【作者】 李滨；

【导师】 吕志平；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 研究目的在不影响慢性肝纤维化病程自然发展前提下,分别阻断支配大鼠肝脏的交感神经（sympathetic nerve）、迷走神经（vagus nerve）以降低自主神经系统（autonomic nervous system）的活性,观察自主神经系统对肝卵圆细胞（hepaticoval cell,HOC）活化的调控作用。根据HOC活化细胞数同肝纤维化发展程度病理分级的关系,探讨HOC在慢性肝纤维化病程中所起的确切作用,为以HOC为靶细胞的治疗开拓思路。观察大黄蟅虫丸对慢性肝纤维化大鼠模型HOC的活化作用,了解其在治疗肝纤维化中的科学意义,为中医药治疗肝纤维化提供依据。研究方法1.动物分组:取雄性Wistar大鼠96只,体重180±20g,每组12只,随机分为8组,分别为:HOC活化抑制组（迷走神经肝支切断）,HOC活化增强组（化学性肝交感神经阻断）,肝纤维化模型假手术组,肝纤维化模型组,大黄蟅虫丸高剂量组（按成人5倍剂量）,大黄蟅虫丸中剂量组（按成人2.5倍剂量）,大黄蟅虫丸常规剂量组（按成人等效剂量）,空白对照组。各组给予普通饮食,自由饮水。2.预处理:HOC活化抑制组行迷走神经肝支切断术;HOC活化增强组以6-羟基多巴胺（6-hydroxydopamine,6-OHDA）建立肝脏去交感神经动物模型,具体方法:6-OHDA临用时用灭菌生理盐水配制成100ml/L的溶液。Wistar大鼠10%水合氯醛腹腔注射麻醉,固定于大鼠手术板上,腹部剪毛,局部消毒,于腹部正中纵行切开一长3cm切口,用镊子轻轻夹出小肠,理出空肠段系膜,找到肠系膜血管,用4号半头皮针刺入肠系膜主干静脉中,并同时注入2ml 6-OHDA（含200μg）。用棉签压迫针眼约8～10min,待针孔出血停止,将小肠还纳腹腔,逐层缝合腹壁,常规饲养;肝纤维化模型假手术组则于造模前行腹腔切开,随后手术缝合,术后常规护理。此三组处理均在肝纤维化造模前15天进行以尽量减少对实验结果的影响。3.肝纤维化模型的制作:除空白对照组外,其余各组以二甲基亚硝胺（N-Nitrosodimethylamine,DMN）10mg·kg^-1剂量大鼠腹腔注射,每周连续3天,每日1次,共4周。干预方法:大黄蟅虫丸高、中、常规剂量组在造模第一天开始中药干预。大鼠每日灌胃剂量:大黄蟅虫丸高、中、常规剂量组分别含生药3.0g/kg、1.5g/kg、0.6g/kg;其余各组以等量生理盐水灌胃作为对照。4.血清检测:于造模开始后第29日,大鼠以10%水合氯醛（3ml/kg）ip麻醉后,仰卧位固定,打开腹腔,腹主动脉采血,离心获取血清。用放射免疫RIA法检测血清透明质酸（hyaluronic acid,HA）、Ⅲ型前胶原（typeⅢprocollagen,PCⅢ）、层粘连蛋白（Laminin,LN）血清浓度变化。5.肝组织的采集与处理:腹主动脉采血后,脱颈椎处死大鼠。取0.3～0.4cm厚肝组织,40g/L中性甲醛液固定,24h后逐级酒精脱水,二甲苯透明,56℃石蜡包埋,6μm厚切片,用于免疫组化染色。取1.0cm×0.8cm×0.3cm大小肝组织,40g/L中性甲醛液固定,24h后逐级酒精脱水,二甲苯透明,56℃石蜡包埋,4μm厚切片,用于Mallory染色。取50～100mg肝组织放入Trizol中,置入超低温冰箱保存,用于实时荧光定量PCR检测。6.肝纤维化程度鉴定:应用Mallory三步染色法判断各组胶原纤维增生程度。判定标准:0期,正常肝脏,无明显胶原纤维增生;Ⅰ期,胶原纤维增生,中央静脉和门脉区有少量星状胶原纤维束放散,但无间隔形成;Ⅱ期,胶原纤维增生,中央静脉和门脉区结缔组织变厚,由此向四周伸出纤维索,形成不完全间隔;Ⅲ期,胶原纤维大量增生,有个别菲薄的完全间隔形成,或较厚的不完全间隔即将形成假小叶;Ⅳ期,形成的完全间隔较厚,假小叶大量形成。7.卵圆细胞的鉴定与计数:采用免疫组织化学染色,以OV-6作为HOC标志物。免疫组织化学样本制备采用两步法:大鼠肝组织切片脱蜡至水,PBS冲洗、浸泡5分钟,高压热抗原修复,3%H₂O₂室温室温孵育5～10分钟,加抗大鼠OV-6抗体（1:100稀释）孵育,置入湿盒中,4℃过夜;PBS冲洗,滴加二抗;PBS冲洗,加二氨基联苯胺（diaminobenzidine,DAB）显色5～30分钟。苏木精液复染细胞核1分钟;自来水冲洗,盐酸酒精分色1～2次;自来水泡洗5分钟;切片脱水、透明;中性树胶封片。PBS替代一抗作为空白对照染色。取肝组织免疫组化切片,光镜下（10×10）观察OV-6阳性染色细胞的分布,每张切片随机选择5个不同视野,在40×10放大倍数下,应用IPP6.0软件对阳性细胞染色面积进行统计,以5个不同视野面积计数的平均值为每张切片的计数值。8.统计方法:所有数据处理采用SPSS13.0软件完成,数据均以均数±标准差（（?）±s）表示。HOC活化抑制组、HOC活化增强组、肝纤维化模型假手术组、肝纤维化模型组、空白对照组间HOC细胞计数的比较,大黄蟅虫丸高、中、常规剂量组、肝纤维化组模型组、空白对照组对HOC活化的影响结果以及各组间血清学指标的比较,以及各组TIMP-1 mRNA、CTGF mRNA、Notch1 mRNA的差异表达均采用单向方差分析,组间比较如方差齐采用LSD法,方差不齐则采用Dunnett’s T3法;各组间肝纤维化病理分期的比较采用非参数检验之KruskalWallis检验。HOC增生与肝纤维化的相关分析采用Spearman相关分析法。结果一、自主神经系统及大黄蟅虫丸对肝纤维化病理分期的影响:HOC活化增强组（化学性交感神经阻断）纤维化程度较肝纤维化模型假手术组显著降低;HOC活化抑制组（迷走神经肝支切断）则较肝纤维化模型假手术组纤维化程度加重;肝纤维化模型假手术组与肝纤维化模型纤维化程度无差异。大黄蟅虫丸中剂量组纤维化程度明显低于肝纤维化模型组;大黄蟅虫丸高、常规剂量组纤维化程度低于肝纤维化模型组。二、自主神经系统及大黄蟅虫丸对血清HA的影响:HOC活化抑制组（迷走神经肝支切断）与肝纤维化模型假手术组比较无显著性差异（P=0.142）;与肝纤维化模型假手术组相比,HOC活化增强组（化学性交感神经阻断）HA水平显著降低（P=0.000）。与肝纤维化模型组相比,大黄蟅虫丸高剂量（P=0.003）、中剂量（P=0.000）、常规剂量组（P=0.004）均能显著降低HA水平。三、自主神经系统及大黄蟅虫丸对血清PCⅢ的影响:与肝纤维化模型假手术组相比,HOC活化抑制组（迷走神经肝支切断）PCⅢ水平显著升高（P=0.007）,HOC活化增强组显著（P=0.010）降低。与肝纤维化模型组相比,大黄蟅虫丸高剂量（P=0.040）、大黄蟅虫丸中剂量（P=0.003）可显著降低肝纤维化大鼠模型血清PCⅢ水平。四、自主神经系统及大黄蟅虫丸对血清LN的影响:与肝纤维化模型假手术组相比,HOC活化抑制组（迷走神经肝支切断）无显著性差异（P=0.148）;HOC活化增强组（化学性交感神经阻断）LN水平显著降低（P=0.001）。与肝纤维化模型组相比,大黄蟅虫丸中剂量（P=0.001）、常规剂量（P=0.048）可显著降低肝纤维化大鼠模型血清LN水平,大黄蟅虫丸高剂量组（P=0.146）差异无统计学意义。