中药肝复康在肝纤维化发生发展中的调节作用
|
摘要
背景和目的
肝纤维化是肝脏受到慢性损伤时的一个愈合修复过程,又是所有慢性肝病进展成肝硬化的共同病理基础。有效的治疗肝纤维化可以防止各种慢性肝病向肝硬化发展。近年来,国内外在对肝纤维化启动和进展机制的研究方面取得了一定进展。但是在肝纤维化临床治疗领域,并没有取得明显的进步。许多西药由于抗纤维化作用靶位单一,疗效并不理想,而有些药物毒副作用大于治疗作用,不宜用于临床。我国的中药复方制剂具有多种药效成分协同作用,安全性强的特点,具有显著的优势。中药肝复康是本研究室多年来通过反复实验总结出的抗纤维化经验方,即往的动物实验已多次成功证实了肝复康对肝纤维化组织具有预防及治疗作用。本实验拟在肝星状细胞(hepatic stellate cells,HSC)培养体系中加入血小板衍生生长因子(platelet-derived growth factor,PDGF)及中药血清,应用半定量RT-PCR技术,分别测定肝星状细胞经外源性PDGF处理,再通过不同浓度含药血清干预后,其Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化特点;并以Western blot检测不同方式处理后肝星状细胞STAT、ERK、PI3-K蛋白的不同表达情况。从而进一步揭示中药肝复康对肝星状细胞在肝纤维化中的分子调节机制。
在肝纤维化发病的分子机理方面,尽管目前国内外已从基因和转录水平进行了许多成功的研究。然而基因的功能活性主要靠蛋白质来实现,若能直接从蛋白质整体水平入手来研究将更加全面、真实地揭示肝纤维化的演变过程。但迄今为止,有关中药对肝星状细胞的蛋白质组的干预实验尚无报道。本实验将首次开展中药对肝星状细胞和肝组织蛋白质组的干预实验,可望从蛋白质组学角度进一步揭示肝纤维化的发病机制和中药的药理作用,为新药研制提供理论依据。
方法
一、用RT-PCR方法检测Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化将HSC分为5组:即空白对照组:加10%对照血清的DMEM;模型对照组:加10%对照血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康低剂量组:加10%低剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康中剂量组:加10%中剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康高剂量组:加10%高剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM,分别测定肝星状细胞经外源性PDGF处理,再通过不同浓度含药血清干预后,其Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化特点。
二、Western blot检测不同方式处理后STAT、ERK、PI3-K蛋白的不同表达
将HSC随机分为6组,分别为:对照组:加10%正常对照血清的DMEM;模型组:加10%正常对照血清的DMEM;中药组:加10%中剂量药物血清;ERK阻断剂组:加U0126(ERK阻断剂,终浓度为10μmol/L);PI3K阻断剂组:加LY-294002(PI3K阻断剂,终浓度为10μmol/L);STAT阻断剂组:加Parthenolide(JAK-STAT阻断剂,终浓度为50μmol/L)。以上各组均作用30分钟后,除对照组外,其余各组均加入血小板衍生生长因子(终浓度60ng/ml)。反应10分钟后,提取不同分组HSC总蛋白样品,行Western blot检测STAT、ERK、PI3-K蛋白的不同表达。
三、二维电泳技术检测肝组织及肝星状细胞蛋白质分子的动态表达肝星状细胞2-D电泳:制备正常大鼠肝星状细胞、加入血小板衍生生长因子的肝星状细胞及加入肝复康药物血清和血小板衍生生长因子的肝星状细胞,分别设定为正常对照组、模型组及药物组。分别提取以上各组的总蛋白质并进行等电聚焦电泳,随后进行SDS-PAGE电泳。凝胶染色后比对蛋白质斑点,找出上调和下调的蛋白质。
肝脏组织2-D电泳:将对照组、模型组及药物组大鼠肝脏取出,提取组织总蛋白质,分别进行等电聚焦电泳,随后进行SDS-PAGE电泳。凝胶染色后比对蛋白质斑点,找出上调和下调的蛋白质。统计学处理
多组比较用方差分析,两组间比较用t检验。
结果
一、药物血清对肝星状细胞Ⅰ、Ⅲ型胶原mRNA表达的影响
在空白对照组,HSCⅠ、Ⅲ型胶原mRNA表达呈相对低水平;加入对照血清及PDGF刺激的模型对照组,HSC细胞显示出对外源性PDGF刺激因子的敏感反应性,与空白对照组相比Ⅰ、Ⅲ型胶原mRNA表达明显上调(P<0.01﹚;在药物血清观测组,与模型对照组相比,由PDGF刺激诱导的HSCⅠ、Ⅲ型胶原mRNA表达增加明显受到抑制(P<0.01﹚;不同药物剂量产生的干预效果不同,肝复康各剂量之间比较,以中剂量组Ⅰ型胶原mRNA表达降低最明显,优于低剂量组(P<0.01﹚,和高剂量组相比无显著性差异;中剂量组Ⅲ型胶原mRNA表达降低亦最明显,但三个剂量组之间相比无显著性差异。
二、药物血清对肝星状细胞α-SMA mRNA表达的影响
在空白对照组,HSCα-SMA mRNA表达呈相对低水平;而模型对照组α-SMA mRNA表达呈明显升高(P<0.01﹚;药物血清各组则均具有能明显抑制这种升高的作用(P<0.01﹚,其中以中剂量组干预效果最为明显,但三个剂量组之间相比无显著性差异。
三、肝星状细胞STAT、ERK、PI3K蛋白的表达情况
肝星状细胞STAT、ERK、PI3K蛋白表达在不同干预条件下存在差别,表现为对照组、模型组、中药组、阻断剂组各免疫印迹条带的灰度及面积不同。中药可有效抑制STAT、ERK、PI3K蛋白的表达。此外,在分别应用STAT、ERK、PI3K阻断剂后,除相对应的蛋白质表达明显下调外,其余两种蛋白质的表达也有不同程度的下降。
四、HSC蛋白质的差异展示
(一)培养12小时后蛋白质的差异展示
HSC培养12小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数181个,模型组蛋白质斑点数161个,中药治疗组蛋白质斑点数124个。
(二)培养24小时后蛋白质的差异展示
HSC培养24小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数189个,模型组蛋白质斑点数229个,中药治疗组蛋白质斑点数397个。
(三)培养48小时后蛋白质的差异展示
HSC培养48小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数118个,模型组蛋白质斑点数183个,中药治疗组蛋白质斑点数81个。
(四)不同治疗时间蛋白质的差异展示
随着治疗时间的不断延长,HSC的蛋白质表达在不同时间有了不同变化.有的变
化发生在治疗后24小时,有的则发生在治疗后的48小时。
五、肝组织蛋白质的差异展示
