【作者】 陈淼；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 【背景与目的】近年研究表明，细胞凋亡是导致肝脏损伤的一个重要细胞机制之一，内毒素血症作为肝衰竭的第二次打击，在肝衰竭发生发展过程中发挥重要作用。线粒体释放的第二种Caspase激活物smac是2000年发现的一种促凋亡分子。它正常时位于线粒体膜间间隙，在凋亡诱导因子的作用下，可释放入胞浆，与凋亡抑制蛋白IAPs结合并解除其对天冬氨酸特异性半胱氨酸蛋白酶的抑制作用，从而使Caspase9和Caspase3活性增强，促进细胞凋亡发生。本实验采用D-氨基半乳糖氨建立大鼠肝衰竭模型和体外LPS刺激L02肝细胞，探讨促凋亡蛋白smac从线粒体释放及对Caspase9和Caspase3的活性的影响，为肝衰竭内毒素血症防治措施提供新的思路。【实验材料和方法】一．细胞培养L02传代肝细胞株由我校肝病研究所提供，肝细胞培养在含有10％小牛血清高糖的DMEM培养基中，于37℃，5％CO₂细胞培养箱中培养，每隔3-4天对细胞进行传代，分别培养在六孔板中，100ug／ml LPS分别刺激4 h、8 h、16 h、24 h，收集各组的肝细胞进行检测。二．动物模型采用D-GaLN诱导急性肝衰竭大鼠模型，24只wistar健康大鼠随机分为四组，组Ⅰ：空白对照组（生理盐水组），组Ⅱ：100mg／kg D-GaLN模型组，组Ⅲ：200mg／kg D-GaLN模型组，组Ⅳ：300mg／kg D-GaLN模型组。三．标本采集和处理于腹腔注射D-GaLN后24小时，采用6％的水合氯醛腹腔注射麻醉大鼠，在无菌条件下分离出股静脉，切除股静脉并用肝素抗凝管取全血，低温离心取血清，并取肝脏组织，置于液氮中速冻，分别于—70℃保存待测。四．实验方法1．Hoechst和吖啶橙染色荧光显微镜观察L02细胞凋亡。2．流式分析仪检测L02细胞凋亡。3．流式分析仪检测Smac在L02肝细胞膜的表达。4．采用免疫组化方法检测Smac和Caspase9的L02肝细胞的表达。5．caspase3活性检测试剂盒检测L02肝细胞Caspase3活性。6．L02肝细胞线粒体与胞浆分离。7．Western blotting分析L02肝细胞线粒体上smac和细胞中Caspase9的表达。8．采用全自动生化分析系统检测大鼠血清谷丙转氨酶，总胆红素和总蛋白的水平。9．采用HE染色检测大鼠肝组织的病理变化。10．免疫组化方法检测Smac在大鼠肝组织的表达。11．采用鲎试剂基质显色法检测大鼠的血清内毒素水平。12．大鼠肝组织线粒体与胞浆分离。13．caspase3活性检测试剂盒检测大鼠肝组织Caspase3活性。14．Western blotting分析大鼠肝组织线粒体smac和细胞中Caspase9的表达。五．统计学分析方法所有资料采用SPSS13.0软件进行分析，实验数据均数以均数±标准差（x±s）表示，多组间采用方差分析，两组间采用t检验，P＜0.05为差异有统计学意义。【结果】1．荧光显微镜下检测，Hoechst和吖啶橙染色显示L02凋亡细胞细胞核呈致密均匀蓝颜色和绿色荧光。2．正常对照组凋亡率0.30±0.10％，LPS刺激4 h、8 h、16 h、24 h后凋亡率分别2.30±0.45％、4.00±0.89％、6.20±1.17％、18.20±2.26％，呈进行性增高（P＜0.01），24 h期间达到一个凋亡高峰，与正常对照组比较差异有显著意义（P＜0.05）。3．正常对照组Smac表达为0.77±0.14％，LPS刺激4 h、8 h、16 h、24h后Smac在肝细胞膜的表达1.73±0.78％、2.84±0.56％、3.72±1.26％、8.66±1.93％，呈进行性增高（P＜0.05），呈现出时间依赖性。4．通过免疫组织化学的方法分别观察Smac和Caspase9在L02肝细胞的表达情况，免疫组化检测smac和Caspase9结果：smac蛋白和Caspase9蛋白表达阳性，主要定位在胞浆中，呈棕黄色颗粒。Smac和Caspase9蛋白随着LPS刺激时间的增长表达率呈现逐渐增高的趋势（P＜0.01），与正常对照组相比，各刺激组Smac和Caspase9蛋白表达显著增高（P＜0.05）。5．在给予LPS刺激4个小时水平变化不是很明显，只有轻度的升高。而在8h、16h、24h刺激组Caspase3活性显著增高，各组间比较差异有统计学意义（P＜0.05）。6．Western blot检测方法结果表明经过LPS处理4 h、8 h、16 h、24 h后细胞线粒体中Smac的表达量是逐渐降低的，与对照组比较差异具有显著的意义（P＜0.05）。相反，在细胞中caspase9表达量却逐渐升高，与对照组比较也有显著意义（P＜0.05）。7．肝衰竭模型肝组织HE染色未见正常的肝小叶，肝细胞混浊肿胀，并见散在大片坏死，汇管区炎症严重，可见汇管区周围出现大块坏死带。正常组肝组织结构正常。8．通过免疫组织化学的方法观察smac在肝组织中的表达情况，可见smac蛋白在胞浆中呈溶解状棕黄色表达。模型组相对于对照组明显升高。9．组Ⅱ血清ALT水平仅轻度增高，组Ⅲ和组Ⅳ显著增高，其中组Ⅳ高于组Ⅲ。且血清总胆红素随着D-GaLN的剂量的增大而升高，其中组Ⅳ明显升高（P＜0.05），但各组血清总蛋白正常，比较无差异（P＞0.05）。10．组Ⅰ大鼠体内未形成内毒素血症，组Ⅱ、组Ⅲ、组Ⅳ大鼠检测出明显的内毒素血症，且随着D-GaLN的剂量的增大内毒素血症的水平逐渐增高。与组Ⅰ比较具有统计学意义（P＜0.01）。11．caspase3活性在组Ⅱ轻度升高，与组Ⅰ比较具有统计学意义（P＜0.05）。组Ⅲ、组Ⅳ水平明显升高，且随着D-GaLN的剂量的增大caspase3活性逐渐增高，与组Ⅰ比较具有统计学意义（P＜0.01）（见表3）。12．Western blot检测方法结果表明经过D-GaLN诱导急性肝衰竭大鼠模型后，随着D-GaLN剂量的增加，细胞线粒体中Smac的表达量是逐渐降低的，与对照组比较差异具有显著的意义（P＜0.05）。相反，在细胞中caspase9表达量却逐渐升高，与对照组比较也有显著意义（P＜0.05）。【结论】本研究发现促凋亡蛋白smac在肝细胞存在高表达现象，体外实验中证明，内毒素可以直接促进人肝细胞的凋亡，其促进凋亡的通路可能包括了smac通路。并且内毒素可能可以促进smac从线粒体释放到细胞膜参与凋亡，而不仅是释放到胞浆。在肝衰竭模型实验中，单用D-GalN建立大鼠肝衰竭模型，证明D-GalN可以引发大鼠肝衰竭，出现明显的内毒素血症，随着内毒素水平的升高，可以促进smac从线粒体释放出胞浆进一步激活下游启动酶caspase9和效应酶caspase3，更加证明内毒素引发凋亡可能与smac通路有关。总之，线粒体信号通路在内毒素所致的肝细胞凋亡中发挥重要作用，其机制可能是通过促进Smac从线粒体向胞浆和胞膜释放，并活化其下游通路caspase9和caspase3有关。更多还原

【Abstract】 【Background and objective】Recent studies demonstrated that apoptosis is one of the major cellular machanismsleading the liver injury. Endotoxemia, as the second attack, plays an important role in itsdevelopment of serious hepatitis. Smac released from mitochondria is a pro-apoptoticmolecule discovered in 2000. Under normal conditions, it is lo-cated in intermembranespace but is released into the cytoplasm when being stimulated by apoptosis-inducingfactors. After binding with IAPs, it can increase the activity of