五、自主神经系统及大黄蟅虫丸对肝纤维化模型大鼠HOC增殖的影响:与肝纤维化模型假手术组相比,HOC活化抑制组（迷走神经肝支切断）、HOC活化增强组（化学性交感神经阻断）均有显著性差异（P=0.000）,HOC活化抑制与HOC活化增强分别能抑制、促进HOC的增生。与肝纤维化模型组相比,大黄蟅虫丸高、中、常规剂量组均可显著降低DMN肝纤维化大鼠模型HOC增殖（P<0.05）。六、HOC活化增殖与肝纤维分级的相关性分析1.自主神经系统对HOC活化增殖与肝纤维化分级相关性分析将HOC活化抑制组（迷走神经肝支切断）、HOC活化增强组（化学性交感神经阻断）、肝纤维化模型假手术组纳入统计范围。结果显示:r_p＝-0.776,P＝0.000,两者存在显著负相关关系。即随着HOC数量的增多,纤维化程度呈下降趋势。2.大黄蟅虫丸对HOC活化增殖与肝纤维化分级相关性分析将大黄蟅虫丸高、中、常规剂量组、肝纤维化模型组纳入统计范围。结果显示:r_p＝0.602,P＝0.002,两者存在显著正相关关系。即HOC活化数量越少,肝纤维化程度越低。七、自主神经系统及大黄蟅虫丸对肝纤维化模型大鼠TIMP-1 mRNA、CTGFmRNA、Notch1 mRNA表达的影响:与肝纤维化模型假手术组相比,HOC活化抑制组（迷走神经肝支切断）、HOC活化增强组（化学性交感神经阻断）TIMP-1m RNA、CTGF mRNA、Notch1 mRNA的表达均显著下调。与肝纤维化模型组相比,大黄蟅虫丸各剂量组TIMP-1m RNA、CTGF mRNA、Notch1 mRNA的表达均显著下调（P＜0.01）。结论一、交感神经系统（sympathetic nervous system,SNS）抑制剂（SNSIs）可以显著降低DMN肝纤维化大鼠纤维化程度,保护实验动物肝损伤;迷走神经阻断则加重DMN大鼠纤维化、损伤程度。二、SNSIs通过SNS对肝星状细胞（hepatic stellate cell,HSC）和HOC的直接调节影响对损伤肝脏的修复;推测大鼠的肝脏祖细胞可能表达M3型受体,迷走神经肝支通过乙酰胆碱（ACh）结合M3型受体刺激了卵圆细胞的激活。三、大黄蟅虫丸抗纤维化机制之一是抑制了TIMP-1的活性,降低了与MMPs的结合,使MMPs对ECM的降解作用增强;大黄蟅虫丸可以抑制HSC的增殖,促进MMPs对ECM的降解,体现了其“化瘀”的作用。四、大黄蟅虫丸抗纤维化的机制与下调纤维化病程中过度表达的CTGFmRNA有关。CTGF被抑制,其促ECM合成的作用减弱,有利于ECM降解,是“化瘀”的另一种体现;其表达细胞黏附因子的作用减弱,使细胞不易黏附和结合基质蛋白,脱离纤维趋化环境,此即中医“活血”思想的体现。五、大黄蟅虫丸通过下调Notch1 mRNA的表达诱导HOC向肝细胞方向分化。六、在慢性肝维化病理状态下,HOC的激活是一种机体自身适应性反应,便于肝脏的修复;但长期地处于这种状态,HOC的过度活跃、增生导致肝脏结构、功能破坏和肝癌的发生。更多还原

【Abstract】 ObjectiveThe activity of sympathetic nervous system（SNS） and parasympathetic nervous system（PNS） was separately inhibited in order to regulate the accumulation of HOC （hepatic oval cell） in liver fibrosis。Then.the relationship between the degree of activated HOCs and the liver fibrosis was evaluated to determine whether the HOC effects chronic liver fibrosis.HOC activation may be a useful therapeutic approach to cure liver fibrosis.To investigate how the dahuangzhechongwan affect the regulation of HOC in liver fibrosis.Methods1.Animals:96 male Wistar rats,180±20g,were randomly divided into 8 groups, each group contains 12 rats.respectively:the group of inhibited HOC activation （hepatic branch of vagus nerve cut）,the group of enhanced HOC activation（chemical hepatic sympathetic inhibition）,the group of liver fibrosis model with sham operation, the group of liver fibrosis model,the group of dahuangzhechongwan with conventional dose（according to the adult dose）,the group of dahuangzhechongwan with middle dose（2.5 times of the adult dose）,the group of dahuangzhechongwan with high dose（5 times of the adult dose）,control group.Each group was given ordinary feed,free from drinking water.2.Pretreatment:The group of inhibited HOC activation was cut the vagus nerve of liver branch with surgery;The group of enhanced HOC activation was given the 6-hydroxydopamine（6-OHDA） to set up chemical sympathectomy animal model,. Methods:6-OHDA was configured to 100mg/L solution with sterile saline before operation.Rats were anesthetized with 10%chloral hydrate anesthesia,then fixed on the surgery board.After shearing and disinfection,we make a long longitudinal incision about 3cm in the middle of abdomen,then pull out the intestine gently with tweezers.The mesenteric blood vessel was