【作者】 朱洪怡；

【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HSOS模型的建立与评价目的：采用PBS、野百合碱、环磷酰胺、吗替麦考酚酯、云南三七、土三七小鼠灌胃法,比较各组小鼠HSOS发生率,以求成功构建稳定可复制的HSOS建模方法,并给予综合评价。方法：160只清洁级健康雌性昆明小鼠随机分为六组：PBS正常对照组、野百合碱组、环磷酰胺组、吗替麦考酚酯组、云南三七组和土三七组。分别予以野百合碱灌胃7天,环磷酰胺隔日灌胃30天,PBS、吗替麦考酚酯、云南三七、土三七灌胃30天后处死小鼠。检测血液中血细胞计数、肝功能情况；测肝重/体重；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。结果：1.野百合碱组、环磷酰胺组和土三七组分别有23只、5只和24只小鼠成模,PBS正常对照组、云南三七组和MMF组小鼠未诱导出HSOS。2.与PBS正常对照组比较,野百合碱组及土三七组小鼠体重有增加(P<0.05),其中以土三七组小鼠体重增加更为明显；环磷酰胺组小鼠体重明显下降(P<0.05)；而MMF组和云南三七组小鼠体重与PBS正常对照组无明显差异(P>0.05)。3.与PBS正常对照组对比,野百合碱组和土三七组小鼠血细胞中RBC及PLT计数下降,WBC计数增加(P<0.05)；云南三七组RBC计数稍有增加,WBC和PLT计数稍有减少,但差异并无统计学意义(P>0.05)；环磷酰胺组和MMF组小鼠均出现三系减少,且以环磷酰胺组减少得更为明显(P<0.05)。4.与PBS正常对照组比较,各模型组小鼠肝指数、ALT、AST、 TBIL均明显升高,ALB明显下降(P<0.05)；MMF组小鼠肝功能较PBS正常对照组亦存在一定程度损害,但明显轻于环磷酰胺组(P<0.05)；云南三七组小鼠肝功能变化情况与正常对照PBS正常对照组比较无明显差异(P>0.05)。5.根据修改后的Deleve评分标准,野百合碱组小鼠多为轻-中度HSOS改变,土三七组及环磷酰胺组小鼠多表现为中-重度HSOS。结论：1.野百合碱、环磷酰胺、土三七均能成功诱导小鼠HSOS模型,其中环磷酰胺法成模率低于野百合碱和土三七灌胃法,云南三七和MMF未能成功诱导出小鼠HSOS模型。2.野百合碱、土三七诱导的小鼠HSOS模型均符合人类HSOS病理改变,其中野百合碱可诱导急性HSOS模型,土三七可诱导慢性HSOS模型,为HSOS的进一步研究提供了理想动物模型。3.与野百合碱比较,土三七诱导的HSOS动物模型在进行药物建模机制、药物干预研究方面更为理想。4.与土三七比较,云南三七未能成功诱导出小鼠HSOS模型,说明云南三七无明显肝毒性,临床用药相对安全。5.与环磷酰胺比较,吗替麦考酚酯的骨髓抑制及肝毒性均较轻,是相对安全的免疫抑制剂。第二章强的松对土三七诱导HSOS的防治疗效观察目的：探讨不同剂量强的松对土三七诱导小鼠HSOS的防治作用。方法：115只清洁级健康雌性昆明小鼠随机分为四组：PBS正常对照组、土三七组、低剂量强的松干预组(5mg·kg-1·d-1)和高剂量强的松干预组(10mg·kg-1·d-1)分别灌胃30天后处死小鼠。检测血液中血细胞计数、肝功能情况；测肝重/体重；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；并用免疫组织化学法和RT-PCR法检测肝组织的TGF-β1和CTGF表达水平。结果：1.土三七组、低剂量强的松干预组和高剂量强的松干预组分别有24只、5只和7只小鼠成模,PBS正常对照组无一成模。2.与土三七组比较,两组强的松干预组小鼠RBC及PLT计数稍有增加,WBC计数减少,ALB下降明显轻于土三七组,TBIL增高程度亦低于土三七组(P<0.05),ALT和AST无明显差异,不同剂量干预组间无明显差异(P>0.05)；低剂量强的松干预组小鼠肝指数明显下降(P<0.01),但高剂量强的松干预组小鼠肝指数改变不明显(P>0.05)。3.根据修改后的Deleve评分标准,土三七组小鼠多表现为中-重度HSOS,强的松干预组多表现为轻-中度,无重度HSOS发生,其中以低剂量组平均评分更低(P>0.05)。4.肝组织TGF-β1和CTGF表达：免疫组化和RT-PCR均显示两组强的松干预组与土三七组比较,TGF-β1和CTGF表达明显减少(P<0.05),不同剂量干预组间无明显差异(P>0.05)。结论：1.强的松干预可降低土三七诱导的小鼠HSOS发生,改善小鼠一般情况和肝功能水平,减轻肝窦内皮细胞损害及肝窦纤维化等肝脏病理组织学损伤。2.与高剂量强的松(10mg·kg-1·d-1)比较,低剂量强的松(5mg·kg-1·d-1)干预土三七诱导的HSOS小鼠模型,在改善肝窦纤维化方面并无显著差异,但就血药浓度及远期副作用考虑,5mg·kg-1·d-1可能为更为理想的防治剂量。3. TGF-β1和CTGF的表达水平可反映HSOS的病情程度、病理改变及临床转归等情况,对观察疗效、评估预后等有重要意义。第三章强的松对土三七诱导HSOS防治机制的探讨目的：研究强的松对炎性因子及致纤因子的表达及调控机制,从ECM的合成与降解、参与肝窦纤维化形成的细胞因子角度探讨强的松作为HSOS治疗药物的可能性及作用途径和机制,为HSOS的治疗提供进一步的理论和实验依据。方法：115只清洁级健康雌性昆明小鼠随机分为四组：PBS正常对照组、土三七组、低剂量强的松干预组(5mg·kg-1·d-1)和高剂量强的松干预组(10mg·kg-1·d-1)分别灌胃30天后处死小鼠。应用RT-PCR法检测肝组织中MMP-1、TIMP-1、MCP-1、TNF-α、IL-1β和NF-κBp65mRNA的表达,western blot法检测TNF-α、TGF-β1和CTGF蛋白的表达。结果：1. RT-PCR结果：土三七组小鼠肝组织中MMP-1、TIMP-1、 MCP-1、TNF-α、IL-1β、NF-κBp65mRNA的表达较PBS正常对照组显著增高(P<0.01),强的松干预后肝组织中NF-κBp65、TNF-α、IL-1β、 MCP-1mRNA的表达较模型组下降(P<0.05)。与PBS正常对照组比较,土三七组MMP-1/TIMP-1显著减小(P<0.01),强的松干预后MMP-1/TIMP-1较土三七组明显上升(P<0.05)。不同剂量干预组间无明显差异(P>0.05)。2.Western blot结果：土三七组小鼠肝组织中TNA-α、TGF-β1和CTGF蛋白的表达较PBS正常对照组显著增高(P<0.01),强的松干预后肝组织中TNA-α、TGF-β1和CTGF蛋白的表达较模型组下降(P<0.05),且不同剂量干预组间无明显差异(P>0.05)。结论：强的松在土三七诱导HSOS模型过程中,可能通过下调核转录因子NF-κB的表达,调控趋化蛋白MCP-1和炎症因子TNF-α和IL-1p,进而减少HSC的活化,抑制TGF-β1、CTGF、TIMP-1合成,促进MMP-1降解ECM,降低SEC损害及ECM沉积,减轻炎症和纤维化,起到有效防治HSOS的作用。更多还原

