蕲艾提取液对肝纤维化大鼠TGFβ-Smad信号转导通路的影响
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摘要
研究背景与目的：
肝纤维化是肝脏对不同病因所致的慢性损害产生的创伤愈合反应,表现为肝脏细胞外基质各种成分的过度沉积及分布异常,是纤维增生和纤维降解不平衡的结果,是多种慢性肝病向肝硬化发展的中间病理阶段,目前的研究认为肝纤维化病变经过适当的积极治疗有希望延缓甚至逆转,而发展到肝硬化阶段则很难逆转。
中医药治疗肝纤维化具有多靶点、多环节、多途径复合作用的特点,针对肝纤维化发病的复杂机制,体现了中医药的传统优势。蕲艾是湖北省的道地药材,临床研究发现蕲艾有明显的抗肝纤维化的作用,本课题通过建立免疫性肝纤维化动物模型,初步了解蕲艾提取液抑制大鼠肝纤维化的作用,并通过其对大鼠肝组织中TGFβ-Smad信号转导通路中关键的信号传导分子TGFβ1、Smad 3、Smad 4、Smad 7表达水平的变化,以及对抗凋亡基因Bcl-2和细胞周期蛋白的影响,探讨蕲艾提取液抗肝纤维化的分子机理。
方法：
1.采用猪血清诱导肝纤维化模型,将72只Wistar大鼠适应喂养1周后,随机分为正常对照组、肝纤维化模型组、蕲艾提取液低剂量、中剂量和高剂量组及丹参治疗组,每组12只。除正常对照组外,其余各组腹腔注射无菌猪血清0.3ml,每周2次,共10周。蕲艾提取液高、中和低剂量治疗组分别给予6ml/只、3ml/只和1.5ml/只的蕲艾提取液灌胃,每日1次；丹参治疗组给予丹参注射液6ml/只灌胃,每日1次；模型组给予生理盐水6ml/只灌胃,每日1次；正常对照组大鼠按100g体重给予生理盐水0.3ml腹腔注射,每周2次,共10周,并给予生理盐水6ml/只灌胃,每日1次。于10周末最后一次注射猪血清后48h,断头处死各组动物,眼球取血留取足够的血,分离血清,并取一部分肝脏组织,4%福尔马林固定,常规石蜡包埋切片。一部分新鲜肝组织梯度置于-70℃保存,用于实时荧光定量反转录聚合酶链式反应(RT-PCR)法及蛋白印迹实验(Western blot)。
2.血清学检测：各组实验大鼠血清分别检测肝纤维化血清学指标透明质酸(HA)、层粘蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型前胶原(IV-C)的含量,以及脂质过氧化指标超氧化物歧化酶(SOD)、丙二醛(MDA)O ELISA法检测血清中TGFβ1含量。
3.肝组织学检测：采用HE染色和Masson三重胶原染色法观察大鼠肝组织病理学变化。
4.免疫组织化学检测：采用免疫组化SABC法,结合显微镜,观察大鼠肝组织TGFβ1、Smad3、Smad4、Smad7以及Bcl-2、Cyclin D1的表达变化。
5.采用实时荧光定量反转录聚合酶链式反应(RT-PCR)法及蛋白印迹实验(Western blot)分别检测TGFβ1、Smad3、Smad4、Smad7 mRNA和蛋白及Bcl-2、cyclinDl mRNA和蛋白的表达变化。
结果
1.蕲艾提取液抑制大鼠肝纤维化作用的实验研究
与正常对照组比较,模型组中肝纤维化血清学指标HA、PCⅢ、LN、IV-C含量均明显升高(P<0.01),血清TGF-β1水平亦明显升高(P<0.05),血清MDA含量上升、SOD活性显著降低(P<0.01)；与模型组比较,蕲艾提取液治疗各组HA、PCⅢ、LN、IV-C含量均显著降低(P<0.05),血清TGF-β1含量亦明显下降(P<0.05),血清脂质过氧化指标MDA含量显著降低(P＜0.05), SOD活性显著升高(P<0.05),且蕲艾提取液治疗组与正常对照组无显著性差异(P>0.05)。
光镜下观察：模型组大鼠可见肝组织结构部分破坏,肝细胞发生广泛的变性水肿,水样变性甚至气球样变性,还可见程度、范围不等的肝细胞坏死；肝板排列紊乱,部分肝小叶失去正常结构；汇管区纤维隔内有大量嗜酸性细胞、单核淋巴细胞及成纤维细胞浸润,大量胶原沉积,有较明显纤维组织增生,胶原纤维沿汇管区及中央静脉周围向肝小叶内延伸,可见相互连接的纤维分割肝小叶,甚至有部分区域有早期肝硬化表现。蕲艾提取液及丹参治疗各组大鼠肝脏在色泽、质地、大小、表面光滑度方面较模型组有明显改善,细胞变性和坏死程度显著减轻,肝小叶结构破坏不明显,炎性细胞浸润和纤维组织增生减少,中央静脉和汇管区有少量纤维组织延伸,不同剂量蕲艾提取液治疗组间无显著性差异。
2.蕲艾提取液对大鼠肝组织中TGFβ-Smad信号转导通路蛋白表达的影响
免疫组织化学结果显示,TGFβ1、Smad3、Smad4在正常对照组肝细胞内无阳性细胞表达,模型组肝内表达明显增强,主要见于间质细胞、炎性细胞、损伤的肝细胞,阳性物质主要集中在细胞膜上,胞质内有少量表达；蕲艾提取液各剂量组及丹参组阳性细胞数目明显减少,阳性染色程度明显减轻。
RT-PCR及Western blot检测结果显示,TGFβ1、Smad3、Smad4 mRNA及其蛋白,在正常对照组中表达明显低下,而Smad7 mRNA及其蛋白表达则明显升高；蕲艾提取液高、中、低剂量组TGFβ1、Smad 3、Smad4 mRNA及其蛋白与模型组比较表达逐渐减少,Smad7 mRNA及其蛋白表达与模型组比较则逐渐升高,其中中、高剂量组TGFβ1、Smad3、Smad7 mRNA及其蛋白与模型组比较有显著性差异(P<0.05,P<0.01),而与丹参组比较无显著性差异(P>0.05)。
3.蕲艾提取液对抗凋亡基因Bcl-2及细胞周期蛋白Cyclin D1表达的影响
免疫组织化学结果显示,Bcl-2及Cyclin D1蛋白在正常对照组肝细胞内无阳性细胞表达,模型组肝内表达明显增强,主要见汇管区、肝细胞、间质细胞膜上及细胞浆内,阳性物质主要集中在细胞膜上；蕲艾提取液各剂量组及丹参组阳性细胞数目明显减少,阳性染色程度明显减轻。
RT-PCR及Western blot检测结果显示,正常对照组Bcl-2、Cyclin D1 mRNA及蛋白表达较模型组低下,蕲艾提取液各剂量组Bcl-2、Cyclin D1 mRNA及蛋白表达较模型组逐渐减少,其中高剂量组与模型组比较有显著性差异(P<0.05),与正常对照组和丹参组比较无显著性差异(P>0.05)。
结论
1.蕲艾提取液可以明显改善猪血清诱导的大鼠免疫性肝纤维化的肝组织病理学损害,减轻纤维组织增生,明显减少胶原纤维间隔、肝细胞坏死、变性以及炎性细胞的浸润,并能有效降低肝纤维化血清学指标,其抗纤维化的机理可能与抗脂质过氧化有关。
2.蕲艾提取液能够显著抑制大鼠肝组织中TGFβ1、Smad3、Smad4 mRNA及其蛋白表达水平,上调Smad7 mRNA及其蛋白表达水平,表明其可能通过有效调控TGFβ1-Smad信号转导通路,从而抑制大鼠肝纤维化。
3.蕲艾提取液能够降低大鼠肝组织中抗凋亡基因Bcl-2 mRNA及其蛋白表达水平,提示其可能通过有效诱导大鼠HSC凋亡,从而达到抗肝纤维化的作用。
4.蕲艾提取液可能通过下调大鼠肝组织中细胞周期蛋白Cyclin D1 mRNA及其蛋白表达水平,发挥对细胞周期的调控,抑制细胞增殖,而达到抗肝肝纤维化的作用。
Background and Objective:
Hepatic fibrosis is regarded as a pathologically repair reac-tion followed various kinds of chronic liver diseases. Fibrosis mainly presents the imbalance of the ECM compound production and degradation, which makes the ECM deposit pervadingly in liver. Hepatic fibrosis is a common stage of most chronic liver diseases regardless of the etiology, and its progression may lead to hepatic cirrhosis or hepatocelluar carcinoma (HCC). Although hepatic fibro-sis is thought to be a reversible pathological state, there is no established effective therapy for hepatic fibrosis yet.
Because of its special feature of poly-target, poly-lint, poly-channel, and its compound effects, the Chinese medicine can aim to the complicated mechanisms of hepatic fibrosis accordingly, which makes it have traditional and elearly prevalence against the western medicine. The topic is through the establishment of immune animal model of hepatic fibrosis, to get a preliminary understanding the inhibition of Artemisia argyi extracting solution on rat hepatic fibrosis, and through its rat liver tissue TGFβ-Smad signal transduction pathway in the key signal transduction molecules TGFβ1, Smad 3, Smad 4, Smad 7 expression level changes, as well as against the apoptosis gene bcl-2 and cell cycle proteins, to explore Artemisia argyi extracting solution the molecular mechanism of anti-fibrosis.
Methods:
1. Use of pig serum-induced liver fibrosis model,72 Wistar rats were fed 1 week after adaptation,were randomly divided into normal control group, liver fibrosis model group, high dose, middle dose and low-dose treatment of Artemisia argyi extracting solution grou-ps,12 rats in each. In addition to the normal control group, the rest of the rats by intraperitoneal injection of sterile body weight 100g pig serum 0.3ml,2 times per week, a total of 10 weeks,20 times. Artemisia argyi extracting solution large, medium and low-dose treatment group were given 6ml/only,3ml/only and 1.5ml/only with Artemisia argyi extracting solution fed to pray daily 1; model group were given 6ml/only normal saline gavage, every day 1 times; the normal control group was given intraperitoneal injection of saline,
2 times per week,10 weeks a total of 20 times, and to give 6ml/only normal saline gavage, day 1 times. At 10 over the weekend after the last injection of pig serum 48h, animals in each group were decapitated, eyes, enough to take blood specimens from blood, separa-tion of serum, and take part of the liver,4% formalin-fixed, conven-tional paraffin-embedded sections.Part of the gradient of fresh liver tissue placed in -70℃to save, for real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR) method and protein blot (Western blot).
2. Serological test:The rats in each group were detected in serum indicators of liver fibrosis serum hyaluronic acid (HA),laminin (LN),Ⅲprocollagen (PCⅢ),Ⅳprocollagen (Ⅳ-C)content, as well as indicators of lipid peroxidation superoxide dismutase(SOD), malondialdehyde (MDA). ELISA, serum TGFβ1 levels。
3. Liver histology tests:using HE staining and Masson staining of collagen triple-pathological changes in rat liver.
4.Immunohistochemistry:using of immunohistochemistry SABC method, combined with microscope to observe the rat liver tissue TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl of expression.
5. Using real-time fluorescent quantitative reverse transcription-poly mer-ase chain reaction (RT-PCR) method and Western blot test (Western blot) were measured TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl mRNA and protein expression.
Results:
1. Artemisia argyi extract solution inhibited rat liver fibrosis in rats.
Compared with the normal control group, the serum markers of liver fibrosis HA,PCⅢ,LN,Ⅳ-C levels of model group,were signi-ficantly higher(P＜0.01), serum TGF-β1 levels are significantly higher (P＜0.05), increase in serum MDA concentration, SOD activity was significantly decreased(P＜0.01);with the model group,Artemi-sia argyi extracting solution treatment for all groups HA, PCIII, LN,Ⅳ-C levels were significantly lower (P＜0.05), serum TGF-β1 content also decreased significantly(P＜0.05), serum indicators of lipid peroxidation MDA contents were significantly lower(P＜0.05), SOD activity was significantly increased (P＜0.05), and Artemisia argyi extracting solution treatment group and normal control group had no significant difference (P＞0.05).
Optical microscope observation:model group can be seen part of the organizational structure of liver damage, liver cells a wide range of variability of edema, water balloon-like degeneration or degeneration, but also shows the extent, scope, ranging from liver cell necrosis; liver disorder board arrangement, some of loss of normal structure of hepatic lobule; periportal fibrous septum in a large number of eosinophils, monon-uclear cell infiltration of lymphocytes and fibroblasts, a large number of collagen deposition, there are more obvious fibrous tissue hyperplasia, collagen fibers along the periportal areas and around the central vein to the hepatic lobule within the extension,we can see the connection between fiber split hepatic lobule, and even some regions the performance of early liver cirrhosis. Artemisia argyi extracting solution therapy in liver of rats in each group in the color, textu-re, size, surface smoothness in a significant improvement compared with model group, cell degeneration and necrosis significantly reduced hepatic lobule was not obvious structural damage, inflame-matory cell infiltration and fibrous tissue reduced proliferation, the central veins and periportal extension of a small amount of fibrous tissue, different doses of Artemisia argyi extracting solution between the treatment group no significant difference.