found out in jejunal segment mesenteric. Then the superior mesenteric vein was pierced with scalp acupuncture,at the same time,2ml 6-OHDA（containing 200μg） was injected into the vein.After oppressing on the eye of a needle with cotton for about 8～10min,the abdominal wall was sutured the layer-by-layer.Then these rats was bred conventional;Rats of liver fibrosis model with sham operation were treated by abdominal incision and exploratory laparotomy,followed by surgical suture,then was bred conventionally. These three group model was made 15 days before liver fibrosis model in order to minimize the impact on the experimental results.3.Liver fibrosis model:In addition to the control group,the other groups,were injected with DMN（dimethylnitrosamine） of 10mg.kg^-1 intraperitoneal according to the Matsuda’s method.Interventions:Dahuangzhechongwan conventional dose, middle dose and high dose group were intervened on the first day of making liver fibrosis model with the dose of 0.6g/kg,1.5g/kg,3.0g/kg corresponding.The other groups were bred conventionally.4.Serum testing:29 days after injection,all rats were anesthetized with 10% chloral hydrate anesthesia,fixed with the supine position,cut the abdominal cavity. Blood was collected from abdominal aorta,serum was collected through centrifugation.The difference in hyaluronic acid（hyaluronic acid,HA）,Ⅲprocollagen type（typeⅢprocollagen,PCⅢ）,laminin（Laminin,LN） was tested through radioimmunoassay RIA.5.Treatment of liver tissue:After the collection of blood,all rats were killed from cervical dislocation.A liver tissue about 0.3～0.4cm was choosed and fixed by formaldehyde solution of 40g/L.dehydrated through each grade of alcohol after 24h, dealed with dimethylbenzene,paraffin-embedded in 56℃.made a slice of 6μm thick for immunohistochemical staining.Liver tissue of 1.0cm×0.8cm×0.3cm was choosed and fixed by neutral formaldehyde solution of 40g/L,dehydrated through each level of alcohol after 24 h,dealed with dimethylbenzene,embedded in 56℃by paraffin, made a slice of 4μm for Mallory staining.Liver tissue of 50～100mg was mixed with Trizol in order to real time PCR.6.The stage of hepatic fibrosis:A three-step method of collagen fibers staining, Mallory was used to identified the degree of proliferation in each group.Criteria:0 period,normal liver,no significant proliferation of collagen fibers;Ⅰperiod, proliferation of collagen fibers is founded,there is a small amount of stellate collagen fibers in the central vein and portal area,but there is no fibrous septa;Ⅱperiod, proliferation of collagen fibers is founded,connective tissues in portal vein and central vein become thickening,which reach out fiber cables all-around,and become incomplete fibrous septa;Ⅲperiod,substantial proliferation of collagen fibers is founded,some of which forms thin complete fibrous septa,or some thick incomplete fibrous septa is about to forming pseudo lobule;Ⅳperiod,the complete fibrous septa is rather thick,a lot of pseudo lobules were found.7.Identification of the HOC:OV-6 was used to mark the HOC during the period of