造模4周后,模型组与治疗组肝组织总蛋白质存在着一定差异。具体体现为:蛋白质斑点数不同,模型组217;治疗组241。同时,部分蛋白质在两组中表现出不同的丰度。
结论
一、经肝复康含药血清处理,可下调HSC-T6细胞株Ⅰ、Ⅲ型胶原、α-SMA mRNA表达,不同浓度含药血清作用强度也有所不同。其中以中剂量组干预效果最为明显。中药肝复康对肝纤维化具有明显的抑制作用,其作用机制可能是通过非特异性干扰PDGF功能,抑制Ⅰ、Ⅲ型胶原、α-SMA mRNA的转录,以达到抗纤维化的作用。
二、应用中药肝复康可通过干预HSC主要信号转导途径,从而减弱STAT、ERK、PI3-K的表达,以达到抗肝纤维化的目的。同时,在分别应用STAT、ERK、PI3K阻断剂后,除相对应的蛋白质表达明显下调外,其余两种蛋白质的表达也有不同程度的下降,因此,我们猜测在肝星状细胞内Ras/ERK、PI3K和JAK/STAT途径之间存在着相互作用,既cross-talk。
三、在对三个不同时间段的模型组肝星状细胞蛋白质的凝胶图谱比较发现:每一时间段都有与肝纤维化相关的蛋白质表达,并且随着时间的不断延长,蛋白质的数量也有所变化。这些现象提示了肝纤维化的发生与多种蛋白质的作用有关。
四、中药肝复康治疗肝纤维化是通过调节多个蛋白质表达产生的。随着中药治疗时间的不断延长,肝星状细胞中药治疗组的蛋白质斑点也有了一定的变化。体现出中药复方多成分、多环节、多层次、多靶点的综合药理作用。
Background and objective:
Liver fibrosis is characterized by an accumulated deposition of extracellular matrix materials (ECMs) and the resulting reconstruction of liver parenchyma, companying the impairment of normal liver function. A clinical and experimental study has found that liver cells, hepatic stellate cells (HSC), kupffer cells and sinus endothelial cells all take part in the formation of hepatic fibrosis, in which HSC plays a very important role. Activation of HSC is commonly regarded as the major link of hepatic fibrosis and the main resource of synthesis of ECM. The main characteristic of activation of HSC is excessive proliferation of HSC. In addition,α-SMA is regarded as a marker of activation of HSC Activation of HSC can lead to excessive synthesis of collagen. HSC proliferation is stimulated by growth factors derived from hepatocytes, Kupffer cells, infiltrating macrophages, platelets, and activated HSCs themselves in paracrine and autocrine manners. In particular, PDGF(platelet-derived growth factor,PDGF), the most potent mitogen for HSCs, stimulates their DNA synthesis by activating extracellular signal-regulated kinas(ERK), phophatidylinositol 3-kinase (PI3K) and signal transducer and activator of transcription(STAT) pathways. Hence, PDGF play a very important role in proliferation, activation of HSC and synthesis of ECM. Traditional Chinese medicine has shown its own advantage in treating some difficult diseases. Developed by our department compound Ganfukang has been used as the traditional Chinese medicine for treating hepatic fibrosis. Clinical observations in our affiliated hospital have proved its effective. However, further study of its detailed anti-hepatic fibrosis mechanism is still needed. On the basis of theabove mentioned theory and research developments, our study with the cell culture as technical platform, was to observe the influence of serum collected from rats perfused with Ganfukang on activation and proliferation of HSC in vitro, and using transcription-polymerase chain reaction (RT-PCR) and image analysis technology to observe its influence on the expressions ofα-SMA, collagens I, III, using western blotting to observe its influence on the expressions of protein ERK, PI3K and STAT, particularly through disturbing platelet-derived growth factor dependent signaling pathways and cross-talk among these pathways.
