【作者】 刘凤超；

【导师】 贺永文；

【作者基本信息】 华中科技大学 ， 传染病学， 2009， 博士

【摘要】 【背景及目的】急性肝衰竭死亡率高，治愈率低。其主要病理变化是肝细胞的大量死亡，从而导致肝功能严重受损。其临床特点是肝功能迅速恶化，可引起肝性脑病及凝血功能障碍。急性肝衰竭病因复杂，在我国由于病毒性肝炎感染率高，是引起肝衰竭的主要病因，其次可由药物、植物或化学品中毒等引起。研究显示，细胞凋亡在急性肝衰竭的发病过程中起着非常重要的作用。然而，迄今对于急性肝衰竭肝细胞发生大量凋亡的机理尚缺乏足够认识。一般而言细胞凋亡的发生存在二种机制：由携带凋亡信号的配体与细胞膜上的相应受体结合，将凋亡信号传导入细胞内，从而启动一系列的生化反应，最终导致细胞凋亡，该途径即死亡受体途径；或由细胞核内DNA的损伤诱发细胞凋亡的发生。在急性肝衰竭早期是否存在DNA损伤及DNA损伤与肝细胞凋亡的关系研究尚未见相关文献报道。为了探讨其发病机理，本研究利用D-氨基半乳糖加脂多糖建立的小鼠动物模型，以彗星实验、ELLISA、TUNEL等方法探讨肝细胞凋亡的发生机制，包括：1、研究急性肝衰竭模型小鼠肝细胞核DNA损伤的发生及演变。2、研究急性肝衰竭模型小鼠血清TNF-α浓度的变化及与肝细胞凋亡的关系。3、研究急性肝衰竭模型小鼠肝细胞损伤的演变过程。4、研究天晴甘平干预对急性肝衰竭模型小鼠肝细胞损伤的影响及机制。5、研究阿拓莫兰干预对急性肝衰竭模型小鼠肝细胞损伤及凋亡的影响及其机制。【方法】1、腹腔注射D-氨基半乳糖(D—galn)和脂多糖(LPS)，制作急性肝衰竭小鼠模型。2、、采用彗星实验从早期开始检测急性肝衰竭模型小鼠肝细胞核DNA的损伤，用Olive尾矩值表示DNA的损伤程度。3、采用ELISA法检测急性肝衰竭模型小鼠血清TNF-α的浓度。4、采用HE染色的方法检测急性肝衰竭模型小鼠肝组织的细胞形态学改变。5、采用TUNEL法检测急性肝衰竭模型小鼠肝细胞的凋亡，并计算凋亡指数。6、腹腔注射D-氨基半乳糖(D—galn)和脂多糖(LPS)前3h，实验组给予天晴甘平灌胃，分别于注射后不同时间点用彗星实验检测肝细胞核DNA的损伤。7、腹腔注射D-氨基半乳糖(D—galn)和脂多糖(LPS)前2h，实验组给予肌肉注射阿拓模兰，分别于注射D—galn和LPS后不同时间点用彗星实验检测肝细胞核DNA的损伤，用ELISA法检测血清TNF-α浓度，用TUNEL检测肝细胞凋亡。【结果】1、腹腔注射D-Galn和LPS后0.5h，模型组小鼠肝细胞核DNA的Olive尾矩值即明显增大，至1 h，出现细胞核DNA损伤的肝细胞达100％，且随时间延伸，肝细胞核DNA的Olive尾矩值逐渐增加。各组两两之间比较，差异均具统计学意义(P＜0.05)。2、腹腔注射D-Galn和LPS后0.5 h，模型组小鼠血清TNF-α浓度无升高；1h略有升高，但与正常对照组比较，差异无统计学意义；2h以后，升高趋势明显，各组与正常对照组比较，差异均具统计学意义(P＜0.05)。3、腹腔注射D-Galn和LPS 2h，模型组小鼠HE染色结果发现，肝细胞始出现嗜酸性染色增强，至8h肝细胞出现大片坏死，肝窦明显扩张充血并出血。4、腹腔注射D-Galn和LPS后6 h，模型组小鼠TUNEL检测结果发现肝组织始有凋亡细胞出现，至8 h出现大量凋亡细胞。5、腹腔注射D-Galn和LPS后，天晴甘平治疗组与对照组比较Olive尾矩值明显变小，差异具显著意义(P＜0.05)。6、腹腔注射D-Galn和LPS后，阿拓模兰治疗组与对照组比较Olive尾矩值明显变小，差异具统计学意义(P＜0．05)；但血清TNF-α浓度无明显改变；在腹腔注射D-Galn和LPS后的6h时间点，阿拓模兰治疗组的凋亡指数较对照组减少，差异具统计学意义，但在8h时间点，差异无统计学意义。【结论】1、急性肝衰竭模型小鼠肝细胞核DNA较早出现损伤，并随时间延伸逐渐加重，最终可能诱发了肝细胞凋亡。导致急性肝衰竭模型小鼠肝细胞核DNA损伤的原因可能是D-氨基半乳糖和脂多糖的毒性作用。同时，本研究HE染色的结果也提示，急性肝衰竭肝细胞坏死的机理部分也可能与肝细胞核DNA的损伤有关。2、血清TNF-α浓度的升高与肝细胞核DNA损伤的发生及演变不完全同步，但可能具有进一步促进肝细胞核DNA损伤的作用。3、天晴甘平具有减轻急性肝衰竭小鼠肝细胞核DNA的损伤的作用。4、阿拓模兰具有减轻急性肝衰竭小鼠肝细胞核DNA的损伤及细胞凋亡的作用，但这种作用与血清TNF-α浓度的改变无关。更多还原

【Abstract】 【Background and objective】In China, acute hepatic failure is associated with high mortalityand low cure rate. The main pathological change is massive death ofhepatocytes which causes serious damage of liver function. The cliniccharacteristic is that the liver functions rapidly degenerate, leading tohepatic encephalopathy and coagulation disorders. The pathogeny of theacute hepatic failure is complicated. Due to the high incidence of viralhepatitis, infection by hepatitis viruses is still the first cause of the acutehepatic failure until now. Secondly, the acute hepatic failure can becaused by drugs, plants, chemicals and so on. Some results ofexperiments have indicated that the apoptosis plays an important role inthe onset of acute hepatic failure. But until now, the understanding of themechanism of apoptosis of hepatocytes is inadequate. Generally, there two mechanisms underlying