immunohistochemistry.A two-step sample preparation was used for immunohisto-chemistry: Liver tissue slices were fixed with acetone for 10mins,then dealed with H₂O₂ of 3ml/L at room temperature for 30min.The slices were incubated with Anti-rat OV-6 antibody（diluted by 1:100） in wet box,overnight at 4℃temperature; The slices were incubated with horseradish peroxidase（HRP） in wet box for 30mins at 37℃temperature.Then diaminobenzidine（DAB） was added onto it for 3～5mins. The nuclei were stained by hematoxylin for 10s.Sections were sealed by balsam neutral.Instead of first antibody,PBS was used to stain as blank control.We observe the rat liver tissue sections to distribution the cells with OV-6 immunohistochemical staining through light microscopy（10×10）.After five different fields of vision randomly selected we count the cells of positive staining at 40×10 magnification.The average of cell numbers in five different fields is the cell numbers of each section. 8.The statistics method:All data was processed by spss13.0,and was expressed as mean number±standard deviation（（?）±s ）.One Way ANOVA was used to analysis the difference between groups such as the activation of HOC.TIMP-1 mRNA,CTGF mRNA or Notch1 mRNA;Kruskal Wallis was used to analysis the difference of the stage of liver fibrosis.Spearman was used to analysis the relationship between the regulation of HOC and the stage of liver fibrosis.Results1.The statistics shows significant differences between all groups.Compared with group of liver fibrosis model with sham operation,either group of inhibited HOC activation or group of enhanced HOC activation has quite different degree of liver fibrosis.The group of inhibited HOC activation is more serious than the group of liver fibrosis model with sham operation,the group of enhanced HOC activation is less serious than it.The statistics shows no significant differences between the group of liver fibrosis model with sham operation and the group of liver fibrosis model. Compared with the group of liver fibrosis model,the groups of dahuangzhechongwan with different dose is less serious on the degree of liver fibrosis.2.Comparison of HA:In contrast with group of liver fibrosis model with sham operation,the group of enhanced HOC activation has quite different HA.Compared with group of liver fibrosis model,the group of dahuangzhechongwan with three different dose can reduce the degree of HA.3.Comparison of PCⅢ:In contrast with group of liver fibrosis model with sham operation,either group of inhibited HOC activation or group of enhanced HOC activation has quite different PCⅢ.The statistics shows significant differences between these three groups.Compared with the group of liver fibrosis model,either group of dahuangzhechongwan with middle dose or group of dahuangzhechongwan with high dose has quite different PCⅢ.4.Comparison of LN:Compared with the group of liver fibrosis model with sham operation,the group of enhanced HOC activation has less LN.The statistics shows significant differences between these two groups.In contrast with group of liver fibrosis