【作者】 陈哲；

【导师】 霍继荣；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 第一章土三七诱导小鼠肝小静脉闭塞病模型的建立及评价目的：建立土三七诱导的小鼠肝小静脉闭塞病(HVOD)模型。分别用土三七、野百合碱(MCT)、环磷酰胺(CTX)、吗替麦考酚酯(MMF)四种药物诱导小鼠HVOD模型,并用磷酸盐缓冲液(PBS)作为阴性对照组。对各模型组进行比较,以寻求良好的造模方法,为进一步探讨HVOD发病机制提供模型基础。方法：135只小鼠随机分为五组：土三七组、野百合碱组、环磷酰胺组、MMF组和正常对照组。分别予以野百合碱灌胃7天,土三七、环磷酰胺、MMF或PBS灌胃30天后处死小鼠。检测血清中ALT、AST、TBIL、DBIL、ALB及RBC、WBC、PLT计数；留取肝组织行HE染色、Masson染色,用修改后的Deleve评分标准进行病理学评分；用免疫组织化学法检测肝组织血管性血友病因子(vWF)的表达；并用RT-PCR法检测肝组织中内皮素-1(ET-1)、肿瘤坏死因子-a (TNF-a)、白细胞介素-1β(IL-1β) mRNA的表达。结果：1.土三七组、野百合碱组和环磷酰胺组分别有24只、23只和5只小鼠成模,正常对照组和MMF组小鼠未诱导出HVOD。2.与正常对照组比较,各成模小鼠肝重／体重、ALT、AST、TBIL、DBIL均明显升高,ALB明显降低,其差异有统计学意义(P<0.05)。3.土三七组及野百合碱组小鼠WBC计数较正常组升高,RBC、PLT较正常组明显降低(P<0.05)。环磷酰胺组和MMF组小鼠WBC、RBC、及PLT计数均较正常组有所下降(P<0.05),以环磷酰胺组下降更为明显,二组间差异有统计学意义(P<0.05)。4.根据修改后的Deleve评分标准,土三七组及环磷酰胺组成模小鼠多表现为中、重度HVOD,而野百合碱成模小鼠多表现为轻、中度HVOD。免疫组化结果显示：vWF在正常对照组和MMF组肝细胞和血管周围基本不表达,而各成模小鼠肝细胞及肝窦内皮细胞(SEC)胞浆vWF表达明显增多,以肝窦周围及中央静脉区尤为明显。5.RT-PCR法检测肝组织ET-1、TNF-α、IL-1βmRNA结果：土三七组和野百合碱组小鼠肝组织中ET-1、TNF-αIL-1βmRNA表达较正常对照组均明显增多(P<0.05),两组间表达水平无明显差异(P>0.05)。结论：土三七、野百合碱及环磷酰胺均能诱导小鼠HVOD模型,MMF未能诱导出HVOD模型。土三七诱导的小鼠HVOD模型成模率达80%,且为慢性模型,是更适于进一步研究HOVD发病机制的模型。各成模小鼠肝细胞及SEC胞浆中vWF表达明显增多,与文献报道一致。土三七及野百合碱中均含有吡咯烷类生物碱(PAs),二者诱导的HVOD模型中肝组织ET-1、TNF-αIL-1β表达明显增多,提示内皮损伤可能参与了PAs诱导的HVOD的发生发展过程。与环磷酰胺相比,MMF对骨髓的抑制及肝功能的损害程度均较轻,是一种更为安全、理想的免疫抑制剂。第二章川芎嗪对土三七诱导的小鼠肝小静脉闭塞病防治作用的观察目的：探讨不同剂量川芎嗪对土三七诱导的小鼠HVOD的预防性治疗作用。方法：115只小鼠随机分为土三七组、低剂量川芎嗪干预组(100mg·kg-1·d-1)、高剂量川芎嗪干预组(200mg·kg-1·d-1)和正常对照组,灌胃30天后处死小鼠。留取血清及肝组织。测肝重/体重；检测血清ALT、AST、TBIL、DBIL、ALB水平；对肝组织进行HE染色和Masson染色,用修改后的Deleve评分标准进行病理学评分；并用免疫组织化学法检测肝组织纤溶酶原激活物抑制因子-1 (PAI-1)的表达水平。结果：1.土三七组、低剂量川芎嗪干预组和高剂量川芎嗪干预组各有24只、6只、2只小鼠发生HVOD。与土三七组比较,各干预组小鼠肝重／体重,血清ALT、AST、TBIL、DBIL有所下降,ALB有所升高,其差异有统计学意义(P<0.05)。以高剂量干预组改善更为明显,两干预组间差异有统计学意义(P<0.05)。2.各组小鼠肝脏病理评分结果：根据修改后的Deleve评分标准,土三七组小鼠多表现为中、重度HVOD;低剂量川芎嗪干预组多表现为轻度HVOD,仅1只为重度；高剂量川芎嗪干预组2只成模小鼠表现为轻中度HVOD。3.肝组织PAI-1免疫组化结果：土三七组小鼠肝组织中PAI-1表达水平较正常对照组明显增高。与土三七组比较,各干预组PAI-1表达水平均明显降低(P<0.05),以高剂量干预组下降更为明显,两干预组间差异有统计学意义(P<0.05)。结论：1.川芎嗪对土三七诱导的小鼠HVOD有一定的防治作用,能减轻小鼠的临床症状、改善其肝功能,从而延缓病情的进展。高剂量川芎嗪组(200mg·kg-1·d-1)的防治效果较低剂量组(100mg·kg-1·d-1)更为明显。2.各组小鼠肝组织中PAI-1表达水平与肝脏的病变轻重相关,提示PAI-1可作为HVOD病情严重程度和疗效的监测指标。第三章川芎嗪对土三七诱导的小鼠肝小静脉闭塞病防治机制的探讨目的：通过检测和比较各组小鼠肝组织中组织因子(TF)、早期生长反应因子-1 (Egr-1)、核因子-KB p65 (NF-KBp65)在mRNA和蛋白水平的表达来探讨川芎嗪防治土三七诱导的小鼠HVOD的可能机制。方法：115只小鼠随机分为土三七组、低剂量川芎嗪干预组(100mg·kg-1·d-1)、高剂量川芎嗪干预组(200mg·kg-1·d-1)和正常对照组,灌胃30天后处死所有小鼠。应用RT-PCR法和western blot法分别检测肝组织中TF及其下游调节因子Egr-1、NF-K Bp65 mRNA和蛋白水平的表达。结果：1. RT-PCR法检测肝组织TF、Egr-1、NF-KBp65mRNA表达结果：土三七组TF、Egr-1、NF-KBp65mRNA表达水平较正常对照组明显升高(P<0.05),各干预组的表达较土三七组均有所下降(P<0.05),以高剂量川芎嗪干预组(200mg·kg-1·d-1)下降更为明显。2. Western blot法检测肝组织TF、Egr-1、NF-KBp65蛋白表达结果：土三七组TF、Egr-1、NF-KBp65蛋白表达水平较正常对照组均明显增高(P<0.05),各干预组的表达较土三七组均有所下降(P<0.05),以高剂量川芎嗪干预组(200mg·kg-1·d-1)下降更为明显。结论：1.川芎嗪可能通过下调肝组织中TF的表达,抑制凝血系统活性的异常,从而达到防治HVOD的效果,200mg·kg-1·d-1的效果优于100mg·kg-1·d-1。2.川芎嗪干预后小鼠肝组织中Egr-1、NF-KBp65表达量减少,提示川芎嗪可能通过下调转录因子Egr-1、NF-KBp65的表达来降低体内TF水平,200mg·kg-1·d-1的下调作用更为明显。更多还原