【Abstract】 Part1Establishment and evaluation of mice model of hepatic sinusoidal obstruction syndrome induced by Gynura segetumObjective:To establish and evaluate mice model of hepatic sinusoidal obstruction syndrome (HSOS) induced by Gynura segetum. Phosphate buffer (PBS) was used as the control group. Mice models of HSOS 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:160female KunMing mice were randomly divided into six groups. PBS, monocrotaline, cyclophosphamide, mycophenolate mofetil (MMF), Yunnan Sanqi, and gynura segetum was intragastric administered. The mice in cyclophosphamide group were administered every other day, meanwhile other groups were administered every day. They were sacrificed on day30,7,30,30,30,30. The weight of the liver and body were measured; blood samples were collected to determine cytometry, alanine aminotransferase (ALT), aspartate aminotransferase (AST),albumin (ALB), total bilirubin(TBIL). Liver were sectioned and stained using HE and Masson to observe the pathological changes, and the changes on light microscopy were assessed by a modified Deleve scoring system.Results:1.23mice in group monocrotaline,5in group cyclophosphamide, and24in group gynura segetum developed HSOS. Mice in group PBS, Yunnan Sanqi and mycophenolate mofetil failed to show any manifestations of HSOS.2. Compared with the controls, mice in group monocrotaline and group gynura segetum showed increased the weight of body (P<0.05) especially in group gynura segetum. Meanwhile, the body weight were significantly decreased in group cyclophosphamide (P<0.05). There were no significant differences between the MMF and Yunnan Sanqi group compared with the controls (P>0.05)3. Compared with the controls, the value of WBC increased in group monocrotaline and gynura segetum, while the values of RBC and PLT reduced significantly (P<0.05). the value of RBC slightly increased in group Yunnan Sanqi, while the values of WBC and PLT reduced moderatly(P>0.05)without any statistical significance. Hypocytosis was found in mice gavaged with cyclophosphamide and mycophenolate mofetil, with more significant reduction in mice gavaged with cyclophosphamide (P<0.05)4. Compare with PBS group, the model groups had significant increased in the level of liver index, serum ALT, AST, TBIL and decreased in the level of serum ALB (P<0.05). Although, the liver function in group MMF had injured at a certain level compare with PBS group, obviously better than in group cyclophosphamide.5. Most of gynura segetum and cyclophosphamide group were in moderate or severe HSOS, while monocrotaline group were in mild or moderate HSOS by a modified Deleve scoring system.Conclusion:1. Monocrotaline, cyclophosphamide and gynura segetum could induce the model of HSOS successfully. The model induced by gynura segetum and monocrotaline were higher than cyclophosphamide. Yunnan Sanqi and MMF failed to induce the model of HSOS.2. The model induced by gynura segetum exhibited the clinical and histogical features of human HSOS, and this model may provide a new animal model experimental approach for the further study of the human HSOS. Monocrotaline could induce the acute HSOS model, while gynura segetum could induce the chronic ones.3. Compared with monocrotaline, gynura segetum induced more reliable animal models of HSOS in research of mechanism and effects of medicine.4. Compared with gynura segetum, Yunnan Sanqi had no hepatotoxicity, and more safe in clinical use.5. Compared with cyclophosphamide, MMF is considered to be a immunosuppressive safe drugs without obvious hepatotoxicity and bone marrow suppression.Part2Therapeutic effect of prednisone on mice model of hepatic sinusoidal obstruction syndrome induced by Gynura segetumObjective:To investigate the possible therapeutic effect of prednisone with different doses on HSOS induced by Gynura segetum.Methods:115female KM mice were randomly divided into4groups, they were gavaged with30ml·kg-1·d-1PBS (group A),30g·kg-1·d-1Gynura segetum (group B),30g·kg-1·d-1Gynura segetum+5mg·kg-1·d-1prednisone (groupC), or30g·kg-1·d-1Gynura segetum+10mg·kg-1·d-1prednisone (groupD). All the mice were sacrificed on the30day. Blood samples were collected to determine cytometry, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin(TBIL). Liver were sectioned and stained using HE and Masson to observe the pathological changes, and the changes on light microscopy were assessed by a modified Deleve scoring system. The expression of TGF-β1and CTGF was determined by immunohistochemical staining and RT-PCR.Results:1.24mice in group B,5in group C and7in group D developed HSOS. Compared with the controls, they had increased WBC、liver ratio、 TBIL、DBIL、ALT、AST and decreased RBC、PLT、ALB (P<0.05). Administration of prednisone improved the clinical signs and biochemistry parameters without dose-dependent manner.2. Most mice of group B developed moderate or severe HSOS, with those in group C and D were in mild or moderate HSOS by a modified Deleve scoring system. The value of group C was lower (P<0.05).3. Compared with group B, the expression of TGF-β1and CTGF decreased in groups C and D (P<0.05), and there was no significant difference between groups C and D (P>0.05). These changes were in accordance with the histopathologic changes.ConclusionPrednisone with different doses has therapeutic effect on HSOS, improving clinical signs and liver function and independent of dose. The expression of TGF-β1and CTGF is associated with the clinical course and severity of HSOS and response to treatment, so it can be an index for monitoring.Part3The possible mechanism of the effect of prednisone on mice models of HSOS induced by Gynura segetumObjective:To investigate the possible mechanism of the therapeutic effect of prednisone on HSOS induced by Gynura segetum.Methods:115female KM mice were randomly divided into4groups, they were gavaged with30ml·kg-1·d-1PBS (group A),30g·kg-1·d-1Gynura segetum (group B),30g·kg-1·d-1Gynura segetum+5mg·kg-1·d-1prednisone (groupC), or30g·kg-1·d-1Gynura segetum+10mg·kg-1·d-1prednisone (groupD). All the mice were sacrificed on the30day. The livers were harvested. The expression of MMP-1、TIMP-1、MCP-1、 TNF-α、IL-1β and NF-κBp65mRNA were determined by RT-PCR and the expression of TNF-α、TGF-β1and CTGF were determined by western blot.Results:RT-PCR:Compared with the controls, the expression of MMP-1, TIMP-1, MCP-1, TNF-a, IL-1β and NF-KBp65mRNA increased significantly in group B (P<0.01). Prednisone reduced the mRNA expression of MMP-1、TIMP-1、MCP-1、TNF-α、IL-1β and NF-κBP65in group C and D (P<0.05). Compared with the controls, the expression of MMP-1/TIMP-1decreased significantly in group B (P<0.01) while MMP-1/TIMP-1significantly increased (P<0.05). No significant difference between group C and D (P>0.05)Western blot:Compared with the controls, the expression of TNF-α、 TGF-β1and CTGF protein increased significantly in group B (P<0.01) Prednisone reduced the protein expression of TNF-α、TGF-β1and CTGF in group C and D (P<0.05). No significant difference between group C and D (P>0.05)Conclusion:Prednisone has therapeutic effect on HSOS, the possible mechanism may be that prednisone could reduce inflammation and fobrosis by down regulate the expression of NF-κB, reduce MCP-1, TNF-a and IL-1(3, thereby reducing the activation of HSC, inhibit synthesis of TGF-β, CTGF, TIMP-1, promote MMP-1degradating ECM, and reducing SEC damage and ECM deposition.更多还原