【作者】 史华新；

【导师】 向楠；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的：以人正常肝细胞LO2为研究对象,以外周型苯二氮卓受体(peripheral benzodiazepine receptor, PBR)表达水平为研究切入点,以线粒体能量代谢为主线,从“疗效-机制-靶点”不同层次,从肝细胞的功能、凋亡率、线粒体膜电位、胞内Ca2+浓度、线粒体调节蛋白PBR浓度等不同角度,主要研究在乙型肝炎肝硬化患者血清影响下肝细胞LO2的损伤情况以及加味补肝散含药血清的保护作用,系统阐述其线粒体途径的作用机制,为中药复方加味补肝散的临床应用及研究开发提供实验数据和理论依据。方法：1.运用细胞培养技术体外传代培养人正常肝细胞LO2,设定含有不同血清比例的3组培养液模拟乙型肝炎肝硬化患者机体内环境,乙型肝炎肝硬化患者血清(以下简称“患者血清”)和胎牛血清(fetal bovin serum, FBS)的浓度分别为：5%和10%,10%和10%,10%和5%。8h、12h和24h后分别通过MTT法检测肝细胞增殖情况筛选适宜的造模血清浓度。2.确定造模血清浓度后,先将实验分为空白组(不含肝细胞)、正常组和模型组,共3个组。正常组用含10%FBS的培养液培养,空白组、模型组均用含10%患者血清和5%FBS的培养液培养。24h后,光镜观察肝细胞L02形态学改变、定性检测乙型肝炎病毒血清学标志物(HBV-M)、定量检测肝细胞功能,从细胞形态、病原和功能等角度评价肝细胞损伤的体外实验模型。3.模型确定后,再将实验分为正常组、模型组、对照组(还原型谷胱甘肽,GSH,浓度为100μg/ml)、加味补肝散含药血清高剂量组(浓度为20%)、中剂量组(浓度为10%)、低剂量组(浓度为5%),共6个组。24h和48h后分别观察、检测相关指标：(一)光镜观察各组肝细胞LO2的形态改变；(二)全自动生化分析仪检测各组肝细胞功能；(三)流式细胞仪检测各组肝细胞凋亡率；(四)流式细胞仪检测各组肝细胞线粒体膜电位和胞内Ca2+浓度；(五)ELISA法检测线粒体膜PBR浓度水平。结果：1.筛选出适宜造模血清比例：24h后,除正常组外,3个测试组肝细胞形态均出现不同程度的改变：数量减少、体积增大、细胞碎裂、核仁缩小。含10%患者血清和5%FBS组最明显,且改变随着患者血清浓度的增高和作用时间的延长更加明显。MTT法检测结果显示,12h和24h后,该组肝细胞吸光值明显低于正常组(P<0.05),增殖明显受到抑制,提示该组血清比例适宜造模。2.体外实验模型成功：24h后,模型组肝细胞数量减少,部分细胞脱壁,细胞体积肿胀,形态出现改变：颜色变深,颗粒粗糙,核固缩,染色质凝集等；HBsAg阳性率明显高于正常组,差异有显著统计学意义(P<0.01)；肝细胞功能ALT、AST、TBil等指标较正常组、空白组均升高,差异有显著统计学意义(P<0.05)。提示模型能够模拟肝细胞在患者体内的损伤情况。3.对肝功能的影响：24h和48h后,模型组肝细胞功能ALT、AST、TBi1等指标均高于正常组,差异有统计学意义(P<0.05), GSH组以及加味补肝散含药血清高、中、低剂量组ALT、AST、TBi1等指标均低于模型组,差异有统计学意义(P<0.05)。4.对肝细胞凋亡率的影响：模型组肝细胞凋亡率高于正常组,差异有统计学意义(P<0.05), GSH组以及加味补肝散含药血清高、中、低剂量组肝细胞凋亡率均低于模型组,差异有统计学意义(P<0.05)。5.对线粒体膜电位的影响：模型组肝细胞线粒体膜电位Rh123荧光值低于正常组,差异有显著统计学意义(P<0.01), GSH组以及加味补肝散含药血清高、中、低剂量组肝细胞线粒体膜电位Rh123荧光值均高于模型组,差异有统计学意义(P<0.05,P<0.01)。6.对肝细胞胞内Ca2+浓度的影响：模型组肝细胞Ca2+浓度高于正常组,差异有显著统计学意义(P<0.01)。GSH组以及加味补肝散含药血清高、中、低剂量组Ca2+浓度均低于模型组,差异有统计学意义(P<0.05,P<0.01)。7.对线粒体调控蛋白PBR浓度的影响：模型组PBR浓度高于正常组,差异有统计学意义(P<0.05). GSH组以及加味补肝散含药血清高、中剂量组PBR浓度均低于模型组,差异有统计学意义(P<0.05)。结论：1.用含10%患者血清和5%FBS的培养液培养人正常肝细胞LO2,能够抑制肝细胞增殖,造成肝细胞损伤,是体外模拟乙型肝炎肝硬化患者体内肝细胞损伤情况的适宜浓度。2.用该血清比例的培养液培养肝细胞L0224h,能够在体外模拟乙型肝炎肝硬化患者体内肝细胞损伤情况。3.在肝细胞LO2培养液中加入一定比例的肝炎肝硬化患者血清,肝细胞形态发生改变,并诱导线粒体膜PBR表达增加,从而影响线粒体能量代谢,出现肝细胞凋亡和功能损伤。加味补肝散含药血清对肝细胞具有明显的保护作用,其机制也与线粒体能量代谢有关。更多还原

【Abstract】 Objectives:To provide experimental basis for the clinical application of Bugan Powder, studying on the injury of human normal hepatic cells LO2 cultured in serum from hepatitis-B cirrhosis patients and the protective effects of Bugan Powder medicated serum to hepatic cells as well as its mitochondrial mechanism systematically, by researching the mitochondrial energy metabolism via starting from peripheral benzodiazepine receptor (PBR) observing the functional hepatic cells injury, apoptosis rate, mitochondrial transmembrane potential (MTP), calcium concentration within cells, and mitochondrial regulatory protein, for conducting the study in layers of "efficacy-mechanism-target".Methods:1. LO2, subjected to subculture, were put to 3 groups stimulating environment of liver cirrhosis after hepatitis B by composing the nutrient solution in different proportions (in volume) of serum from patients and that from fetal bovin:5% and 10%,10% and 10%, 10% and 5%. Optimal serum concentration for culturing were selected by MTT assay at 8h, 12h and 24 for determining hepatic cell proliferation.2. After determining the optimal serum concentration,3 groups were divided for the study: normal group (cultured in 10% FBS contained nutrient solution), blank group (contained no hepatic cells, subjected to 10% patient serum and 5% FBS), and model group (cultured in 10% patient serum and 5% FBS). LO2 cells morphological changes in optical microscope, qualitative measurement for HBV-M, quantitative detection of liver cell function were observed for assessment for the in vitro model of hepatic