【Abstract】 Part 1 Establishment and evaluation of mice model of hepatic veno-occlusive disease induced by Gynura segetumObjective:To establish and evaluate mice model of hepatic veno-occlusive disease (HVOD) induced by Gynura segetum. Phosphate buffer (PBS) was used as the control group. Mice models of HVOD induced by Gynura segetum, monocrotaline, cyclophophamide and mycophenolate mofetil were evaluated in order to choose a more favourable model for further research about the disease.Methods:135 female Kunming (KM) mice were randomly classified into 5 groups, they were gavaged with 30g·kg-1·d-1 Gynura segetum(group A),150mg·kg-1·d-1 monocrotaline(group B),30mg·kg-1·d-1 cyclophosphamide(group C),100mg·kg-1·d-1 mycophenolate mofetil(group D) or 30ml·kg-1·d-1 PBS (group E). Mice were sacrificed 7 days (group B) or 30 days (group A、C、D、E) later. Blood samples were collected, liver function and blood cell counts were measured. Histopathologic changes of liver tissues were assessed by a modified Deleve scoring system. The expression of vWF was detected by immunohistochemistry while the expression of ET-1、TNF-aand IL-1βmRNA were investigated by RT-PCR.Results:1.24 mice in group A,23 in group B and 5 in group C developed HVOD. Mice in group D and E failed to show any manifestations of HVOD.2.Compared with the controls, mice with HOVD showed increased liver ratio、serum total bilirubin(TBIL)、direct bilirubin(DBIL)、transaminase and decreased albumin(ALB) (P<0.05). There were no significant differences between the models. Compared with the controls, the value of WBC increased in group A and B, while the values of RBC and PLT reduced significantly. Cytopenia was found in mice gavaged with cyclophosphamide and mycophenolate mofetil, with more significant reduction in mice gavaged with cyclophosphamide (P<0.05).3.When evaluating with modified scoring system, most of the mice in group A and C belonged to moderate or severe HVOD, and most of the mice in group B belonged to mild or moderate HVOD.4.1mmunohistochemical staining demonstrated high dense deposition of vWF in liver tissues with HVOD. Moreover, when compared with the controls, the expression of ET-1、TNF-a and IL-1βmRNA were increased significantly in mice with HVOD (P<0.05)Conclusion:Compared with cyclophosphamide, Gynura segetum and monocrotaline induced more reliable models of HVOD. The increased expression of ET-1、TNF-a and IL-1βsuggested that injury to sinusoidal endothelial cells and hepatocytes may be involved in the development of HVOD associated with pyrrolizidine alkaloids. As one of the most frequently used immunosuppressive drugs in solid organ transplant, mycophenolate mofetil is considered to be a safe agent without obvious toxicity to liver and myelosuppression. Part 2 Therapeutic effect of ligustrazine