The results showed that the cells were pretreated with amiloride hydrochloride for 30 min. The uptake rate decreased significantly. In addition, James green B was used to stain mouse liver mitochondria, and its diameter, membrane permeability and swelling degree were measured. The results of laser particle size analyzer showed that the diameter of the extracted mitochondria was between 531.2 and 955.4 nm, and the swelling degree of mitochondria showed that the absorbance of the solution 540 nm was stable and the membrane structure of mitochondria was stable. Finally, we determined that the appropriate concentration of APAP was 10 mM, for 24 h, and successfully established the cell model of APAP induced liver injury, and preliminarily studied the protective effect of mouse liver mitochondria on the cells damaged by APAP. The cells were divided into 3 groups: normal control group, model group and exogenous mitochondrial protection group. The apoptosis rate, the contents of glutamate-pyruvic transaminase (alanine aminotransferase,ALT), glutamic oxalacetic transaminase (aspartate aminotransferase,AST), glutathione (glutathione,GSH), adenosine triphosphate (adenosine triphosphate,ATP) in three groups were measured. Changes of mitochondrial morphology and membrane potential. The results showed that compared with the normal control group, the apoptosis rate of the model group was increased, the content of ALT,AST in the cell culture medium was significantly increased, the content of ATP,GSH in the cell was significantly decreased, and the mitochondrial membrane potential was significantly decreased and vacuolated. Compared with the model group, the apoptosis rate of exogenous mitochondrial protection group decreased, the content of ALT,AST in cell culture medium decreased, the content of ATP,GSH in cell increased, the mitochondrial membrane potential recovered and the degree of mitochondrial vacuolation decreased. In this study, we found that HepG2 cells could absorb exogenous mitochondria within 10 min, and the mechanism of uptake might be giant cell drink. Our extracted mitochondria are in normal state and can be used for functional detection. Exogenous mitochondria can protect hepatocytes from APAP damage.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
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相关期刊论文 前3条
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