cells injury.3. After confirming the model, the experiment were conducted in 6 groups:normal group, model group, control group (GSH), high, medium, low doses groups of Bugan Powder. The concentration of GSH was set to 100μg/ml according to literatures. The volume proportion of Bugan Powder medicated serum in the nutrient solutions were respectively 20%,10% and 5%. Indexes were observed at 24h and 48h, as follows:(1) morphological changes of LO2 by optical microscope, (2) hepatic cell function indexes by automatic biochemical analyzer, (3) cell apopotosis in groups with FCM, (4) mitochondrial transmembrane potential and calcium concentration in cells were determined by FCM, (5) the quantity of PBR expression in hepatic cells with ELISA.Results:1. Cultured cells were observed in fragmentation, enlarged volume, nucleolus shrinking and cell count decreasing, with the most obvious in group 3 (containing 10% patient serum and 5% FBS) and being more obvious with the patient serum volume proportion increased and time prolonged. MTT showed the light absorption in group 3 was significantly lower than that in normal group (P<0.05), the proliferation was obviously restrained, implying that serum proportion in group 3 was suitable for modeling.2. Morphological changes as shrinking volume, chromatic agglutination, karyopyknosis at 24h after cultured respectively, with higher HBsAg positive rate that that in normal group (P<0.01), and ALT, AST and TBil higher than that in normal group, were observed. ALT, AST, TBil in model group were all higher than that in normal group of significant difference (P<0.05). This implied that this model could reflect cell injury in patient liver.3. For the liver cell function, at 24h and 48h, the ALT, AST, TBil were higher than that in normal group with significant differences (P<0.05). These indexes in GSH group and the Bugan Powder groups were all lower than that in model group of statistical significance (P<0.05).4. Model group showed increase in apoptosis when compared with normal group (P<0.05), while the GSH group and Bugan Powder groups were observed lower than that in model group (P<0.05), both of the two differences were of statistical significance.5. On MTP, Rh123 OD value in the model group was declining when compared with normal group(P<0.01), with which compared higher values were observed in Bugan Powder groups. There were significant differences in the above comparisons (P<0.05, P<0.01).6. The fluorescence value of Ca2+ in model group was higher than in normal group(P<0.01), while the opposite was observed in GSH group and Bugan Powder groups. The differences were of statistical significance (P<0.05,P<0.01).7. The model group showed an increase in PBR comparing with normal group (P<0.05), also significantly higher that than in GSH group and Bugan Powder groups, of significant differences (P<0.05).Conclusions:1. The environment where hepatic cells injury occurs could be simulated in vitro by putting the cells in the nutrient solution composed of 5% FCS and 10% hepatitis-B cirrhosis serum, which is suitable for reflecting the damage to hepatic cells caused by hepatitis B virus.2. The serum proportion mentioned above can be used for in vitro stimulating hepatic cells injury in hepatitis-B cirrhosis patients by putting LO2 into this proportioned nutrient solution for 24h.3. Hepatic cells appeared morphological changes in patient serum and functional damage was observed. The increase of hepatic cells apoptosis may be related to mitochondria, and PBR may play a role in its regulation, which may also serve the mechanism of the obvious protective effects of Bugan Powder medicated serum on hepatic cell injury.更多还原