apoptosis. One of the two mechanism is thedeath receptor pathway, in which, after the ligands carrying apoptosissignals bind to the relevant receptors on the cell membrane, the apoptosissignals are transmitted into the cells to activate biochemical reactions,which finally cause apoptosis. The other is the intrinsic or mitochondrialpathway, in which apoptosis can be induced by DNA damages in the cellnucleus. Whether there are DNA damages in the early acute hepaticfailure, and the relationship between DNA damages and apoptosis havenot been reported in literature. In order to research the pathogenesisunderlying the acute hepatic failure, this study will probe into themechanisms underlying apoptosis of hepatocytes in the animal model byusing comet assay, ELLISA, TUNEL and other methods, which isestablished by intraperitoneal injection with D-Galactosamine (D-gain)and lipopolysaccharide. The objectives are as fellows:1.To research the occurrence and development of DNA damages innucleus of hepatocytes of mice with the acute hepatic failure model.2. To study the relationship between variation of TNF-αconcentration ofmice blood serum and hepatocyte apoptosis in the acute hepatic failuremodel.3. To examine the changes in the damaged hepatocytes during the acutehepatic failure. 4. To research the effect of Diammonium Glycyrrhizinate Enteric-coatedCapsules on the damage of heaptocytes of mice with the acute hepaticfailure and the relevant mechanism.5. To study the effect of reduced glutathione on the damage and apoptosisof heaptocytes of mice with the acute hepatic failure and the relevantmechanisms.【Method】1. Inject intraperitoneally with D-Galactosamine (D-galn) andlipopolysaccharide to establish the mouse model of acute liver failure.2. Conduct comet assay to detect DNA damages in nucleus of hepatocytesof mice with the acute hepatic failure from earlier time point afterinjection with D-Galactosamine (D-galn) and lipopolysaccharide. TheOliver tail moment value is used to indicate the extent DNA damage.3. Adopt ELISA to measure the serum TNF-αconcentration of mice withthe acute hepatic failure.4. Employ HE staining method to observe the changes in cellularmorphology of mice hepatic tissue in the acute hepatic failure model.5. Use TUNEL method to detect hepatocyte apoptosis of mice in theacute hepatic failure model, and calculate the apoptosis index.6. Administer intragastrically with Diammonium GlycyrrhizinateEnteric-coated Capsules to the experimental group of mice, and three hours later inject intraperitoneally with D—galn and LPS, then use cometassay to detect DNA damages in nucleus of hepatocytes of theexperimental group of mice at different time points after injection, useTUNEL to detect hepatocyte apoptosis.7. Perform intramuscular injection with induced glutathione, and twohours later, inject intraperitoneally with D—galn and LPS, and then usecomet assay to detect DNA damages in nucleus of hepatocytes atdifferent time points after injection, use ELISA to measureTNF-αconcentration, adopt TUNEL to detect hepatocyte apoptosis.