【作者】 陈军梅；

【导师】 张赤志；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2005， 博士

【摘要】 目的 A型肝性脑病(hepatic encephalopathy,HE)是急性肝功能衰竭并发的大脑功能障碍,该病来势凶险,生存率仅为20%。目前,氨中毒学说被公认为仍处于HE众多发病机制假说的中心地位。通常认为它是通过干扰脑的能量代谢引起HE的发生。近年来,国内外学者认为,氨导致星形胶质细胞结构功能失常,可能是HE发病的主要机理。祖国医学称本病为“肝厥”。根据中医理论与临床经验,导师张赤志教授认为本病病因病机为邪毒直犯肝脏,肝失疏泄,脾失健运,肠道传导失司,腑浊内生,化热生痰,上扰神明;病位在肝、心脑与胃;结合“肝与大肠相通,肝病宜疏通大肠”和“上病下治”治疗原则,立通下解毒法;选用酒制大黄和乳酸菌素组成乳黄片治疗HE,临床和前期动物实验均显示有很好的降氨作用,能明显改善HE症状与体征,提高存活率。为进一步明确乳黄片抗HE的作用位点与机制,本课题从动物和细胞实验两方面研究该药对星形胶质细胞结构和蛋白表达的影响。 方法 (1)200±20gSPF级雄性Wistar大鼠96只,随机分为6组,每组16只:正常对照组(A组)、模型组(B组)、乳果糖组(C组)、乳黄片小中大剂量组(D、E、F组)。大鼠禁食过夜(14h以上),除A组外其余各组采用目前应用最成熟的硫代乙酰胺(TAA)诱导法(TAA250mg/kg灌胃两次,每次间隔24h)复制A型HE动物模型。在TAA首次造模前2h,A、B组每只大鼠灌胃生理盐水1ml/100g体重,其余各组给等体积/重量比的相应药物。以后每8h重复给药一次。自首次TAA造模至实验结束,每隔8h皮下注射3ml 10%葡萄糖、2ml含等量生理盐水与20μmol/lKCl溶液的混合液以防止低血糖。实验30h后行脑电图(EEG)检测,之后处置动物取材。实验过程中,密切观察大鼠行为学改变。(2)眼眶取血,检测血清ALT和血氨浓度。(3)伊文思蓝(EB)渗出法检测血脑屏障(BBB)通透性。透射电镜观察星形胶质细胞在内的BBB超微结构变化。(4)小脑延髓池处抽取脑脊液,检测脑脊液氨浓度。取大脑皮层组织,制成匀浆,高效液相色谱法检测谷氨酰胺含量。取整个脑组织,干湿重法检测脑组织含水量比。(5)免疫组织化学法观察大脑皮层组织星形胶质细胞结构蛋白-胶质纤维酸性蛋白(GFAP)的表达。(6)半定量逆转录-多聚酶链法(RT-PCR)检测大鼠皮层脑组织谷氨酸转运体GLT-1mRNA的表达情况。(7)制备乳黄片脑脊液,作用暴露于5mM浓度氯化铵的星形胶质细胞CRL-2541 48h,MTT法检测其活性,免疫细胞化学法检测GFAP表达情况。 结果 (1)乳黄片可明显改善A型HE大鼠行为异常和EEG变化,显著降低血清ALT与血氨水平(P<0.01),提高存活率;大、中、小剂量组疗效均优于乳果糖组,且以中剂量组最佳;(2)乳黄片能明显降低A型HE大鼠脑组织EB含量(P<0.01),显著改善星形胶质细胞在内的BBB超微结构受损情况,以中剂量组疗效最优;(3)乳黄片可显著降低A型HE大鼠脑脊液氨浓度、脑组织谷氨酰胺含量及脑含水量比(P<0.01),以中剂量组下降最明显;(4)乳黄片能显著上调A型HE大鼠大脑皮层组织星形胶质细胞GFAP的表达,升高湖北中医学院博士学位论文其阳性细胞数和平均光密度(P<0.01),以中剂量上升最显著;(5)乳黄片能明显上调A型HE大鼠大脑皮层组织星形胶质细胞上谷氨酸转运体GLT一lmRNA的表达,以中剂量上升最明显;(6)乳黄片脑脊液能显著升高smM浓度氨环境中星形胶质细胞CRL一2541的吸光度,上调GFAP的表达。结论(1)高氨是A型HE发病的病因基础之一,其对星形胶质细胞结构和蛋白的损害在HE的发病机理中占有重要作用。其中,氨及其代谢产物一谷氨酞胺的过量堆积可引起星形胶质细胞结构损害和GFAP蛋白表达缺失,导致血脑屏障完整性受损,通透性增加和血管源性脑水肿的发生;继发的谷氨酸转运体蛋白(如GLT一1)基因表达缺失,可导致谷氨酸大量积于突触间隙,引起神经元毒性和脑水肿的发生。(2)乳黄片有良好防治A型HE的作用,可能是通过以下途径实现的:保肝护肝,降低氨(血和脑脊液氨)与谷氨酞胺浓度,进入血脑屏障,改善星形胶质细胞结构受损与蛋白表达缺失情况。(3)乳黄片抗HE的作用位点可能为星形胶质细胞。关键词:肝性脑病/A型@乳黄片星形胶质细胞实验研究更多还原