cysteine-containingaspartate-specific proteases (caspase9 and caspase3). Though liver cells con-ain largeamount of smac, however, its roles in liver apoptosis, especially in the endotoxin-inducedliver apoptosis have not been well studied.This study, by using D-galactosamine(D-GalN)-induced rat model of hepatic failure and LPS-stimulated L02 hepatic cells,examined smac, a pro-apoptotic protein released from mitochondria, and its effects on theactivity of caspase9 and caspase3, with an attempt to explore a new approach for thecontrol of endotoxemia.【Methods】Liver Cell CultureLiver cells were cultured in the DMEM containing 10% calf serum at 37℃in a 5%CO2 in an incubator. Cell passaging was conducted each 3 to 4 days in a 6-mesh plate. Thecells I each group were stimulated with 10 mg/mL LPS for 4, 8, 16 and 24 h and harvestedfor further test. Animal ModelAcute hepatic failure was induced by i.p. injection of D-GaLN in rats. A total of 24healthy Wistar rats were divided into 4 groups at random: group 1 (blank control group, inwhich normal saline was given), group 2 (model group, in which 100 mg／kg D-GaLN wasi.p. administered), group 3 (model group, in which 200 mg/kg D-GaLN was i.p. injected)and group 4 (model group, in which 300 mg／kg D-GaLN was given).Sample CollectionAfter 24 h, 6% chloral hy-drate was i.p. injected for anesthesia. Femoral vein wasseparated under aseptic condition and severed. Then the whole blood was collected byemploying heparinized tube. The hepatic tissues were taken, put into liquid ni-trogen andstored at -70℃for later use.Experiment method1. The L02 cells were examined by Fluorescent Microscopy after Hoechst andacridine orange Staining.2. Flow Cytometric Examination of L02 Cell Apoptosis.3. Flow Cytometric Detection of Smac on L02 Liver Cell Membrane.4. Determination of Smac and Caspase9 By using immunohistochemical technique.5. Detection of Caspase3 Activity of on L02 Liver Cell.6. Separation of Mitochondria and Cell Plasma of L02 Liver Cell.7. Determination of Smac in mitochondria and Caspase9 in L02 Liver Cell by WesternBlotting.8. All rats’s Blood sample were collected to examine alanine ALT,TBIL and TP.9. Determination of All rats’s hepatic tissues by Hematoxytin (HE) staining. 10. Immunohistochemical Detection of Smac in All rats’s hepatic tissues.11. The level of endotoxin in plasma detect by Limulus amebocyte lysate.12. Separation of Mitochondria and Cell Plasma of rats’s hepatic tissues.13. Detection of Caspase3 Activity of rats’s hepatic tissues.14. Determination of Smac in mitochondria and Caspase9 in rats’s hepatic tissues.Statistics analysisSPSS version 13.0 was used to perfom statistics analysis.【Results】1.Under the fluorescent microscope, L02 apoptotic cells showed evenly distributed blueand green florescence within nucleus.2.The late-stage apoptosis rate of the normal control group was0.30±0.10%. The late-stageapoptosis rate after stimulation with LPS for 4、8、16 and 24h was2.30±0.45%、4.00±0.89%、6.20±1.17% and 18.20±2.26%, respectively. There were signifi-cantdifferences in the apoptosis rate (P＜0.05) between normal control and model groups(P＜0.01).3. The rate of smac expression was 0.77±0.14% in the con-trol group and the rates of smacexpression on liver cell membrane, after stimulation with 4, 8, 16 and 24h, were1.73±0.78%、2.84±0.56%、3.72±1.26%、8.66±1.93%, respectively. The rate increasedprogressively over time.4.The expression of smac protein and caspase9 were positive, and they were