2. Artemisia argyi extracting solution expect rat liver tissue TGFβ-Smad signal transduction pathway protein expression.
Immunohistochemistry results showed that, TGFβ1, smad3, smad4 liver cells in the normal group no positive cells, liver expression was significantly increased in model group, mainly seen in interstitial cells, inflammatory cells, injury of liver cells, the positive substances mainly concentrated on the membrane, there is a little expression in the cytoplasm; Artemisia argyi extracting solution each dose group significantly reduced the number of positive cells, positive staining is reduced significantly.
RT-PCR and Western blot test results showed, TGFβ1, smad3, smad4 mRNA and protein expression in the normal control group, significantly lower, while the smad7 mRNA and protein expression was significantly increased; Artemisia argyi extracting solution high, medium and low dose group TGFβ1, smad3, smad4 mRNA and protein expression compared with model group gradually reduced, smad7 mRNA and protein expression compared with model group were increased gradually, in which middle and high dose group TGFβ1, smad3, smad7 mRNA and protein and model group and there was significant difference (P＜0.05, P＜0.01), compared with the SM group was no significant difference (P＞0.05). 3. Artemisia argyi extracting solution against apoptosis gene Bcl-2 and cell cycle protein expression of cyclinDl.
Immunohistochemistry results showed that, Bcl-2 and cyclinDl protein in normal liver cells there is no positive cells, liver expression was significantly increased in model group, mainly seen in portal area,hepatic cells and stromal cells'membrance and cytoplasm, positive substance is mainly concentrated in the membr-ance; Artemisia argyi extracting solution each dose group signifi-cantly reduced the number of positive cells, positive staining is reduced significantly.
RT-PCR and Western blot test results showed that the normal control group, Bcl-2, CyclinDl mRNA and protein expression compared with model group, low, Artemisia argyi extracting solution every dose group Bcl-2, CyclinD1 mRNA and protein expression compared with model group gradually reduced, which high-dose group compared with the model group were significantly different (P＜0.05), with the normal group and the SM group showed no significant difference (P＞0.05).
Conelusions:
1. Artemisia argyi extracting solution can significantly improve the rat liver fibrosis histopathological damage induced by immune pig serum, reduce fibrous tissue proliferation and decreased collagen fibers interval,liver cell necrosis,degeneration,and inflammatory cell infiltration,and can effectively reduce serum markers of liver fibrosis and its mechanism may be anti-fibrosis and anti-lipid peroxidation, reducing the concentration of serum TGF-β1.
2. Artemisia argyi extracting solution can significantly inhibit rat liver tissue TGFβ1, Smad3, Smad4 protein and mRNA expression levels, increase the level of Smad7 protein and mRNA expression, indicating that it can effectively regulate TGFβ-Smad signal transduction pathway, thereby suppressing rat liver fibrosis.
3. Artemisia argyi extracting solution can reduce the rat liver tissue of anti-apoptotic gene Bcl-2 protein and mRNA expression levels, suggesting that it can effectively induce apoptosis in rat HSC to achieve the anti-fibrosis.
4. Artemisia argyi extracting solution could be reduced in rat liver tissue of cyclin CyclinDl protein and mRNA expression levels to play on cell cycle regulation, so that stagnation in the G0/G1 phase cell replication, inhibit cell proliferation.
肝纤维化是肝脏对不同病因所致的慢性损害产生的创伤愈合反应,表现为肝脏细胞外基质各种成分的过度沉积及分布异常,是纤维增生和纤维降解不平衡的结果,是多种慢性肝病向肝硬化发展的中间病理阶段,目前的研究认为肝纤维化病变经过适当的积极治疗有希望延缓甚至逆转,而发展到肝硬化阶段则很难逆转。
中医药治疗肝纤维化具有多靶点、多环节、多途径复合作用的特点,针对肝纤维化发病的复杂机制,体现了中医药的传统优势。蕲艾是湖北省的道地药材,临床研究发现蕲艾有明显的抗肝纤维化的作用,本课题通过建立免疫性肝纤维化动物模型,初步了解蕲艾提取液抑制大鼠肝纤维化的作用,并通过其对大鼠肝组织中TGFβ-Smad信号转导通路中关键的信号传导分子TGFβ1、Smad 3、Smad 4、Smad 7表达水平的变化,以及对抗凋亡基因Bcl-2和细胞周期蛋白的影响,探讨蕲艾提取液抗肝纤维化的分子机理。
方法：
1.采用猪血清诱导肝纤维化模型,将72只Wistar大鼠适应喂养1周后,随机分为正常对照组、肝纤维化模型组、蕲艾提取液低剂量、中剂量和高剂量组及丹参治疗组,每组12只。除正常对照组外,其余各组腹腔注射无菌猪血清0.3ml,每周2次,共10周。蕲艾提取液高、中和低剂量治疗组分别给予6ml/只、3ml/只和1.5ml/只的蕲艾提取液灌胃,每日1次；丹参治疗组给予丹参注射液6ml/只灌胃,每日1次；模型组给予生理盐水6ml/只灌胃,每日1次；正常对照组大鼠按100g体重给予生理盐水0.3ml腹腔注射,每周2次,共10周,并给予生理盐水6ml/只灌胃,每日1次。于10周末最后一次注射猪血清后48h,断头处死各组动物,眼球取血留取足够的血,分离血清,并取一部分肝脏组织,4%福尔马林固定,常规石蜡包埋切片。一部分新鲜肝组织梯度置于-70℃保存,用于实时荧光定量反转录聚合酶链式反应(RT-PCR)法及蛋白印迹实验(Western blot)。
2.血清学检测：各组实验大鼠血清分别检测肝纤维化血清学指标透明质酸(HA)、层粘蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型前胶原(IV-C)的含量,以及脂质过氧化指标超氧化物歧化酶(SOD)、丙二醛(MDA)O ELISA法检测血清中TGFβ1含量。
3.肝组织学检测：采用HE染色和Masson三重胶原染色法观察大鼠肝组织病理学变化。
4.免疫组织化学检测：采用免疫组化SABC法,结合显微镜,观察大鼠肝组织TGFβ1、Smad3、Smad4、Smad7以及Bcl-2、Cyclin D1的表达变化。
5.采用实时荧光定量反转录聚合酶链式反应(RT-PCR)法及蛋白印迹实验(Western blot)分别检测TGFβ1、Smad3、Smad4、Smad7 mRNA和蛋白及Bcl-2、cyclinDl mRNA和蛋白的表达变化。
结果
1.蕲艾提取液抑制大鼠肝纤维化作用的实验研究
与正常对照组比较,模型组中肝纤维化血清学指标HA、PCⅢ、LN、IV-C含量均明显升高(P<0.01),血清TGF-β1水平亦明显升高(P<0.05),血清MDA含量上升、SOD活性显著降低(P<0.01)；与模型组比较,蕲艾提取液治疗各组HA、PCⅢ、LN、IV-C含量均显著降低(P<0.05),血清TGF-β1含量亦明显下降(P<0.05),血清脂质过氧化指标MDA含量显著降低(P＜0.05), SOD活性显著升高(P<0.05),且蕲艾提取液治疗组与正常对照组无显著性差异(P>0.05)。
光镜下观察：模型组大鼠可见肝组织结构部分破坏,肝细胞发生广泛的变性水肿,水样变性甚至气球样变性,还可见程度、范围不等的肝细胞坏死；肝板排列紊乱,部分肝小叶失去正常结构；汇管区纤维隔内有大量嗜酸性细胞、单核淋巴细胞及成纤维细胞浸润,大量胶原沉积,有较明显纤维组织增生,胶原纤维沿汇管区及中央静脉周围向肝小叶内延伸,可见相互连接的纤维分割肝小叶,甚至有部分区域有早期肝硬化表现。蕲艾提取液及丹参治疗各组大鼠肝脏在色泽、质地、大小、表面光滑度方面较模型组有明显改善,细胞变性和坏死程度显著减轻,肝小叶结构破坏不明显,炎性细胞浸润和纤维组织增生减少,中央静脉和汇管区有少量纤维组织延伸,不同剂量蕲艾提取液治疗组间无显著性差异。
2.蕲艾提取液对大鼠肝组织中TGFβ-Smad信号转导通路蛋白表达的影响
免疫组织化学结果显示,TGFβ1、Smad3、Smad4在正常对照组肝细胞内无阳性细胞表达,模型组肝内表达明显增强,主要见于间质细胞、炎性细胞、损伤的肝细胞,阳性物质主要集中在细胞膜上,胞质内有少量表达；蕲艾提取液各剂量组及丹参组阳性细胞数目明显减少,阳性染色程度明显减轻。
RT-PCR及Western blot检测结果显示,TGFβ1、Smad3、Smad4 mRNA及其蛋白,在正常对照组中表达明显低下,而Smad7 mRNA及其蛋白表达则明显升高；蕲艾提取液高、中、低剂量组TGFβ1、Smad 3、Smad4 mRNA及其蛋白与模型组比较表达逐渐减少,Smad7 mRNA及其蛋白表达与模型组比较则逐渐升高,其中中、高剂量组TGFβ1、Smad3、Smad7 mRNA及其蛋白与模型组比较有显著性差异(P<0.05,P<0.01),而与丹参组比较无显著性差异(P>0.05)。
3.蕲艾提取液对抗凋亡基因Bcl-2及细胞周期蛋白Cyclin D1表达的影响
免疫组织化学结果显示,Bcl-2及Cyclin D1蛋白在正常对照组肝细胞内无阳性细胞表达,模型组肝内表达明显增强,主要见汇管区、肝细胞、间质细胞膜上及细胞浆内,阳性物质主要集中在细胞膜上；蕲艾提取液各剂量组及丹参组阳性细胞数目明显减少,阳性染色程度明显减轻。
RT-PCR及Western blot检测结果显示,正常对照组Bcl-2、Cyclin D1 mRNA及蛋白表达较模型组低下,蕲艾提取液各剂量组Bcl-2、Cyclin D1 mRNA及蛋白表达较模型组逐渐减少,其中高剂量组与模型组比较有显著性差异(P<0.05),与正常对照组和丹参组比较无显著性差异(P>0.05)。
结论
1.蕲艾提取液可以明显改善猪血清诱导的大鼠免疫性肝纤维化的肝组织病理学损害,减轻纤维组织增生,明显减少胶原纤维间隔、肝细胞坏死、变性以及炎性细胞的浸润,并能有效降低肝纤维化血清学指标,其抗纤维化的机理可能与抗脂质过氧化有关。
2.蕲艾提取液能够显著抑制大鼠肝组织中TGFβ1、Smad3、Smad4 mRNA及其蛋白表达水平,上调Smad7 mRNA及其蛋白表达水平,表明其可能通过有效调控TGFβ1-Smad信号转导通路,从而抑制大鼠肝纤维化。
3.蕲艾提取液能够降低大鼠肝组织中抗凋亡基因Bcl-2 mRNA及其蛋白表达水平,提示其可能通过有效诱导大鼠HSC凋亡,从而达到抗肝纤维化的作用。
4.蕲艾提取液可能通过下调大鼠肝组织中细胞周期蛋白Cyclin D1 mRNA及其蛋白表达水平,发挥对细胞周期的调控,抑制细胞增殖,而达到抗肝肝纤维化的作用。
Background and Objective:
Hepatic fibrosis is regarded as a pathologically repair reac-tion followed various kinds of chronic liver diseases. Fibrosis mainly presents the imbalance of the ECM compound production and degradation, which makes the ECM deposit pervadingly in liver. Hepatic fibrosis is a common stage of most chronic liver diseases regardless of the etiology, and its progression may lead to hepatic cirrhosis or hepatocelluar carcinoma (HCC). Although hepatic fibro-sis is thought to be a reversible pathological state, there is no established effective therapy for hepatic fibrosis yet.
Because of its special feature of poly-target, poly-lint, poly-channel, and its compound effects, the Chinese medicine can aim to the complicated mechanisms of hepatic fibrosis accordingly, which makes it have traditional and elearly prevalence against the western medicine. The topic is through the establishment of immune animal model of hepatic fibrosis, to get a preliminary understanding the inhibition of Artemisia argyi extracting solution on rat hepatic fibrosis, and through its rat liver tissue TGFβ-Smad signal transduction pathway in the key signal transduction molecules TGFβ1, Smad 3, Smad 4, Smad 7 expression level changes, as well as against the apoptosis gene bcl-2 and cell cycle proteins, to explore Artemisia argyi extracting solution the molecular mechanism of anti-fibrosis.