【Abstract】 Objective Hepatic encephalopathy (HE) of type A is a kind of encephalopathies associated with acute liver function failure. It is dangerous and the survival rate of it is only 20%. At present, ammonia intoxication is still in the center of the pathogeneses of HE. Usually, it was thought that ammonia caused HE by interacting brain energy. Recently, the domestic and abroad scholars think the main pathogenesis of HE maybe the disorders of astroglia structure and functions caused by ammonia. In Traditional Chinese Medicine (TCM), it was called "GanJue" . Based on TCM theories and clinical experiences, my tutor -professor Zhang ChiZhi think that the pathogenesis of Ganjue is the result of heat and phlegm troubling mind which caused by the disorders of liver, speel and intestine. The sick position of it is liver, heart, brain and stomach. According to TCM therapeutic principle of "liver and large intestine are collected with each other, diseases of liver are fit to clean large intestine " and "be ill upper, treat down" , professor Zhang uses wined Rhubarb and Lactohacillin to make RuHuang Pill. Clinic and animal studies showed it had good effects on the treatment of HE. It could decline ammonia concentration, obviously ameliorative the symptoms and signs of HE, increase survive rate. The study is to observe the effects of RuHuang pill on the structure and protein expressions of astroglia, to reveal the function point and mechanism of it. Methods(l)96 male Wistar rats weighted 200±20g of SPF grade were randomly divided into six groups: Normal control group (group A), model group (group B), lactulose group (group C), RuHuang Pill group of small dose (group D), RuHuang Pill group of middle dose (group E), RuHuang Pill group of large dose (group F). After fasted overnight for over 14h, all rats except group A were induced to be model of HE of type A by administration with TAA 250mg/ kg (twice, per 24 h once). Before two hours of administration with TAA, each rat in group A and group B were given normal saline 1ml/100g weight, and the left were given the same volume/weight medicine respectively, then redone every 8h. From the first administration with TAA to the end of experiment, every rat was injected mixture solution contained 10% glucose 3ml, saline 1ml, and 20umol/l KC1 lml every 8h to avoid hypoglucose. At 30h of the experiment, electroencephalogram (EEG) examination was done in each group, and then all rats were sacrificed. During the experiment, the behaviors of all rats were observed closely. (2) Blood from eye socket was used to examine the concentrations of serum ALT and blood ammonia. (3) The permeability of blood-brain barrier (BBB) was detected by Evans Blue (EB) leakage method, and the ultrastruncture of BBB, which contained astroglia, was observed under electron microscopy. (4) The cerebrospinal fluid collected from cerebellommedullary cistern was used to determine the concentrations ofammonia. Cerebral cortexes homogenized were used to examine the contents of glutamine by high performance liquid chromatography. Whole brain tissues were used to determine the percentages of brain water content by dry-wet method. (5)The expressions of glial fibrillary acidic protein (GFAP) in cerebral cortex, which was an important structure protein of astroglia, were observed via immunocytochemistry. (6)The expressions of GLT-1mRNA in cerebral cortex, which was a kind of astroglia glutamate transporters, were detected by reverse transcription-coupled polymerase chain reaction (RT-PCR). (7) Cerebrospinal fluid of RuHuang Pill was prepared to effect astroglia CRL-2541 exposed to 5mM concentration of NH4C1. After 48h treatment, the MTT tests were performed to observe the effect of drug on the rate of CRL-2541 survival, immunocytochemistries were performed to examine the expressions of GFAP. Result (1) RuHuang pill could obviously ameliorative the disorders of behaviors and EEGs of rats with HE of type A, decline the concentrations of serum ALT and blood ammonia(P<0.01), increase survive rates. Three dose groups of RuHuang P更多还原