【摘要】：對(duì)乙酰氨基酚(acetaminophen,APAP)是一種廣泛使用的解熱鎮(zhèn)痛藥。治療劑量的APAP療效明確,用藥安全。但過(guò)量服用APAP可以引起嚴(yán)重的肝損傷,表現(xiàn)為肝細(xì)胞壞死。APAP引起肝細(xì)胞損傷的重要靶標(biāo)是線(xiàn)粒體。APAP所致肝細(xì)胞毒性可能是通過(guò)干擾細(xì)胞線(xiàn)粒體的生物合成破壞線(xiàn)粒體結(jié)構(gòu)和功能,進(jìn)而破壞肝細(xì)胞的功能。盡管乙酰半胱氨酸是臨床上治療APAP中毒的標(biāo)準(zhǔn)藥物,但其給藥方式、劑量、時(shí)間仍存在爭(zhēng)議。因此,尋找減輕APAP肝毒性的途徑非常重要。本文中我們以構(gòu)建了APAP致肝損傷的細(xì)胞模型,研究外源線(xiàn)粒體對(duì)APAP損傷的細(xì)胞的影響。首先我們構(gòu)建了線(xiàn)粒體可以表達(dá)綠色熒光蛋白的HepG2細(xì)胞,并從此細(xì)胞中提取綠色熒光線(xiàn)粒體,同時(shí)采用線(xiàn)粒體特異性的紅色熒光染料(MitoTracker Red CMXRos)對(duì)正常的HepG2細(xì)胞中提取的線(xiàn)粒體進(jìn)行染色,獲得紅色熒光線(xiàn)粒體。將制備好的熒光線(xiàn)粒體與HepG2細(xì)胞共孵育后,實(shí)時(shí)觀察細(xì)胞攝取線(xiàn)粒體的情況,結(jié)果顯示細(xì)胞在10 min內(nèi)可以攝取外源線(xiàn)粒體。然后用巨胞飲抑制劑—鹽酸阿米洛利、網(wǎng)格蛋白抑制劑—高滲蔗糖溶液預(yù)處理細(xì)胞30 min后,測(cè)定細(xì)胞對(duì)外源線(xiàn)粒體的攝取率,結(jié)果顯示當(dāng)鹽酸阿米洛利預(yù)處理細(xì)胞后,攝取率顯著降低。此外,我們用詹姆斯綠B對(duì)小鼠肝臟線(xiàn)粒體染色,并測(cè)定其直徑、膜通透性及腫脹度。激光粒度儀測(cè)定結(jié)果顯示,我們提取的線(xiàn)粒體的直徑主要分布在531.2?955.4 nm之間,線(xiàn)粒體腫脹度檢測(cè)結(jié)果顯示,線(xiàn)粒體溶液540 nm的吸光度較穩(wěn)定,線(xiàn)粒體的膜結(jié)構(gòu)穩(wěn)定。最后,我們確定APAP合適的作用濃度為10 mM、損傷時(shí)間為24h,成功建立APAP致肝損傷的細(xì)胞模型,并初步探討小鼠肝臟線(xiàn)粒體對(duì)APAP損傷的細(xì)胞的保護(hù)作用。將細(xì)胞分為3組,分別為正常對(duì)照組、模型組和外源線(xiàn)粒體保護(hù)組。我們檢測(cè)了3組細(xì)胞的凋亡率、細(xì)胞培養(yǎng)液中谷丙轉(zhuǎn)氨酶(alanine aminotransferase,ALT)、谷草轉(zhuǎn)氨酶(aspartate aminotransferase,AST)、細(xì)胞中谷胱甘肽(glutathione,GSH)、三磷酸腺苷(adenosine triphosphate,ATP)的含量、線(xiàn)粒體形態(tài)和膜電位的變化。檢測(cè)結(jié)果顯示,與正常對(duì)照組相比,模型組細(xì)胞的凋亡率增加;細(xì)胞培養(yǎng)液中ALT、AST的含量顯著增加;細(xì)胞中ATP、GSH的含量顯著降低;線(xiàn)粒體膜電位顯著降低且發(fā)生空泡化。與模型組相比,外源線(xiàn)粒體保護(hù)組細(xì)胞的凋亡率減少;細(xì)胞培養(yǎng)液中ALT、AST的含量降低;細(xì)胞中ATP、GSH的含量增加;線(xiàn)粒體膜電位有所恢復(fù);線(xiàn)粒體空泡化程度減小。本研究發(fā)現(xiàn),HepG2細(xì)胞在10 min內(nèi)可以攝取外源線(xiàn)粒體,攝取機(jī)制可能是巨胞飲。我們提取的線(xiàn)粒體狀態(tài)正常,可用于功能性檢測(cè)。外源線(xiàn)粒體對(duì)APAP損傷的肝細(xì)胞具有保護(hù)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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R96

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本文編號(hào)：2402036