Methods:
1. Use of pig serum-induced liver fibrosis model,72 Wistar rats were fed 1 week after adaptation,were randomly divided into normal control group, liver fibrosis model group, high dose, middle dose and low-dose treatment of Artemisia argyi extracting solution grou-ps,12 rats in each. In addition to the normal control group, the rest of the rats by intraperitoneal injection of sterile body weight 100g pig serum 0.3ml,2 times per week, a total of 10 weeks,20 times. Artemisia argyi extracting solution large, medium and low-dose treatment group were given 6ml/only,3ml/only and 1.5ml/only with Artemisia argyi extracting solution fed to pray daily 1; model group were given 6ml/only normal saline gavage, every day 1 times; the normal control group was given intraperitoneal injection of saline,
2 times per week,10 weeks a total of 20 times, and to give 6ml/only normal saline gavage, day 1 times. At 10 over the weekend after the last injection of pig serum 48h, animals in each group were decapitated, eyes, enough to take blood specimens from blood, separa-tion of serum, and take part of the liver,4% formalin-fixed, conven-tional paraffin-embedded sections.Part of the gradient of fresh liver tissue placed in -70℃to save, for real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR) method and protein blot (Western blot).
2. Serological test:The rats in each group were detected in serum indicators of liver fibrosis serum hyaluronic acid (HA),laminin (LN),Ⅲprocollagen (PCⅢ),Ⅳprocollagen (Ⅳ-C)content, as well as indicators of lipid peroxidation superoxide dismutase(SOD), malondialdehyde (MDA). ELISA, serum TGFβ1 levels。
3. Liver histology tests:using HE staining and Masson staining of collagen triple-pathological changes in rat liver.
4.Immunohistochemistry:using of immunohistochemistry SABC method, combined with microscope to observe the rat liver tissue TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl of expression.
5. Using real-time fluorescent quantitative reverse transcription-poly mer-ase chain reaction (RT-PCR) method and Western blot test (Western blot) were measured TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl mRNA and protein expression.
Results:
1. Artemisia argyi extract solution inhibited rat liver fibrosis in rats.
Compared with the normal control group, the serum markers of liver fibrosis HA,PCⅢ,LN,Ⅳ-C levels of model group,were signi-ficantly higher(P＜0.01), serum TGF-β1 levels are significantly higher (P＜0.05), increase in serum MDA concentration, SOD activity was significantly decreased(P＜0.01);with the model group,Artemi-sia argyi extracting solution treatment for all groups HA, PCIII, LN,Ⅳ-C levels were significantly lower (P＜0.05), serum TGF-β1 content also decreased significantly(P＜0.05), serum indicators of lipid peroxidation MDA contents were significantly lower(P＜0.05), SOD activity was significantly increased (P＜0.05), and Artemisia argyi extracting solution treatment group and normal control group had no significant difference (P＞0.05).
Optical microscope observation:model group can be seen part of the organizational structure of liver damage, liver cells a wide range of variability of edema, water balloon-like degeneration or degeneration, but also shows the extent, scope, ranging from liver cell necrosis; liver disorder board arrangement, some of loss of normal structure of hepatic lobule; periportal fibrous septum in a large number of eosinophils, monon-uclear cell infiltration of lymphocytes and fibroblasts, a large number of collagen deposition, there are more obvious fibrous tissue hyperplasia, collagen fibers along the periportal areas and around the central vein to the hepatic lobule within the extension,we can see the connection between fiber split hepatic lobule, and even some regions the performance of early liver cirrhosis. Artemisia argyi extracting solution therapy in liver of rats in each group in the color, textu-re, size, surface smoothness in a significant improvement compared with model group, cell degeneration and necrosis significantly reduced hepatic lobule was not obvious structural damage, inflame-matory cell infiltration and fibrous tissue reduced proliferation, the central veins and periportal extension of a small amount of fibrous tissue, different doses of Artemisia argyi extracting solution between the treatment group no significant difference.
2. Artemisia argyi extracting solution expect rat liver tissue TGFβ-Smad signal transduction pathway protein expression.
Immunohistochemistry results showed that, TGFβ1, smad3, smad4 liver cells in the normal group no positive cells, liver expression was significantly increased in model group, mainly seen in interstitial cells, inflammatory cells, injury of liver cells, the positive substances mainly concentrated on the membrane, there is a little expression in the cytoplasm; Artemisia argyi extracting solution each dose group significantly reduced the number of positive cells, positive staining is reduced significantly.
RT-PCR and Western blot test results showed, TGFβ1, smad3, smad4 mRNA and protein expression in the normal control group, significantly lower, while the smad7 mRNA and protein expression was significantly increased; Artemisia argyi extracting solution high, medium and low dose group TGFβ1, smad3, smad4 mRNA and protein expression compared with model group gradually reduced, smad7 mRNA and protein expression compared with model group were increased gradually, in which middle and high dose group TGFβ1, smad3, smad7 mRNA and protein and model group and there was significant difference (P＜0.05, P＜0.01), compared with the SM group was no significant difference (P＞0.05). 3. Artemisia argyi extracting solution against apoptosis gene Bcl-2 and cell cycle protein expression of cyclinDl.
Immunohistochemistry results showed that, Bcl-2 and cyclinDl protein in normal liver cells there is no positive cells, liver expression was significantly increased in model group, mainly seen in portal area,hepatic cells and stromal cells'membrance and cytoplasm, positive substance is mainly concentrated in the membr-ance; Artemisia argyi extracting solution each dose group signifi-cantly reduced the number of positive cells, positive staining is reduced significantly.
RT-PCR and Western blot test results showed that the normal control group, Bcl-2, CyclinDl mRNA and protein expression compared with model group, low, Artemisia argyi extracting solution every dose group Bcl-2, CyclinD1 mRNA and protein expression compared with model group gradually reduced, which high-dose group compared with the model group were significantly different (P＜0.05), with the normal group and the SM group showed no significant difference (P＞0.05).
Conelusions:
1. Artemisia argyi extracting solution can significantly improve the rat liver fibrosis histopathological damage induced by immune pig serum, reduce fibrous tissue proliferation and decreased collagen fibers interval,liver cell necrosis,degeneration,and inflammatory cell infiltration,and can effectively reduce serum markers of liver fibrosis and its mechanism may be anti-fibrosis and anti-lipid peroxidation, reducing the concentration of serum TGF-β1.
2. Artemisia argyi extracting solution can significantly inhibit rat liver tissue TGFβ1, Smad3, Smad4 protein and mRNA expression levels, increase the level of Smad7 protein and mRNA expression, indicating that it can effectively regulate TGFβ-Smad signal transduction pathway, thereby suppressing rat liver fibrosis.
3. Artemisia argyi extracting solution can reduce the rat liver tissue of anti-apoptotic gene Bcl-2 protein and mRNA expression levels, suggesting that it can effectively induce apoptosis in rat HSC to achieve the anti-fibrosis.
4. Artemisia argyi extracting solution could be reduced in rat liver tissue of cyclin CyclinDl protein and mRNA expression levels to play on cell cycle regulation, so that stagnation in the G0/G1 phase cell replication, inhibit cell proliferation.
引文
[1]但杰,高鹏,申力,等.TGFβ1和CTGF在非酒精性脂肪性肝炎肝组织中的表达及意义[J].世界华人消化杂志,2008；16(32)：3637-3641.
[2]Ihn H.Pathogenesis of fibrosis:role of TGF-beta and CTGF[J]. CurrOpin Rheumatol,2002;14 (6):681-685.
[3]严蓉,陈峰.Smad蛋白-肝纤维化时TGFβ1信号转导的关键物质[J].国际流行病学传染病学杂志.2007；34：191-195.
[4]Friedman SL.Hepatic fibrosis-Overview[J]. Toxicology,2008; 254(3):120-129.
[5]席文娜,孙水林,李方春.α-2干扰素对大鼠肝组织bc1-2基因表达的影响及意义[J].世界华人消化杂志,2009；17(31)：3237-3240
[6]赵宏贤,陈霞,郭勇,等.粉防己碱对大鼠肝星状细胞增殖及细胞周期D1、增殖细胞核抗原表达的调控[J].时珍国医国药,2008；19(1)：108-110
[7]贺骏,阮秋蓉,杨秀萍,等.Pin1和Cyclin D1在人类5种常见肿瘤中的表达及其意义[J].中国组织化学与细胞化学杂志,2006；15(6)：608-612.
[8]李红雨,徐茜,朱涛,等.宫颈癌细胞株和宫颈上皮组织中Pinl和CyclinD1的表达及临床意义[J].癌症,2006；25(3)：367-372.
[9]Calbo J, ParrenoM, Sotillo E. et al.G1 cyclin/cyclin dependent kinase-coordinated phosphorylation of endogenous pocket pro-teins differentially regulates their interactions with E2F4 and E2FI and gene expression [J]. J Biol Chem.2002; 277:50-63.
[10]Lents N H, Keenan SM, Bellone C. et al. Stimulation of the Raf/ MEK/ERK cascade is necessary and sufficient for activation and Thr 160 phosphorylation of a nuclear-targeted CDK2 [J]. J Biol Chem.2002; 277:47-69.
[11]杨冬娣,刘金元.青蒿琥酯对肝纤维化小鼠肝星状细胞凋亡及其相关蛋白的影响[J].长春中医药大学学报,2009；25(3)：323-324.
[12]Lamireau T, Desmouliere A,Bioula-Sage P. et al. Mechanisms of hepatic fibrogenesis[J]. Arch Pediatr.2002; 9(4):392-405.
[13]孟爱红,陈芝芸,严茂祥.银杏叶提取物对肝纤维化大鼠肝细胞凋亡的影响[J].中华中医药学刊,2009；27(10)：2095-2097.
[14]Mutluay R,Ozenirler S,Poyraz A. The expression of bcl-2 in chronic liver diseases [J]. Saudi Med J.2005; 26(5):1245-1249.
[15]Novo E, Marra F, Zamara E, et al. Overexpression of Bcl-2 by activated human hepatic stellate cells:resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans [J].Gut.2006; 55:1174-1182.
[16]关幼波.肝病杂病论[M].北京世界图书出版公司,1994：81.
[17]盛国光.慢性乙型肝炎中医病因病机的探讨[J].中西医结合肝病杂志,1997；7(2)：126-127.
[18]刘绍能,刘为民.从络脉理论探讨肝纤维化证治规律[J].中国中医药信息杂志,2003；10(7)：3.
[19]刘为民,姚乃礼.络病理论与肝纤维化临床[J].中国药学报,2003；31(1)：2.
[20]李校天,杨书良,王军民,等.丹参对肝星状细胞丝裂原活化蛋白激酶通路的抑制作用[J].解放军医学杂志,2006；31(1)
[21]薛冬英,洪嘉禾,徐列明.丹参酚酸B对大鼠肝星状细胞中丝裂原激活的蛋白激酶通路的抑制作用[J].中华肝脏病杂志,2004；12(8)：471-474.
[1]Guyot C, Lepreux S, Combe C. et al. Hepatic fibrosis and cirrho-sis:the(myo)fibroblastic cell subpopulations involved[J]. Int J Biochem Cell Boil,2006; 8(2):135-151.
[2]Hui AY,Friedman SL.Molecular basis of hepatic fibrosis[J]. Expert Rev Mol Med,2003; 5(5):1-23.
[3]GressnerOA,Weiskirchen R,GressnerAM. Biomarkers of hepatic fibrosis, fibrogenesis and genetic predisposition pending bet-ween fiction and reality[J].J Cell Mol Med,2007; 11 (5):1031-1051.
[4]陆伦根,曾民德.肝纤维化非创伤性诊断研究进展[J].中华医学杂志,2005;85(15)：1078-1080.
[5]Mardini H, Recod C. Dection assessment and monitoring of hepatic fibrosis:biochemistry or biopsy?[J]Ann Clin Biochen,2005;42 (6):441-447.
[6]陆伦根,曾民德.肝纤维化的诊断和评估[J].中华肝脏病杂志,2005；13(8)：603-604.
[7]Hu YY, Liu P, Liu C. et al. The value of serum markers in evaluating liver fibrotic changes [J]. Zhonghua Gan Zang Bing Za Zhi,2006; 14(3):174-177.
[8]SCHIAVON LL,NARCISO,SCHIAVON JL, et al. Serum levels of YKL 240 and hyaluronic acid as noninvasive markers of liver fibrosis in haemodialysis patients with chronic hepatitis C virus infection[J].J Viral Hepat,2008; 15 (9):2666-2674.
[9]程书权,张自富,赵永伟.血清HA、PLD、PCIII、LN、CIV联合检测对慢性肝病的诊断价值探讨[J].中国实用内科杂志,1999；19(1)：28-30.
[10]刘仁才,刘龙秀,张德远,等.血清HA、PLD、PCIII、LN、CIV含量与慢性肝炎炎症活动度及纤维化程度的关系[J].中国传染病杂志,1999；17(2)：12-123.
[11]Lee KS, Lee SJ, Park HJ, et al.Oxidative stress effect on the activation of hepatic stellate cells [J]. Yonsei Med J,2001, 42(1):1-8.