on mice model of hepatic veno-occlusive disease induced by Gynura segetumObjective:To investigate the possible therapeutic effect of ligustrazine with different doses on HVOD induced by Gynura segetum.Methods:115 female KM mice were randomly divided into 4 groups, they were gavaged with 30g·kg-1·d-1 Gynura segetum (group A), 30g·kg-1·d-1 Gynura segetum+100mg·kg-1·d-1 ligustrazine (group B), 30g·kg-1·d-1 Gynura segetum+200mg·kg-1·d-1 ligustrazine (groupC) or 30ml·kg-1·d-1 PBS (groupD).30 days later, all the mice were sacrificed. Blood samples and livers were harvested. Weight ratio of liver to body and liver function were measured. Liver sections were evaluated by light microscopy and the expression of PAI-1 was determined by immunohistochemical staining.Results:1.24 mice in group A,6 in group B and 2 in group C developed HVOD. Compared with the controls, they had increased liver ratio、TBIL、DBIL、ALT、AST and decreased ALB (P<0.05). Administration of ligustrazine improved the clinical signs and biochemistry parameters in a dose-dependent manner. Compared with group A, the value of scoring in microscopy decreased in groups B and C.2. Compared with group A, the expression of PAI-1 decreased in groups B and C (P<0.05), and there were significant differences between groups B and C (P<0.05). These changes were in accordance with the histopathologic changes.Conclusion Ligustrazine with different doses has therapeutic effect on HVOD, improving clinical signs and liver function in a dose -dependent manner. The expression of PAI-1 is associated with the clinical course and severity of HVOD and response to treatment, so it can be an index for monitoring.Part 3 The possible mechanism of the effect of ligustrazine on mice models of hepatic veno-occlusive disease induced by Gynura segetumObjective:To investigate the possible mechanism of the therapeutic effect of ligustrazine on HVOD induced by Gynura segetum.Methods:115 female KM mice were randomly divided into 4 groups, they were gavaged with 30g·kg-1·d-1 Gynura segetum (group A), 30g·kg-1·d-1 Gynura segetum+100mg·kg-1·d-1 ligustrazine (group B), 30g·kg-1·d-1 Gynura segetum+200mg·kg-1·d-1 ligustrazine (groupC) or 30ml·kg-1·d-1 PBS (groupD).30 days later, all the mice were sacrificed and livers were harvested. The expression of TF、Egr-1、NF-K Bp65 were determined by RT-PCR and western blot.Results:Compared with group D, the expression of TF、Egr-1、NF-KBp65 increased significantly in group A (P<0.05).Administration of ligustrazine reduced the expression of TF、Egr-1、NF-K Bp65 in a dose-dependant manner, these findings were confirmed by both RT-PCR and western blot.Conclusion:Ligustrazine has therapeutic effect on HVOD, the possible mechanism may be that ligustrazine could reduce the expression of TF by down regulate the expression of transcription factors:Egr-1 and NF-KBp65.更多还原