【Results】1. The DNA Olive tail moment values of the hepatocytes weresignificantly greater, 0.5 hour after intraperitoneal injection ofD-GaIN/LPS, and increased with time. Comparison between any twogroups showed that the differences were statistically significant (P＜0.05).The ratio of damaged hepatocytes was 100 percent at 1 hour time pointafter injection.2. 0.5 hour after intraperitoneal injection with D—galn and LPS, theTNF-αconcentration of mice in experimental group did not increase. Itincreased slightly till 1 hour. After two hours, the tendency of increasingis obvious, and the difference between experimental group and controlgroup is significant (P＜0.05). 3. 2 hours after intraperitoneal injection with D—galn and LPS, HEstaining results showed enhanced acidophilic staining of hepatocytes inexperimental group. Till 8 hours, the massive necrosis of hepatocytesoccured, and the hepatic sinusoid obviously expanded, became hyperemiaand hemorrhage.4. Until 6 hour time point after intraperitoneal injection with D—galn andLPS, the TUNEL results showed apoptotic cells in the experimentalgroup. Till 8h, the massive apoptosis occured.5. After intraperitoneal injection with D—galn and LPS, the Olive tailmoment values of the experimental group treated by DiammoniumGlycyrrhizinate Enteric-coated Capsules decreased significantly(P＜0.05).6. After intraperitoneal injection with D—galn and LPS, the Olive tailmoment values of the experimental group decreased significantly ,towhich induced glutathione was administered(P＜0.05), and the apoptosisindex of the administered group is significantly lower than that of thecontrol group, but the TNF-αconcentration of blood serum did not changesignificantly.【Conclusion】1. DNA damage in nucleus of hepatocytes of mice with acute hepaticfailure occurs earlier, and expands with time, which maybe induce apoptosis at the end. The toxicity of D—galn and LPS maybe cause theDNA damage. The necrosis showed by HE may also be related to DNAdamage.2. TNF-αconcentration in blood serum did not increase synchronouslywith DNA damage in nucleus of hepatocytes of mice with acute hepaticfailure, but the increase of TNF-αconcentration in blood serum mayfurther lead to the DNA damage.3. Diammonium Glycyrrhizinate Enteric-coated Capsules can reduceDNA damage in nucleus of hepatocytes mice with the acute hepaticfailure.4. Induced glutathione can reduce DNA damage in nucleus of hepatocytesand apoptosis in liver of mice with the acute hepatic failure, but this is notrelated to variation of serum TNF-αconcentration.更多还原