[12]Hoek JB,Pastorino JG, Ethanol. Oxidative stress and cytokine induced liver cell injury [J]. Alcohol,2002; 27:63-68.
[13]Adams LA, Lindor KD. Nonalcoholic fatty liver disease[J]. Ann Epidemiol,2007; 17:863-869.
[14]戴颖,朱萱.基质金属蛋白酶抑制因子-1与肝纤维化[J].中国全科医学杂志,2008；11(5)：906-908.
[15]Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis [J]. J Hepatol,2001; 35 (2):297-306.
[16]Ozaras R,Tahan V,Aydin S,et al.N-acetylcysteine attenuates alcohol induced oxidative stress in the rat [J]. World J Gastroe-nterol,2003; 9(4):125-128.
[17]郭安平,李俊,吕雄文,等.来氟米特对急性化学性肝损伤小鼠肝Cyt.P450酶含量及CYP2E1 mRNA表达的影响[J].安徽医科大学学报, 2008；43(1)：55-61.
[18]Shi HY,Xu JW, Ren XX. Effect of genistein on hepatic stellate cell proliferation and lipid peroxidation in vitro[J]. Nanfang Yike Daxue Xuebao,2008; 28(11):2066-2068.
[19]何燕,胡志峰,李平,等.柴胡皂苷抗肝纤维化大鼠脂质过氧化作用的研究[J].中国中药杂志,2008；33(8)：915-919.
[20]高春芳.转化生长因子β1与肝纤维化[J].中华医学杂志,2005；85(15)：1074,
[21]丁宁.肝纤维化形成机制的研究进展[J].临床肝胆病杂志,2009；25(1)：73-74.
[1]Friedman SL. Liver fibrosis-from bench to beside[J]. J Hepatoly, 2003; 38(Suppl.1):38.
[2]过建春,陈群伟,包剑锋,等.转化生长因子β1在肝纤维化发病中的作用[J].国际消化病杂志,2009；29(2)：86-88.
[3]俞蕾敏,吕宾.TGF-β smad信号转导通路与肝纤维化的关系[J].国际消化病杂志,2008；28(5)：397-400.
[4]申月明,朱萱.肝星状细胞激活与信号转导[J].世界华人消化杂志,2007；15(8)：873-878.
[5]罗云,戴立里,沈鼎明,等.肝星状细胞与肝纤维化的研究进展[J].中华肝脏病杂志,2000；8(4)：251-252.
[6]唐桂连.肝星状细胞与肝纤维化[J].实用医技杂志,2007；6 (1)：116-117.
[7]Schuppan D, Porov Y. Hepatic fibrosis:From bench to beside [J]. J Gastroenterol Hepatol,2002; 17(3):300-305.
[8]Mauviel A.Transforming growth factor-bata:a key mediator of fibrosis[J].Methods Mol Med,2005; 117 (2005):69-80.
[9]卢平,周力,张永宏.转化生长因子β1-Smad信号传导与肝纤维化的关系[J].临床肝胆病杂志,2006；22(1)：69-71.
[10]刘莲,康海燕,叶立红,等.肝组织转化生长因子β1与肝纤维化的关系研究[J].临床肝胆病杂志,2009；25(6)：446-448.
[11]Sato M, Suzuki S, Senoo H. Hepatic stellate cells:unique charact-eristics in cell biology and phenotype[J]. Cell structure and function,2003; 28(2):105-112.
[12]Liu C, Gaca MD, Swenson ES, et al.Smads2 and 3 are differenti-ally activated by transforming growth factor-β (TGF-β) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-β independent [J].J Biol Chem,2003; 278:11721-11728.
[13]Uemura M, Swenson ES, Gaca MD, et al.Smad2 and Smad3 play dif-ferent roles in rat hepatic stellate cell function and smo-oth muscle actin organization [J]. Mol Biol Cell,2005,1(9): 4214-4224.
[14]RobertA B, Piek E, Bottinger EP, et al. Is Smad3 a major player in signal transduction pathways leading to fibrogenesis [J]. Chest,2001,120(Suppl.1):43S-47S.
[15]王瑾,马韵.肝脏纤维化形式和逆转的研究进展[J].医学研究杂志,2009；38(3)：88-89.
[16]孙校男,王先开,娄国强.Smad蛋白与肝纤维化[J].国外医学·流行病学传染病学分册,2005；32(4)：243-245.
[17]何燕,肖诚,胡志峰,等.Smad蛋白及其对中医药防治肝纤维化作用的研究进展[J].中国中医基础医学杂志,2007；13(5)：399-402.
[18]吴凌康,厉有名.Smads家族在肝纤维化中的作用.浙江医学,2005,27(4)：319-321.
[19]Seyhan H, Hamzavi J, Wiercinska E, et al. Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling [J]. J Cell Mol Med,2006; 10(4):922-932.
[20]Ten Dijke P, Hill CS. New insights into TGF β-Smad signalling [J]. Trend Biochem Sci,2004; 29(5):265-273.
[21]Uemura M, wenson ES, Gaca MD, et al. Smad2 and Smad3 play different roles in rat hepatic stellate cell function and smooth muscle actin organization[J].Mol Biol Cell.2005; 16 (9):4214-4224.
[22]Flanders KC. Smad3 as a mediator of the fibrotic response [J]. Int J Exp Pathol.2004; 85 (2):47-64.
[23]Dooley S, Hamzavi J, Breit kopf K, et al. Smad7 prevent activation of hepatic stellate cells and liver f ibrosis in rats [J]. Gastro-enterology,2003; 125(1):178-191.
[24]严蓉,陈峰.Smad蛋白-肝纤维化时TGF β1信号转导的关键物质[J].国际流行病学传染病学杂志,2007；34(3)：191-195.
[25]Tahashi Y,Matsuzaki K, Date M, et al. Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury [J]. Hepatology,2002; 35:49-61.
[26]DK Lee, Shee Park, Y Yi, et al.The hepatitis B virus encoded oncoprotein pX amplifies TGF-β family signaling through direct interaction with Smad4:potential mechanism of hepatitis B virus-induced liver fibrosis [J]. Genes Dev,2001; 15(4):455-466.
[27]W Zhang, J Ou, Y Inagaki,et al. Synergistic Cooperation between Sp1 and Smad3/Smad4 Mediates Transforming Growth Factor β1 Stimulation of α2 (Ⅰ)-Collagen (COL1A2)Transcription[J]. J Biol Chem,2000; 275(50):39237-39245.
[28]郭宝良,闫朝岐,仲雷,等.Smad4 siRNA对转化生长子p诱导Ⅰ型胶原表达的作[J].中国组织工程研究与临床康复,2009；13(11)：2062-2065.
[1]Bataller R, Brenner DA. Liver fi brosis [J]. J Clin Invest, 2005; 115:209-218.
[2]李石.肝纤维化发生机制[J].中华消化杂志,1999；19：48-50.
[3]席文娜,孙水林,李方春,等.α-2a干扰素对大鼠肝组织bc1-2基因表达的影响及意义[J].世界华人消化杂志,2009；17(31)： 3237-3240. [4] Holley SL,Mathias C, Jahnke V, et al. Association of cyclinDl polymorphism with increased susceptibility to oral squamous cell carcinoma [J]. Oral Oncol,2005; 41 (2):156-160.
[5]Harper N, Hughes M, MacFarlane M, et al. Fas-associated death dom-ain protein and caspase-8 are not recruited to the tumor necros-is factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis [J].J Biol Chem,2003; 278:25534-25541
[6]Jaeschke H, Gores GJ, Cederbaum AI, et al. Mechanisms of hepatoto xicity[J].Toxicol Sci,2002; 65:166-176.
[7]Baskin-Bey ES,Gores GJ. Death by association:BH3 domain only proteins and liver injury[J]. Am J Physiol Gastrointest Liver Physiol,2005; 289:987-990.
[8]Oltvai ZN, Milliman CL, Korsmeyer SJ.Bcl-2 heterodimerizes in-vivo with a conserved homolog, Bax, that accelerates programmed cell death [J]. Cell,1993; 74:609-619.
[9]Rogerio F, Jordao H JR, Vieira AS, et al. Bax and Bcl-2 expression and TUNEL labeling in lumbar enlargement of neonatal rats after sciatic axotomy and melatonin treatment [J]. Brain Res,2006,11 (12):80-90.
[10]Higuchi M,Aggarwal BB, Yeh ET. Activation of CPP32-like pro-tease in tumor necrosis factorinduced apoptosis is depend-ent on mitochondrial function[J].J Clin Invest,1997;99:1751-1758.
[11]孙怀宇,王军,王鹏.Bcl-2家族和Bcl-x1对细胞凋亡调控的研究进展[J].中国煤炭工业医学杂志,2009；12(4)：675-677.
[12]孙桦.靶向bcl-2基因的siRNA对肝星状细胞凋亡的影响[J].上海交通大学学报(医学版),2008；28(7)：845-849.
[13]Motokura T, Arnold A. Cycl inD and oncogenesis [J]. CurrOpin Genet Dev.1993; 3(1):5-10.
[14]Umekita Y,OHI Y.Overexpression of cyclin D1 predicts for poor prognosis in estrogen receptor-negative breast cancer patien-ts[J].Int J Cancer,2002; 98 (3):415-418.
[15]张伟,杜成友,徐尔侃.肝癌临床发展与cyclinDl表达DNA含量及细胞凋亡发生的关系[J].第三军医大学学报,2005；27(5)：46.
[16]Loyer P, Trembley JH, KatonaR, et al.Role of CDK/cyclin comple-xes in transcription and RNA splicing. Cell Signa[J],2005; 17(9):1033-1051.
[17]Hirsch FR, Witta S, Biomarkers for Prediction of sensitivity to EGFR inhibitiors in non-small Cell lung caneer.CurrOpin Onc-ol [J].2005; 17 (2):118-122.
[18]Chenchen Li,Xinmei Li.The different roles of cyclin D1-CDK4 in STEP and mGluR-LTD during the Postnatal development in mice hippocampus area CA1[J].BMC Dev Biol,2007; 7:57.
[19]Holley SL,Heighway J,Hoban PR. Induced expression of human CCND1 altemative transeripts in mouse Cyl-1 knockout fibrob-lasts high lights functional differences [J]. Int J Cancer,2005; 114(3):364-370.
[1]Friedman S L,Bansal M B.Reversal of hepatic fibros is-fact or fantasy[J].Hepatology,2006;43(2 Suppl 1):S82-S88.
[2]姚真敏,吕圭源,赵治友.鳖甲煎丸抗肝纤维化作用的实验研究[J].浙江中医学院学报,1997；21(1)：45-46.
[3]任新周,彭勃,袁词.丙肝康对大鼠肝纤维化抑制作用及其机理探讨[J].江苏中医,2000；21(11)：48-52.
[4]姚立,姚真敏,余涛.鳖甲煎口服液对大鼠肝纤维化的影响[J].中药药理与临床,2002；18(6)：527-530.
[5]王晓玲,刘平,刘成海.丹酚酸A对成纤维细胞活力、增殖及胶原合成的影响[J].中西医结合肝病杂志,2000；10(1)：24-25.
[6]周正,陈捷,周萍.抗纤软肝冲剂药物血清对肝星状细胞增殖的影响[J].中药材,2001；24(11)：809-810.
[7]郭传勇,申振宇,钟敏章,等.中药清肝饮对大鼠肝贮脂细胞和成纤维细胞增殖效应的调控作用[J].中国中医药科技,2000；7(5)：285-286
[8]申凤俊,朱跃科,阴宏,等.DMN肝纤维化模型肝组织Ⅰ、Ⅲ胶原和TIMP-1mRNA表达及复方861的干预作用[J].临床和实验医学杂志,2002；1(3)：150-153.
[9]刘成,刘平,刘成海,等.扶正化瘀方对大鼠肝星状细胞、肝细胞、成纤维细胞胶原合成的影响[J].中国中西医结合脾胃杂志,1998；6(1)：29-31.
[10]邱德凯,李海,曾民德,等.虫草多糖脂质体对大鼠肝纤维化胶原酶mRNA变化的影响[J].新消化病学杂志,1997；5(7)：417-421.
[11]朱冰,刘殿武,房文亮,等.抗纤1号对实验性肝纤维化大鼠组织Ⅰ、Ⅲ型胶原及转移生长因子β1mRNA的影响[J].中国中医基础医学杂志,2003；9(1)：21-23.
[12]李平,朱清静,张建军,等.软肝冲剂对大鼠肝星状细胞增殖及分泌TGE-β1影响的血清药理学研究[J].中国中医药科技,2001；8(5)：292-293.
[13]杨文卓,曾明德,范竹萍,等.氧化苦参碱防治二甲基亚硝胺诱导的大鼠肝纤维化的实验研究[J].中华消化杂志；2003；23(3)：165-167.
[14]舍世锋,贤樟,光东.不同剂量丹参注射液抗肝纤维化临床研究[J].中国中西医结合杂志,2004；24(1)：17-20.