mainly locatedin cytoplasma, presenting as brown particles. The expression rates of smac and caspase9increased with the time of stimulation with LPS (P＜0.01). Compared to the normal controlgroup, the expressions of smac and caspase9 in the model groups were significantlystronger (P＜0.05).Spearman level analysis revealed that expression levels of smac and caspase9 in the different model groups were positively related.5.With the normal control group, there existed no sig-nificant difference in caspase3activity among different time points. At the 4th h, there was no difference in the caspase3activity between the model group and the normal control group. However, the caspase3activity increased significantly in the model group at the 8th h, 16th h and 24th hascompared with model group at the 4th and the normal control groups at all time points(P＜0.05).6.Western blot shows that The expession of smac expression in mitochondria weredecreased progressively over time after stimulation with 4, 8, 16 and 24h, there wasdifference between the model group and the normal control group(P＜0.05).and Theexpression of caspase9 in cells were increased progressively, there was difference betweenthe model group and the normal control group(P＜0.05).7.Smac protein was stained brown cytoplasma. With HE staining, no normal liver lobulewas found. And the cells were swelling and their contour was blurred, with some spottynecrosis. Around the portal area, there are serious inflammation and strips of massivenecrosis.8.Smac protein was stained brown cytoplasma in hepatic failure tissues.9.Blood serum ALT level in group 2 was increased slightly while it was substantiallyelevated in group 3 and group 4. Total blood serum bilirubin increased with the dosage ofD-GaLN in the model groups. Total blood serum protein in all the groups was normal withno significant difference found among them (P＞0.05).10. Rats in group 1 did not develop endotoxemia, while group 2、group 3 and group 4showed obvious symptoms of endotoxemia, which increased with the dosage of D-GaLN(P＜0.01).11. Activity of caspase3 in group 2 was increased slightly while it was substantiallyelevated in group 3 and group 4(P＜0.01).12. Western blot shows that The expession of smac expression in mitochondria were decreased progressively After the treatment with D-GaLN, there was difference betweenthe model group and the normal control group(P＜0.05).and The expression of caspase9 inHepatic Tissues were increased progressively, there was difference between the modelgroup and the normal control group(P＜0.05).【Conclusion】Smac were highly expessioned in hepatic cells, in vitro, endotoxin can ruducesapoptosis through Smac Apoptosis Channel. And Smac not only can be released into theCell Membrane but also can released into the Cell cytoplasm when being stimulated. Invivo, D-GalN alone was used for the establishment of hepatic failure model, and our resultsshowed that could induce obvious symptoms of endotoxemia and hepatic failure in rats,and demonstrated that endotoxin could induce apoptosis by promoting the release of smacfrom mitochondria and increasing the activity of start-up enzyme caspase9 and effectenzyme caspase3.Mitochondria signal channel plays an important role in hepatic apoptosisinduced by endotoxin. Mitochondria signal channel plays an important role in theendotoxin-induced apoptosis of hepatic cells by promoting the release of caspasee9 andcaspase3 from mitochondria to cytoplasma and Membrane.更多还原