model,either group of dahuangzhechongwan with middle dose or group of dahuangzhechongwan with conventional dose has quite different LN.5.Comparison of activated HOCs:In contrast with group of liver fibrosis model with sham operation,either group of inhibited HOC activation or group of enhanced HOC activation has quite different activated HOCs.The statistics shows significant differences between these three groups.The group of dahuangzhechongwan with middle dose has less activated HOCs than that of the group of liver fibrosis model.6.Spearman correlation analysis between HOC activation and liver fibrosis period:Overall correlation analysis taking into account with all groups except control group shows there is no correlation between HOC activation and liver fibrosis period, whose rp is -0.117 and the P is 0.459.Correlation analysis in the effects of sympathetic nervous system and parasympathetic nervous system（taking into account of group of inhibited HOC activation,group of enhanced HOC activation and group of liver fibrosis model with sham operation） shows there is obvious negative correlation between activated HOCs and liver fibrosis period,which is more activated HOCs,less liver fibrosis（rp＝-0.776, P＝0.000）.Correlation analysis in the effects of dahuangzhechongwan with different doses （taking into account three groups of dahuangzhechongwan with different doses and group of liver fibrosis model） show there is obvious positive correlation between activated HOCs and liver fibrosis period,which is less activated HOCs,less liver fibrosis（rp＝0.602,P＝0.002）.7.In contrast with group of liver fibrosis model with sham operation,either group of inhibited HOC activation or group of enhanced HOC activation has quite different TIMP-1 mRNA,CTGF mRNA,Notch1 mRNA.Compared with the group of liver fibrosis model,the group of enhanced HOC activation has quite different TIMP-1 mRNA,CTGF mRNA,Notch1 mRNA. Conclusion1.SNSIs can protect experimental animals from liver fibrosis.Inhibiting the hepatic branch of vagus nerve can increase the degree of fibrosis.2.SNS has direct impect on HSC and HOC,which will influence the liver repairement.Receptor Type M3 can also be expressed by HPC of rats.Vagus nerve affect the proliferation of HOC through M3 Recepter of Ach.3.Dahuangzhechongwan can inhibit the energy of TIMP-1 and reducing the combination with MMPs resulted in strenthing MMPs’S degration to ECM.The growth of HSC can be inhibited by dahuangzhechongwan.In that case,it can not only reduce the origin of TIMP-1 but also accelerate MMPs’s degration to ECM.4.Dahuangzhechongwan is related to CTGF mRNA which was over expressed though the process of adjustment the combination of ECM can be weakened through being inhibited CTGF,but it was good to degration which is another reflection of promoting blood circulation.5.Dahuangzhechongwan can induce HOC to diversify into liver cell through down-regulating the expression of Notch1 mRNA.6.The activation of HOC maybe an organic responsive reaction under the chronic liver fibrosis.Excessive activation of HOC may lead to cancer.更多还原