[15]郑磊,戴立里.丹参素对转化生长因子β1刺激肝星状细胞信号传导的影响[J].重庆医科大学学报,2009；34(2)：182-185.
[16]王丽娜,陶艳艳,李书,等.丹酚酸B盐对大鼠纤维化肝组织脂质过氧化及其MMP-2活性的影响[J].中国中医杂志,2010；35(1)：71-75.
[17]陈在忠,王红.川芎嗪抗肝纤维化作用的实验研究[J].中西医结合肝 病杂志,2007；7(3)：156-158.
[18]王文丽,李孝生,陈爽.川芎嗪对肝星状细胞增殖及结缔组织生长因子表达的影响[J].中国生物制品学杂志,2009；22(1)：65-68.
[19]李涛,范好,刘芳.川芎嗪对肝纤维化大鼠Fas和FasL表达的影响[J].现代中西医结合杂志,2008；17(10)：1468-1469.
[20]陈爽,李孝生,王文丽.川芎嗪对转化生长因子β1刺激肝星状细胞CTG及Ⅰ型胶原表达的影响[J].生命科学研究,2009；13(2)：146-151.
[21]孙玉凤,冯志杰,孙泽明,等.黄芪抗肝纤维化的实验研究[J].河北中医药学报,2008；23(1)：9-10.
[22]程鹏,刘素侠,孙晋浩.黄芪抗肝纤维化的作用与转化生长因子-β1及干扰素Y的关系[J].临床军医杂志,2000；28(3)：22-23.
[23]宋少刚,杨雁,陈敏珠.黄芪提取物对大鼠肝星状细胞增殖及产生胶原的影响[J].中国临床药理学与治疗学,2001；6(2)：111-113.
[24]王要军,权启镇,孙自勤.黄茂对实验性肝纤维化组织ICAMI表达影响的免疫组化研究[J].中国临床药理学与治疗学,2000；5(1)：49-51.
[25]颜吉丽,李华,范钰.虫草多糖对大鼠肝星状细胞核因子-κB活性和肿瘤坏死因数-a表达的影响[J].复旦学报(医学版),2003;30(1)：27-29.
[26]刘玉侃,沈薇,张霞.虫草菌丝对实验性肝纤维化的防治作用及其机制研究[J].中国新药与临床杂志,2004；23(3)：139-143.
[27]孙保木,宣红萍,李风华,等.虫草菌丝提取物对大鼠脂肪性肝炎与肝纤维化的影响[J].中国新药与临床杂志,2008；27(1)：6-11.
[28]王宪波,刘平,唐志鹏,等.虫草菌丝提取物对二甲基亚硝胺大鼠肝窦内皮细胞损伤和表型改变的影响[J].北京中医药大学学报,2008；31(7)：448-451.
[29]周维,胡艳,张红卫,等.羧甲基茯苓多糖对肝纤维化大鼠TGF β-Smad信号转导的影响[J].中国民族民间医药,2009；20(2)：16-18.
[30]杨甫昭,张晓彬,冯英菊,等.泽兰水提物对四氯化碳致小鼠肝纤维化的防治作用[J].中国实验方剂学杂志,2008；14(7)：50-51.
[31]张彦亮,倪海滨,杨大明,等.大黄素抗肝纤维化的细胞学研究[J].江苏医药.2009；35(6)：691-693.
[32]万晓强,郑紫丹,尚军洁.大黄酸对免疫性肝纤维化大鼠形成的影响[J].重庆医学,2009；38(10)：204-206.
[33]吴繁荣,陈飞虎,胡伟,等.鬼针草总黄酮抗大鼠免疫性肝纤维化作用及部分机制研究[J].中国药理学通报,2008；24(6)：753-756.
[341闫波,陈飞虎,吴繁荣,等.鬼针草总黄酮对肝纤维化大鼠治疗作用及机制探讨[J].中国药理学通报,2008；24(12)：1640-1642.
[35]孙旭群1,陈飞虎2,吴繁荣2鬼针草总黄酮对大鼠免疫性肝纤维化治疗作用及抗氧化机制[J].安徽医科大学学报,2009；44(4)462-465.
[36]陈小亮,李俊,邓子煜,等.苦参素对肝纤维化大鼠肝脏TGF-β1的调节作用[J].中国药理学通报,2009；25(6)：761-764.
[37]颜寒斌.苦参素治疗慢性乙型肝炎肝纤维化的临床疗效研究[J].吉林医学,2009；30(8)：1672-1673.
[38]鄂裘恺,郁林曦,朱歆鑫,等.苦参素对实验性肝纤维化的干预作用[J].南通医学院学报,2009；29(2)：95-97.
[39]张汝建,卢兴梅,国力,等.苦参素对肝星状细胞增殖及凋亡的影响[J].泰山医学院学报,2009；30(1)：23-24.
[40]张汝建,王天才,杨明峰,等.苦参素对HSC-T6整合素αV表达的影响[J].胃肠病学和肝病学杂志,2008；17(9)：737-739.
[41]曹力波,李兵,李佐军,等.水飞蓟素对肝纤维化小鼠的保护作用及机制探讨[J].中国药理学通报.2009；25(6)：794-796.
[42]宋维芳,许瑞龄,王登妮,等.水飞蓟宾对大鼠实验性肝纤维化发生发展作用机制的研究[J].山西医科大学学报,2009；40(5)：423-426.
[43]徐宏平,何航.水飞蓟宾对人肝星状细胞LX-2凋亡及α1(Ⅰ)胶原mRNA达的影响[J].郑州大学学报,2009；44(3)：597-599.
[44]Lee SJ, Kim YG, Kang KW, et al. Effects of colchicine on liver functions of cirrhotic rats:beneficial effects result from stellate cell inactivation and inhibition of TGF betal expre-ssion[J].Chem Biol Interact,2004; 147(1):9-21.
[45]石小枫,刘祀,刘林,等.三七皂甙抗实验性肝纤维化的研究[J].中药药理与临床,2004；20(1)：12-14.
[46]薛青,薛绍礼,汪亚松,等.三七总苷对体外培养细胞胶原合成模型的影响.医药导报,2008,5(27)：517-519.
[47]王怡,薛绍礼,张 菁,等.三七总皂苷对传代肝星状细胞增殖的影响[J].淮海医药,2008；26(1)：1-3.
[48]张桂灵,石小枫,冉长清,等.三七总皂苷对抗免疫性肝纤维化的实验研究[J].第三军医大学学报,2007,29(23)：2212-2214.
[49]A. Laxova,M. Earley, P. Farrel, et a. Results of DNA based cys-tic fibrosis newborn screening proficiency testing [J]. Journal of Cystic Fibrosis,2008; 6(2):S14.
[50]张荣华,李景怡,陈如泉,等.三七总皂苷对肝纤维化大鼠肝脏超微结构的影响[J].第三军医大学学报,2005；27(24)：2410-2413.
[51]舒建昌,叶国荣,吕霞,等.姜黄素治疗肝纤维化及其作用机制的初步研究[J].中华肝脏病杂志,2007；15(10)：753-757.
[52]舒建昌,邓亮,叶国荣,等.姜黄素治疗大鼠肝纤维化及其对肝组织Ⅰ、Ⅲ型胶原的影响[J].国际内科学杂志,2008；35(7)：379-382.
[53]何航,华海婴,戈士文,等.姜黄素对四氯化碳诱导大鼠肝纤维化的影响[J].郑州大学学报,2009；44(5)：986-989.
[54]何航,徐宏平,华海婴.姜黄素对四氯化碳诱导肝纤维化大鼠肝组织TGF-β1及Caspase-3表达的影响[J].第四军医大学学报,2009；30(2)：100-103.
[55]胡泰洪,徐敏,蒋祥虎.姜黄素对实验性肝纤维化大鼠肝组织中NF-κB及ERK-1 m RNA表达的影响[J].江苏中医药,2009；41(9)
[56]朱宝和,贺德,黄林,等.姜黄素抑制大鼠肝纤维化的研究[J].中国普通外科杂志,2009；18(7)：723-726.
[57]郑培永,邢练军,尤圣富,等.清肝活血方对肝星状细胞TGFβ1表达及细胞内信号传递的影响[J].江苏中医药,2008；40(7)：84-86.
[58]昭铭,江锋,叶永安,等.抗纤抑癌方对肝纤维化大鼠肝组织基质金属蛋白酶表达的影响廖[J].中医药临床杂志,2009；21(2)：163-65.
[59]崔红燕,刘成海,孙保木,等.扶正化淤方对四氯化碳肝纤维化大鼠基膜型基质金属蛋白酶活性的影响[J].时珍国医国药,2009；(8).
[60]房志鑫,李旭波,刘菁菁,等.蓝玉簪颗粒对大鼠肝纤维化的治疗作用[J].世界华人消化杂志；11-16.
[61]贺卫和,刘勇,杨岩涛,等.保肝颗粒抗大鼠肝纤维化的实验研究[J].中华中医药学刊,2008；26(11)：2502-2503.
[62]贺松其,文彬,吕志平,等.保肝宁对肝纤维化模型大鼠肝组织形态学及JAK2、STAT3蛋白磷酸化水平的影响[J].上海中医药杂志,2008；42(5)：75-79.
[63]叶放,薛博瑜,周珉,等.紫七软肝片对复合因素致大鼠肝纤维化模型的动态影响[J].中华中医药学刊,2009,27(3)：526-529.
[64]王振常,王丽,毛德文,等.壮肝逐瘀煎对肝纤维化大鼠转化生长因子β1表达的影响.河南中医,2009；29(8)：751-752.
[65]王振常,王丽,毛德文,等.壮肝逐瘀煎对肝纤维化大鼠肝星状细胞凋亡的影响[J].中华中医药学刊,2008；26(10)：
[66]林寿宁,王振常,梁尧,等.壮肝逐瘀煎对肝纤维化大鼠TGF2β1及TβRIPⅡmRNA表达的影响[J].中国中医基础医学杂志,2008；14(5)：357-358.
[67]林寿宁,何磊,王振常,等.壮肝逐瘀煎对肝纤维化大鼠TβRI/Ⅱ mRNA表达的影响[J].江苏中医药,2008；40(7)：80-82.
[68]王振常,王丽,毛德文,等.壮肝逐瘀煎对肝纤维化大NF-κBmRNA表达的影响[J].辽宁中医药大学学报,2008；10(7)：134-136.
[69]谢玉梅,聂青和,康文臻,等.中药双甲五灵冲剂对免疫性肝纤维化大鼠抗肝纤维化的分子机制研究[J].胃肠病学和肝病学杂志,2009；18(2)：154-158.
[70]李长秦,袁晓栋,孙守才,等.增损甘露消毒丹对肝纤维化大鼠血清PCⅢ、IV-C影响的实验研究[J].陕西中医学院学报,2009；32(3)：49-52.
[71]李常青,李钢,苏俊芳,等.叶下珠复方Ⅱ号对二甲基亚硝胺诱导大鼠肝纤维化形成的抑制作用[J].辽宁中医杂志,2009；36(8)：1413-1415.
[72]赵辉平,王胜春,胡咏武,等.五灵胶囊调TGF-β/Smad信号通路蛋白对胶原Ⅰ、Ⅲ型表达的影响.国际消化病杂志,2009,29(4)：289-292
[73]赵辉平,王胜春,胡咏武,等.五灵胶囊对肝纤维化大鼠肝组织TGF-β /Smad及Ras/ERK信号通路蛋白的影响赵辉平[J].胃肠病学和肝病学杂志,2009；18(5)：392-396.
[74]王妍,姜慧卿.肝纤维化药物治疗新进展[J].临床荟萃,2009；24(4)：357-359.
[75]肝纤维化的基因治疗研究进展[J].国际消化病杂志,2009；29(1)：45-47.
[76]Jiang W, Yang CQ, Liu WB, et al. Blockage of t ransforming growth factor β receptors prevents progression of pig serum induced rat liver fibrosis[J].World J Gast roenterol,2004; 10:1634-1638.
[77]Cui X, Sakata R, Ueno T, et al. Inhibitory effect of a solublet ransforming growt h factor2h type Ⅱ receptor on the acti-vation of rat hepatic stellate cells in primary culture[J]. J Hepatol,2003; 39:731-737.
[78]Nakamuta M,Morizono S,Tsuruta S,et al. Remote delivery and expression of soluble type Ⅱ TGF-preceptor in muscle pre- vents hepatic fibrosis in rats[J].Int J Mol Med,2005; 16(1): 59-64.
[79]Inagald Y, Kushida M, Higashi K, et al. Cell type-specific interv-ention of transforming growth factor β/Smad signaling suppre-sses collagen gene expression and hepatic fibrosis in mice [J]. Gast roenterology,2005; 129 (1):259-268.
[80]Weng HL, Wang BE, Jia JD, et al. Effect of interferon gamma onhepa-tic fibrosis in chronic hepatitis B virus infection:a randomiz-ed cont rolled study[J]. Clin Gast roenterol Hepatol,2005; 3 (8):819-828.
[81]Chen M, Wang GJ,Diao Y,et al.Adeno2associated virus mediated interferon-γ inhibits the Progression of hepatic fibrosis in vit ro and in vivo [J]. World J Gastroenterol,2005; 11(26):4045-4051.
[82]王吉耀,吴盛迪.抗肝纤维化治疗研究进展[J].肝脏,2009；14(1)：71-73.
[83]姜藻,尤大钰.16-16二甲基前列腺E2,秋水仙碱和硫酸锌抗肝纤维化的实验研究[J].中华消化杂志,1994；14(3)：153-154.
[84]Okuno M, et al.Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-beta in liver stellate cells [J]. Hepatology,1997; 26:913.
[85]李立新,陈大志,贺强,等.血管紧张素受体反义基因对肝星状细胞增殖及Ⅰ型胶原基因表达的影响[J].中华肝脏病杂志,2007；15(10)：789-790.
[86]包小洁.肝素抗炎抗纤维化的研究进展[J].临床和实验医学杂志,2007；6(1)：163-1641.
[87]Ueno T, Sujakuk, Tamaki S, et al. OK-432 treatment increases matrixmetal Loproteinase-9 production and improves dimethyl nitrosamine-induced liver cirrhosis in rats [J].Int J Mol Med,1999; 3(5):497-503.
[88]Hironaka K, Sakaida l,Matsumura Y, et al. Enhanced interstitial collagenase(matrix metalloproteinase-13)production of kupff-er cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis[J]. Biochem Biophys Res Commun,2000; 267(1): 290-295.
[89]刘克洲,侯伟,等.肝纤维化的治疗策略.中国中西医结合杂志,2006,1(1)：82-91.
[90]Siegmund SV, Uchinami H, Osawa Y, et al. Anandamide induces necrosis in primary hepatic stellate cells [J].Hepatology, 2005; 41:1085-1095.
[91]Du SL,Pan H, Lu WY, et al.Cyclic Arg-Gly-Asp peptide-labeled liposomes for targeting drug therapy of hepatic fibrosis in rats[J].J Pharmacol Exp Ther,2007; 322:560-568.
[92]Wang J Y,Song ZJ. Targeting binding of fluorescence labeled cyclic RGD peptides to activated hepatic stellate cells via integrin AVB3 receptor [J]. Hepatology,2007;46 (4suppl):2-6.
[93]Ueki T, Okamoto E, Takeuchi M, et al. Persective on postgenome medicine:Gene therapy for liver cirrhosis[J]. Nippon Rinsho, 2001; 59(1):152-156.
[94]Xue F,Takahara T,Yata Y,et al. Hepatocyte growth factor gene therapy acceleratesregeneration incirrhoticmouse livers after hepatectomy [J]. Gut,2003; 52 (5):694-700.
[95]Matsuno Y, Iwata H, Umeda Y, et al. Hepatocyte growth factor gene transfer into the liver via the portal vein using electropo-ration attenuates rat liver cirrhosis[J]. Gene Ther,2003;10 (18):1556-1569.
[96]Kim W H,Matsumoto K, Bessho K, et al. Growth inhibition and apoptosis in liver myofibroblasts promoted by hepatocyte growth factor leads to resolution from liver cirrhosis [J]. Am J Pathol,2005; 166 (4):1017-1028.
[97]Kusumoto K,Ido A, Moriuchi A, et al. Repeated intravenous injection of recombinant human hepatocyte growth factor amel-iorates liver cirrhosis but causes albuminuria in rats[J].Int J Mol Med,2006; 17 (3):503-509.
[2]Ihn H.Pathogenesis of fibrosis:role of TGF-beta and CTGF[J]. CurrOpin Rheumatol,2002;14 (6):681-685.
[3]严蓉,陈峰.Smad蛋白-肝纤维化时TGFβ1信号转导的关键物质[J].国际流行病学传染病学杂志.2007；34：191-195.
[4]Friedman SL.Hepatic fibrosis-Overview[J]. Toxicology,2008; 254(3):120-129.
[5]席文娜,孙水林,李方春.α-2干扰素对大鼠肝组织bc1-2基因表达的影响及意义[J].世界华人消化杂志,2009；17(31)：3237-3240
[6]赵宏贤,陈霞,郭勇,等.粉防己碱对大鼠肝星状细胞增殖及细胞周期D1、增殖细胞核抗原表达的调控[J].时珍国医国药,2008；19(1)：108-110
[7]贺骏,阮秋蓉,杨秀萍,等.Pin1和Cyclin D1在人类5种常见肿瘤中的表达及其意义[J].中国组织化学与细胞化学杂志,2006；15(6)：608-612.
[8]李红雨,徐茜,朱涛,等.宫颈癌细胞株和宫颈上皮组织中Pinl和CyclinD1的表达及临床意义[J].癌症,2006；25(3)：367-372.
[9]Calbo J, ParrenoM, Sotillo E. et al.G1 cyclin/cyclin dependent kinase-coordinated phosphorylation of endogenous pocket pro-teins differentially regulates their interactions with E2F4 and E2FI and gene expression [J]. J Biol Chem.2002; 277:50-63.
[10]Lents N H, Keenan SM, Bellone C. et al. Stimulation of the Raf/ MEK/ERK cascade is necessary and sufficient for activation and Thr 160 phosphorylation of a nuclear-targeted CDK2 [J]. J Biol Chem.2002; 277:47-69.
[11]杨冬娣,刘金元.青蒿琥酯对肝纤维化小鼠肝星状细胞凋亡及其相关蛋白的影响[J].长春中医药大学学报,2009；25(3)：323-324.
[12]Lamireau T, Desmouliere A,Bioula-Sage P. et al. Mechanisms of hepatic fibrogenesis[J]. Arch Pediatr.2002; 9(4):392-405.
[13]孟爱红,陈芝芸,严茂祥.银杏叶提取物对肝纤维化大鼠肝细胞凋亡的影响[J].中华中医药学刊,2009；27(10)：2095-2097.
[14]Mutluay R,Ozenirler S,Poyraz A. The expression of bcl-2 in chronic liver diseases [J]. Saudi Med J.2005; 26(5):1245-1249.
[15]Novo E, Marra F, Zamara E, et al. Overexpression of Bcl-2 by activated human hepatic stellate cells:resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans [J].Gut.2006; 55:1174-1182.
[16]关幼波.肝病杂病论[M].北京世界图书出版公司,1994：81.
[17]盛国光.慢性乙型肝炎中医病因病机的探讨[J].中西医结合肝病杂志,1997；7(2)：126-127.
[18]刘绍能,刘为民.从络脉理论探讨肝纤维化证治规律[J].中国中医药信息杂志,2003；10(7)：3.
[19]刘为民,姚乃礼.络病理论与肝纤维化临床[J].中国药学报,2003；31(1)：2.
[20]李校天,杨书良,王军民,等.丹参对肝星状细胞丝裂原活化蛋白激酶通路的抑制作用[J].解放军医学杂志,2006；31(1)
[21]薛冬英,洪嘉禾,徐列明.丹参酚酸B对大鼠肝星状细胞中丝裂原激活的蛋白激酶通路的抑制作用[J].中华肝脏病杂志,2004；12(8)：471-474.
[1]Guyot C, Lepreux S, Combe C. et al. Hepatic fibrosis and cirrho-sis:the(myo)fibroblastic cell subpopulations involved[J]. Int J Biochem Cell Boil,2006; 8(2):135-151.
[2]Hui AY,Friedman SL.Molecular basis of hepatic fibrosis[J]. Expert Rev Mol Med,2003; 5(5):1-23.
[3]GressnerOA,Weiskirchen R,GressnerAM. Biomarkers of hepatic fibrosis, fibrogenesis and genetic predisposition pending bet-ween fiction and reality[J].J Cell Mol Med,2007; 11 (5):1031-1051.
[4]陆伦根,曾民德.肝纤维化非创伤性诊断研究进展[J].中华医学杂志,2005;85(15)：1078-1080.
[5]Mardini H, Recod C. Dection assessment and monitoring of hepatic fibrosis:biochemistry or biopsy?[J]Ann Clin Biochen,2005;42 (6):441-447.
[6]陆伦根,曾民德.肝纤维化的诊断和评估[J].中华肝脏病杂志,2005；13(8)：603-604.
[7]Hu YY, Liu P, Liu C. et al. The value of serum markers in evaluating liver fibrotic changes [J]. Zhonghua Gan Zang Bing Za Zhi,2006; 14(3):174-177.
[8]SCHIAVON LL,NARCISO,SCHIAVON JL, et al. Serum levels of YKL 240 and hyaluronic acid as noninvasive markers of liver fibrosis in haemodialysis patients with chronic hepatitis C virus infection[J].J Viral Hepat,2008; 15 (9):2666-2674.
[9]程书权,张自富,赵永伟.血清HA、PLD、PCIII、LN、CIV联合检测对慢性肝病的诊断价值探讨[J].中国实用内科杂志,1999；19(1)：28-30.
[10]刘仁才,刘龙秀,张德远,等.血清HA、PLD、PCIII、LN、CIV含量与慢性肝炎炎症活动度及纤维化程度的关系[J].中国传染病杂志,1999；17(2)：12-123.
[11]Lee KS, Lee SJ, Park HJ, et al.Oxidative stress effect on the activation of hepatic stellate cells [J]. Yonsei Med J,2001, 42(1):1-8.
[12]Hoek JB,Pastorino JG, Ethanol. Oxidative stress and cytokine induced liver cell injury [J]. Alcohol,2002; 27:63-68.
[13]Adams LA, Lindor KD. Nonalcoholic fatty liver disease[J]. Ann Epidemiol,2007; 17:863-869.
[14]戴颖,朱萱.基质金属蛋白酶抑制因子-1与肝纤维化[J].中国全科医学杂志,2008；11(5)：906-908.
[15]Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis [J]. J Hepatol,2001; 35 (2):297-306.
[16]Ozaras R,Tahan V,Aydin S,et al.N-acetylcysteine attenuates alcohol induced oxidative stress in the rat [J]. World J Gastroe-nterol,2003; 9(4):125-128.
[17]郭安平,李俊,吕雄文,等.来氟米特对急性化学性肝损伤小鼠肝Cyt.P450酶含量及CYP2E1 mRNA表达的影响[J].安徽医科大学学报, 2008；43(1)：55-61.
[18]Shi HY,Xu JW, Ren XX. Effect of genistein on hepatic stellate cell proliferation and lipid peroxidation in vitro[J]. Nanfang Yike Daxue Xuebao,2008; 28(11):2066-2068.
[19]何燕,胡志峰,李平,等.柴胡皂苷抗肝纤维化大鼠脂质过氧化作用的研究[J].中国中药杂志,2008；33(8)：915-919.
[20]高春芳.转化生长因子β1与肝纤维化[J].中华医学杂志,2005；85(15)：1074,
[21]丁宁.肝纤维化形成机制的研究进展[J].临床肝胆病杂志,2009；25(1)：73-74.
[1]Friedman SL. Liver fibrosis-from bench to beside[J]. J Hepatoly, 2003; 38(Suppl.1):38.
[2]过建春,陈群伟,包剑锋,等.转化生长因子β1在肝纤维化发病中的作用[J].国际消化病杂志,2009；29(2)：86-88.
[3]俞蕾敏,吕宾.TGF-β smad信号转导通路与肝纤维化的关系[J].国际消化病杂志,2008；28(5)：397-400.
[4]申月明,朱萱.肝星状细胞激活与信号转导[J].世界华人消化杂志,2007；15(8)：873-878.
[5]罗云,戴立里,沈鼎明,等.肝星状细胞与肝纤维化的研究进展[J].中华肝脏病杂志,2000；8(4)：251-252.
[6]唐桂连.肝星状细胞与肝纤维化[J].实用医技杂志,2007；6 (1)：116-117.
[7]Schuppan D, Porov Y. Hepatic fibrosis:From bench to beside [J]. J Gastroenterol Hepatol,2002; 17(3):300-305.
[8]Mauviel A.Transforming growth factor-bata:a key mediator of fibrosis[J].Methods Mol Med,2005; 117 (2005):69-80.
[9]卢平,周力,张永宏.转化生长因子β1-Smad信号传导与肝纤维化的关系[J].临床肝胆病杂志,2006；22(1)：69-71.
[10]刘莲,康海燕,叶立红,等.肝组织转化生长因子β1与肝纤维化的关系研究[J].临床肝胆病杂志,2009；25(6)：446-448.
[11]Sato M, Suzuki S, Senoo H. Hepatic stellate cells:unique charact-eristics in cell biology and phenotype[J]. Cell structure and function,2003; 28(2):105-112.
[12]Liu C, Gaca MD, Swenson ES, et al.Smads2 and 3 are differenti-ally activated by transforming growth factor-β (TGF-β) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-β independent [J].J Biol Chem,2003; 278:11721-11728.
[13]Uemura M, Swenson ES, Gaca MD, et al.Smad2 and Smad3 play dif-ferent roles in rat hepatic stellate cell function and smo-oth muscle actin organization [J]. Mol Biol Cell,2005,1(9): 4214-4224.
[14]RobertA B, Piek E, Bottinger EP, et al. Is Smad3 a major player in signal transduction pathways leading to fibrogenesis [J]. Chest,2001,120(Suppl.1):43S-47S.
[15]王瑾,马韵.肝脏纤维化形式和逆转的研究进展[J].医学研究杂志,2009；38(3)：88-89.
[16]孙校男,王先开,娄国强.Smad蛋白与肝纤维化[J].国外医学·流行病学传染病学分册,2005；32(4)：243-245.
[17]何燕,肖诚,胡志峰,等.Smad蛋白及其对中医药防治肝纤维化作用的研究进展[J].中国中医基础医学杂志,2007；13(5)：399-402.
[18]吴凌康,厉有名.Smads家族在肝纤维化中的作用.浙江医学,2005,27(4)：319-321.
[19]Seyhan H, Hamzavi J, Wiercinska E, et al. Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling [J]. J Cell Mol Med,2006; 10(4):922-932.
[20]Ten Dijke P, Hill CS. New insights into TGF β-Smad signalling [J]. Trend Biochem Sci,2004; 29(5):265-273.
[21]Uemura M, wenson ES, Gaca MD, et al. Smad2 and Smad3 play different roles in rat hepatic stellate cell function and smooth muscle actin organization[J].Mol Biol Cell.2005; 16 (9):4214-4224.
[22]Flanders KC. Smad3 as a mediator of the fibrotic response [J]. Int J Exp Pathol.2004; 85 (2):47-64.
[23]Dooley S, Hamzavi J, Breit kopf K, et al. Smad7 prevent activation of hepatic stellate cells and liver f ibrosis in rats [J]. Gastro-enterology,2003; 125(1):178-191.
[24]严蓉,陈峰.Smad蛋白-肝纤维化时TGF β1信号转导的关键物质[J].国际流行病学传染病学杂志,2007；34(3)：191-195.
[25]Tahashi Y,Matsuzaki K, Date M, et al. Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury [J]. Hepatology,2002; 35:49-61.
[26]DK Lee, Shee Park, Y Yi, et al.The hepatitis B virus encoded oncoprotein pX amplifies TGF-β family signaling through direct interaction with Smad4:potential mechanism of hepatitis B virus-induced liver fibrosis [J]. Genes Dev,2001; 15(4):455-466.
[27]W Zhang, J Ou, Y Inagaki,et al. Synergistic Cooperation between Sp1 and Smad3/Smad4 Mediates Transforming Growth Factor β1 Stimulation of α2 (Ⅰ)-Collagen (COL1A2)Transcription[J]. J Biol Chem,2000; 275(50):39237-39245.
[28]郭宝良,闫朝岐,仲雷,等.Smad4 siRNA对转化生长子p诱导Ⅰ型胶原表达的作[J].中国组织工程研究与临床康复,2009；13(11)：2062-2065.
[1]Bataller R, Brenner DA. Liver fi brosis [J]. J Clin Invest, 2005; 115:209-218.
[2]李石.肝纤维化发生机制[J].中华消化杂志,1999；19：48-50.
[3]席文娜,孙水林,李方春,等.α-2a干扰素对大鼠肝组织bc1-2基因表达的影响及意义[J].世界华人消化杂志,2009；17(31)： 3237-3240. [4] Holley SL,Mathias C, Jahnke V, et al. Association of cyclinDl polymorphism with increased susceptibility to oral squamous cell carcinoma [J]. Oral Oncol,2005; 41 (2):156-160.
[5]Harper N, Hughes M, MacFarlane M, et al. Fas-associated death dom-ain protein and caspase-8 are not recruited to the tumor necros-is factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis [J].J Biol Chem,2003; 278:25534-25541
[6]Jaeschke H, Gores GJ, Cederbaum AI, et al. Mechanisms of hepatoto xicity[J].Toxicol Sci,2002; 65:166-176.
[7]Baskin-Bey ES,Gores GJ. Death by association:BH3 domain only proteins and liver injury[J]. Am J Physiol Gastrointest Liver Physiol,2005; 289:987-990.
[8]Oltvai ZN, Milliman CL, Korsmeyer SJ.Bcl-2 heterodimerizes in-vivo with a conserved homolog, Bax, that accelerates programmed cell death [J]. Cell,1993; 74:609-619.
[9]Rogerio F, Jordao H JR, Vieira AS, et al. Bax and Bcl-2 expression and TUNEL labeling in lumbar enlargement of neonatal rats after sciatic axotomy and melatonin treatment [J]. Brain Res,2006,11 (12):80-90.
[10]Higuchi M,Aggarwal BB, Yeh ET. Activation of CPP32-like pro-tease in tumor necrosis factorinduced apoptosis is depend-ent on mitochondrial function[J].J Clin Invest,1997;99:1751-1758.
[11]孙怀宇,王军,王鹏.Bcl-2家族和Bcl-x1对细胞凋亡调控的研究进展[J].中国煤炭工业医学杂志,2009；12(4)：675-677.
[12]孙桦.靶向bcl-2基因的siRNA对肝星状细胞凋亡的影响[J].上海交通大学学报(医学版),2008；28(7)：845-849.
[13]Motokura T, Arnold A. Cycl inD and oncogenesis [J]. CurrOpin Genet Dev.1993; 3(1):5-10.
[14]Umekita Y,OHI Y.Overexpression of cyclin D1 predicts for poor prognosis in estrogen receptor-negative breast cancer patien-ts[J].Int J Cancer,2002; 98 (3):415-418.
[15]张伟,杜成友,徐尔侃.肝癌临床发展与cyclinDl表达DNA含量及细胞凋亡发生的关系[J].第三军医大学学报,2005；27(5)：46.
[16]Loyer P, Trembley JH, KatonaR, et al.Role of CDK/cyclin comple-xes in transcription and RNA splicing. Cell Signa[J],2005; 17(9):1033-1051.
[17]Hirsch FR, Witta S, Biomarkers for Prediction of sensitivity to EGFR inhibitiors in non-small Cell lung caneer.CurrOpin Onc-ol [J].2005; 17 (2):118-122.
[18]Chenchen Li,Xinmei Li.The different roles of cyclin D1-CDK4 in STEP and mGluR-LTD during the Postnatal development in mice hippocampus area CA1[J].BMC Dev Biol,2007; 7:57.
[19]Holley SL,Heighway J,Hoban PR. Induced expression of human CCND1 altemative transeripts in mouse Cyl-1 knockout fibrob-lasts high lights functional differences [J]. Int J Cancer,2005; 114(3):364-370.
[1]Friedman S L,Bansal M B.Reversal of hepatic fibros is-fact or fantasy[J].Hepatology,2006;43(2 Suppl 1):S82-S88.
[2]姚真敏,吕圭源,赵治友.鳖甲煎丸抗肝纤维化作用的实验研究[J].浙江中医学院学报,1997；21(1)：45-46.
[3]任新周,彭勃,袁词.丙肝康对大鼠肝纤维化抑制作用及其机理探讨[J].江苏中医,2000；21(11)：48-52.
[4]姚立,姚真敏,余涛.鳖甲煎口服液对大鼠肝纤维化的影响[J].中药药理与临床,2002；18(6)：527-530.
[5]王晓玲,刘平,刘成海.丹酚酸A对成纤维细胞活力、增殖及胶原合成的影响[J].中西医结合肝病杂志,2000；10(1)：24-25.
[6]周正,陈捷,周萍.抗纤软肝冲剂药物血清对肝星状细胞增殖的影响[J].中药材,2001；24(11)：809-810.
[7]郭传勇,申振宇,钟敏章,等.中药清肝饮对大鼠肝贮脂细胞和成纤维细胞增殖效应的调控作用[J].中国中医药科技,2000；7(5)：285-286
[8]申凤俊,朱跃科,阴宏,等.DMN肝纤维化模型肝组织Ⅰ、Ⅲ胶原和TIMP-1mRNA表达及复方861的干预作用[J].临床和实验医学杂志,2002；1(3)：150-153.
[9]刘成,刘平,刘成海,等.扶正化瘀方对大鼠肝星状细胞、肝细胞、成纤维细胞胶原合成的影响[J].中国中西医结合脾胃杂志,1998；6(1)：29-31.
[10]邱德凯,李海,曾民德,等.虫草多糖脂质体对大鼠肝纤维化胶原酶mRNA变化的影响[J].新消化病学杂志,1997；5(7)：417-421.
[11]朱冰,刘殿武,房文亮,等.抗纤1号对实验性肝纤维化大鼠组织Ⅰ、Ⅲ型胶原及转移生长因子β1mRNA的影响[J].中国中医基础医学杂志,2003；9(1)：21-23.
[12]李平,朱清静,张建军,等.软肝冲剂对大鼠肝星状细胞增殖及分泌TGE-β1影响的血清药理学研究[J].中国中医药科技,2001；8(5)：292-293.
[13]杨文卓,曾明德,范竹萍,等.氧化苦参碱防治二甲基亚硝胺诱导的大鼠肝纤维化的实验研究[J].中华消化杂志；2003；23(3)：165-167.
[14]舍世锋,贤樟,光东.不同剂量丹参注射液抗肝纤维化临床研究[J].中国中西医结合杂志,2004；24(1)：17-20.
[15]郑磊,戴立里.丹参素对转化生长因子β1刺激肝星状细胞信号传导的影响[J].重庆医科大学学报,2009；34(2)：182-185.
[16]王丽娜,陶艳艳,李书,等.丹酚酸B盐对大鼠纤维化肝组织脂质过氧化及其MMP-2活性的影响[J].中国中医杂志,2010；35(1)：71-75.
[17]陈在忠,王红.川芎嗪抗肝纤维化作用的实验研究[J].中西医结合肝 病杂志,2007；7(3)：156-158.
[18]王文丽,李孝生,陈爽.川芎嗪对肝星状细胞增殖及结缔组织生长因子表达的影响[J].中国生物制品学杂志,2009；22(1)：65-68.
[19]李涛,范好,刘芳.川芎嗪对肝纤维化大鼠Fas和FasL表达的影响[J].现代中西医结合杂志,2008；17(10)：1468-1469.
[20]陈爽,李孝生,王文丽.川芎嗪对转化生长因子β1刺激肝星状细胞CTG及Ⅰ型胶原表达的影响[J].生命科学研究,2009；13(2)：146-151.
[21]孙玉凤,冯志杰,孙泽明,等.黄芪抗肝纤维化的实验研究[J].河北中医药学报,2008；23(1)：9-10.
[22]程鹏,刘素侠,孙晋浩.黄芪抗肝纤维化的作用与转化生长因子-β1及干扰素Y的关系[J].临床军医杂志,2000；28(3)：22-23.
[23]宋少刚,杨雁,陈敏珠.黄芪提取物对大鼠肝星状细胞增殖及产生胶原的影响[J].中国临床药理学与治疗学,2001；6(2)：111-113.
[24]王要军,权启镇,孙自勤.黄茂对实验性肝纤维化组织ICAMI表达影响的免疫组化研究[J].中国临床药理学与治疗学,2000；5(1)：49-51.
[25]颜吉丽,李华,范钰.虫草多糖对大鼠肝星状细胞核因子-κB活性和肿瘤坏死因数-a表达的影响[J].复旦学报(医学版),2003;30(1)：27-29.
[26]刘玉侃,沈薇,张霞.虫草菌丝对实验性肝纤维化的防治作用及其机制研究[J].中国新药与临床杂志,2004；23(3)：139-143.
[27]孙保木,宣红萍,李风华,等.虫草菌丝提取物对大鼠脂肪性肝炎与肝纤维化的影响[J].中国新药与临床杂志,2008；27(1)：6-11.
[28]王宪波,刘平,唐志鹏,等.虫草菌丝提取物对二甲基亚硝胺大鼠肝窦内皮细胞损伤和表型改变的影响[J].北京中医药大学学报,2008；31(7)：448-451.
[29]周维,胡艳,张红卫,等.羧甲基茯苓多糖对肝纤维化大鼠TGF β-Smad信号转导的影响[J].中国民族民间医药,2009；20(2)：16-18.
[30]杨甫昭,张晓彬,冯英菊,等.泽兰水提物对四氯化碳致小鼠肝纤维化的防治作用[J].中国实验方剂学杂志,2008；14(7)：50-51.
[31]张彦亮,倪海滨,杨大明,等.大黄素抗肝纤维化的细胞学研究[J].江苏医药.2009；35(6)：691-693.
[32]万晓强,郑紫丹,尚军洁.大黄酸对免疫性肝纤维化大鼠形成的影响[J].重庆医学,2009；38(10)：204-206.
[33]吴繁荣,陈飞虎,胡伟,等.鬼针草总黄酮抗大鼠免疫性肝纤维化作用及部分机制研究[J].中国药理学通报,2008；24(6)：753-756.
[341闫波,陈飞虎,吴繁荣,等.鬼针草总黄酮对肝纤维化大鼠治疗作用及机制探讨[J].中国药理学通报,2008；24(12)：1640-1642.
[35]孙旭群1,陈飞虎2,吴繁荣2鬼针草总黄酮对大鼠免疫性肝纤维化治疗作用及抗氧化机制[J].安徽医科大学学报,2009；44(4)462-465.
[36]陈小亮,李俊,邓子煜,等.苦参素对肝纤维化大鼠肝脏TGF-β1的调节作用[J].中国药理学通报,2009；25(6)：761-764.
[37]颜寒斌.苦参素治疗慢性乙型肝炎肝纤维化的临床疗效研究[J].吉林医学,2009；30(8)：1672-1673.
[38]鄂裘恺,郁林曦,朱歆鑫,等.苦参素对实验性肝纤维化的干预作用[J].南通医学院学报,2009；29(2)：95-97.
[39]张汝建,卢兴梅,国力,等.苦参素对肝星状细胞增殖及凋亡的影响[J].泰山医学院学报,2009；30(1)：23-24.
[40]张汝建,王天才,杨明峰,等.苦参素对HSC-T6整合素αV表达的影响[J].胃肠病学和肝病学杂志,2008；17(9)：737-739.
[41]曹力波,李兵,李佐军,等.水飞蓟素对肝纤维化小鼠的保护作用及机制探讨[J].中国药理学通报.2009；25(6)：794-796.
[42]宋维芳,许瑞龄,王登妮,等.水飞蓟宾对大鼠实验性肝纤维化发生发展作用机制的研究[J].山西医科大学学报,2009；40(5)：423-426.
[43]徐宏平,何航.水飞蓟宾对人肝星状细胞LX-2凋亡及α1(Ⅰ)胶原mRNA达的影响[J].郑州大学学报,2009；44(3)：597-599.
[44]Lee SJ, Kim YG, Kang KW, et al. Effects of colchicine on liver functions of cirrhotic rats:beneficial effects result from stellate cell inactivation and inhibition of TGF betal expre-ssion[J].Chem Biol Interact,2004; 147(1):9-21.
[45]石小枫,刘祀,刘林,等.三七皂甙抗实验性肝纤维化的研究[J].中药药理与临床,2004；20(1)：12-14.
[46]薛青,薛绍礼,汪亚松,等.三七总苷对体外培养细胞胶原合成模型的影响.医药导报,2008,5(27)：517-519.
[47]王怡,薛绍礼,张 菁,等.三七总皂苷对传代肝星状细胞增殖的影响[J].淮海医药,2008；26(1)：1-3.
[48]张桂灵,石小枫,冉长清,等.三七总皂苷对抗免疫性肝纤维化的实验研究[J].第三军医大学学报,2007,29(23)：2212-2214.
[49]A. Laxova,M. Earley, P. Farrel, et a. Results of DNA based cys-tic fibrosis newborn screening proficiency testing [J]. Journal of Cystic Fibrosis,2008; 6(2):S14.
[50]张荣华,李景怡,陈如泉,等.三七总皂苷对肝纤维化大鼠肝脏超微结构的影响[J].第三军医大学学报,2005；27(24)：2410-2413.
[51]舒建昌,叶国荣,吕霞,等.姜黄素治疗肝纤维化及其作用机制的初步研究[J].中华肝脏病杂志,2007；15(10)：753-757.
[52]舒建昌,邓亮,叶国荣,等.姜黄素治疗大鼠肝纤维化及其对肝组织Ⅰ、Ⅲ型胶原的影响[J].国际内科学杂志,2008；35(7)：379-382.
[53]何航,华海婴,戈士文,等.姜黄素对四氯化碳诱导大鼠肝纤维化的影响[J].郑州大学学报,2009；44(5)：986-989.
[54]何航,徐宏平,华海婴.姜黄素对四氯化碳诱导肝纤维化大鼠肝组织TGF-β1及Caspase-3表达的影响[J].第四军医大学学报,2009；30(2)：100-103.
[55]胡泰洪,徐敏,蒋祥虎.姜黄素对实验性肝纤维化大鼠肝组织中NF-κB及ERK-1 m RNA表达的影响[J].江苏中医药,2009；41(9)
[56]朱宝和,贺德,黄林,等.姜黄素抑制大鼠肝纤维化的研究[J].中国普通外科杂志,2009；18(7)：723-726.
[57]郑培永,邢练军,尤圣富,等.清肝活血方对肝星状细胞TGFβ1表达及细胞内信号传递的影响[J].江苏中医药,2008；40(7)：84-86.
[58]昭铭,江锋,叶永安,等.抗纤抑癌方对肝纤维化大鼠肝组织基质金属蛋白酶表达的影响廖[J].中医药临床杂志,2009；21(2)：163-65.
[59]崔红燕,刘成海,孙保木,等.扶正化淤方对四氯化碳肝纤维化大鼠基膜型基质金属蛋白酶活性的影响[J].时珍国医国药,2009；(8).
[60]房志鑫,李旭波,刘菁菁,等.蓝玉簪颗粒对大鼠肝纤维化的治疗作用[J].世界华人消化杂志；11-16.
[61]贺卫和,刘勇,杨岩涛,等.保肝颗粒抗大鼠肝纤维化的实验研究[J].中华中医药学刊,2008；26(11)：2502-2503.
[62]贺松其,文彬,吕志平,等.保肝宁对肝纤维化模型大鼠肝组织形态学及JAK2、STAT3蛋白磷酸化水平的影响[J].上海中医药杂志,2008；42(5)：75-79.
[63]叶放,薛博瑜,周珉,等.紫七软肝片对复合因素致大鼠肝纤维化模型的动态影响[J].中华中医药学刊,2009,27(3)：526-529.
[64]王振常,王丽,毛德文,等.壮肝逐瘀煎对肝纤维化大鼠转化生长因子β1表达的影响.河南中医,2009；29(8)：751-752.
[65]王振常,王丽,毛德文,等.壮肝逐瘀煎对肝纤维化大鼠肝星状细胞凋亡的影响[J].中华中医药学刊,2008；26(10)：
[66]林寿宁,王振常,梁尧,等.壮肝逐瘀煎对肝纤维化大鼠TGF2β1及TβRIPⅡmRNA表达的影响[J].中国中医基础医学杂志,2008；14(5)：357-358.
[67]林寿宁,何磊,王振常,等.壮肝逐瘀煎对肝纤维化大鼠TβRI/Ⅱ mRNA表达的影响[J].江苏中医药,2008；40(7)：80-82.
[68]王振常,王丽,毛德文,等.壮肝逐瘀煎对肝纤维化大NF-κBmRNA表达的影响[J].辽宁中医药大学学报,2008；10(7)：134-136.
[69]谢玉梅,聂青和,康文臻,等.中药双甲五灵冲剂对免疫性肝纤维化大鼠抗肝纤维化的分子机制研究[J].胃肠病学和肝病学杂志,2009；18(2)：154-158.
[70]李长秦,袁晓栋,孙守才,等.增损甘露消毒丹对肝纤维化大鼠血清PCⅢ、IV-C影响的实验研究[J].陕西中医学院学报,2009；32(3)：49-52.
[71]李常青,李钢,苏俊芳,等.叶下珠复方Ⅱ号对二甲基亚硝胺诱导大鼠肝纤维化形成的抑制作用[J].辽宁中医杂志,2009；36(8)：1413-1415.
[72]赵辉平,王胜春,胡咏武,等.五灵胶囊调TGF-β/Smad信号通路蛋白对胶原Ⅰ、Ⅲ型表达的影响.国际消化病杂志,2009,29(4)：289-292
[73]赵辉平,王胜春,胡咏武,等.五灵胶囊对肝纤维化大鼠肝组织TGF-β /Smad及Ras/ERK信号通路蛋白的影响赵辉平[J].胃肠病学和肝病学杂志,2009；18(5)：392-396.
[74]王妍,姜慧卿.肝纤维化药物治疗新进展[J].临床荟萃,2009；24(4)：357-359.
[75]肝纤维化的基因治疗研究进展[J].国际消化病杂志,2009；29(1)：45-47.
[76]Jiang W, Yang CQ, Liu WB, et al. Blockage of t ransforming growth factor β receptors prevents progression of pig serum induced rat liver fibrosis[J].World J Gast roenterol,2004; 10:1634-1638.
[77]Cui X, Sakata R, Ueno T, et al. Inhibitory effect of a solublet ransforming growt h factor2h type Ⅱ receptor on the acti-vation of rat hepatic stellate cells in primary culture[J]. J Hepatol,2003; 39:731-737.
[78]Nakamuta M,Morizono S,Tsuruta S,et al. Remote delivery and expression of soluble type Ⅱ TGF-preceptor in muscle pre- vents hepatic fibrosis in rats[J].Int J Mol Med,2005; 16(1): 59-64.
[79]Inagald Y, Kushida M, Higashi K, et al. Cell type-specific interv-ention of transforming growth factor β/Smad signaling suppre-sses collagen gene expression and hepatic fibrosis in mice [J]. Gast roenterology,2005; 129 (1):259-268.
[80]Weng HL, Wang BE, Jia JD, et al. Effect of interferon gamma onhepa-tic fibrosis in chronic hepatitis B virus infection:a randomiz-ed cont rolled study[J]. Clin Gast roenterol Hepatol,2005; 3 (8):819-828.
[81]Chen M, Wang GJ,Diao Y,et al.Adeno2associated virus mediated interferon-γ inhibits the Progression of hepatic fibrosis in vit ro and in vivo [J]. World J Gastroenterol,2005; 11(26):4045-4051.
[82]王吉耀,吴盛迪.抗肝纤维化治疗研究进展[J].肝脏,2009；14(1)：71-73.
[83]姜藻,尤大钰.16-16二甲基前列腺E2,秋水仙碱和硫酸锌抗肝纤维化的实验研究[J].中华消化杂志,1994；14(3)：153-154.
[84]Okuno M, et al.Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-beta in liver stellate cells [J]. Hepatology,1997; 26:913.
[85]李立新,陈大志,贺强,等.血管紧张素受体反义基因对肝星状细胞增殖及Ⅰ型胶原基因表达的影响[J].中华肝脏病杂志,2007；15(10)：789-790.
[86]包小洁.肝素抗炎抗纤维化的研究进展[J].临床和实验医学杂志,2007；6(1)：163-1641.
[87]Ueno T, Sujakuk, Tamaki S, et al. OK-432 treatment increases matrixmetal Loproteinase-9 production and improves dimethyl nitrosamine-induced liver cirrhosis in rats [J].Int J Mol Med,1999; 3(5):497-503.
[88]Hironaka K, Sakaida l,Matsumura Y, et al. Enhanced interstitial collagenase(matrix metalloproteinase-13)production of kupff-er cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis[J]. Biochem Biophys Res Commun,2000; 267(1): 290-295.
[89]刘克洲,侯伟,等.肝纤维化的治疗策略.中国中西医结合杂志,2006,1(1)：82-91.
[90]Siegmund SV, Uchinami H, Osawa Y, et al. Anandamide induces necrosis in primary hepatic stellate cells [J].Hepatology, 2005; 41:1085-1095.
[91]Du SL,Pan H, Lu WY, et al.Cyclic Arg-Gly-Asp peptide-labeled liposomes for targeting drug therapy of hepatic fibrosis in rats[J].J Pharmacol Exp Ther,2007; 322:560-568.
[92]Wang J Y,Song ZJ. Targeting binding of fluorescence labeled cyclic RGD peptides to activated hepatic stellate cells via integrin AVB3 receptor [J]. Hepatology,2007;46 (4suppl):2-6.
[93]Ueki T, Okamoto E, Takeuchi M, et al. Persective on postgenome medicine:Gene therapy for liver cirrhosis[J]. Nippon Rinsho, 2001; 59(1):152-156.
[94]Xue F,Takahara T,Yata Y,et al. Hepatocyte growth factor gene therapy acceleratesregeneration incirrhoticmouse livers after hepatectomy [J]. Gut,2003; 52 (5):694-700.
[95]Matsuno Y, Iwata H, Umeda Y, et al. Hepatocyte growth factor gene transfer into the liver via the portal vein using electropo-ration attenuates rat liver cirrhosis[J]. Gene Ther,2003;10 (18):1556-1569.
[96]Kim W H,Matsumoto K, Bessho K, et al. Growth inhibition and apoptosis in liver myofibroblasts promoted by hepatocyte growth factor leads to resolution from liver cirrhosis [J]. Am J Pathol,2005; 166 (4):1017-1028.
[97]Kusumoto K,Ido A, Moriuchi A, et al. Repeated intravenous injection of recombinant human hepatocyte growth factor amel-iorates liver cirrhosis but causes albuminuria in rats[J].Int J Mol Med,2006; 17 (3):503-509.