With the rapid development of proteomic methods, it is possible to find and identify the direct sites of Chinese medicine by using comparative proteomic methods. Two-dimensional gel electrophoresis (2-DE) is currently the most common analytical technique available for the study of protein expression patterns. Our study is to observe the influence of Chinese medicine on liver tissues and HSC by using 2-DE. In this way, we can supply the theory basement for drug development.
Materials and methods:
1 Cell culture and grouping for RT-PCR
According to intervening factors, HSCs were divided into 5 groups, i.e. control group, model group, high dosage serum group, medium dosage serum group and low dosage serum group. Upper culture medium was removed after cultivated for 24 h. Control group serum:10% normal SD rat serum +DMEM, model group serum: 10% normal SD rat serum +DMEM+PDGF(final concentration 60ng/ml), high dosage group serum: 10% high dosage serum +DMEM+PDGF(final concentration 60ng/ml), medium dosage group serum: 10% medium dosage serum + DMEM + PDGF(final concentration 60ng/ml) and low dosage group serum: 10% low dosage serum +DMEM+PDGF(final concentration 60ng/ml) were accordingly added. Total RNA was extracted from HSCs. Expression of mRNA was determined by reverse transcription polymerase chain reaction
2 Cell culture and grouping for western blotting
HSC cells were randomly assorted for six groups, those were control group(10% normal SD rat serum +DMEM), model group(10% normal SD rat serum +DMEM), Chinese herb-treated (with 10%medium dosage serum )group, ERK blocker-treated (U0126, final concentration 10μmol/L)group, PI3K blocker-treated (LY-294002, final concentration 10μmol/L )group and STAT blocker-treated (Parthenolide, final concentration 50μmol/L )group, After the treatments of PDGF (10% normal SD rat serum +DMEM+PDGF:final concentration 60ng/ml),and Ganfukang (10% medium dosage serum + DMEM + PDGF:final concentration 60ng/ml) at defined concentration and time(12h, 24h,48h respectively). Cells of control, PDGF-treated or drug-treated, were harvested. Immunoblotting was used to determine the expressions of STAT, ERK and PI3K.
3 Two-dimensional gel electrophoresis
HSCs were assorted for three groups, those were control group, model group, Chinese herb-treated group; Live tissues were divided into 3 groups. Those were control group, model group and Chinese herb-treated group. All protein from those groups was extracted. The method of O’Farrell, with modification, was followed . For the first-dimension, isoelectric focusing was performed by using a vertical mini-IEF system (Jim-X Company) and using analytical SDS-PAGE carried out the second-dimension separation.
Statistical analysis
Values reported in the figures represent means±SD of three or more independent samples. The results were analyzed by the unpaired Student’s t-test. Statistical significance was set at P<0.05.
Results
1 Effect of each group serum on collagen I, III mRNA expression of HSC in rats
By using RT-PCR, we investigated effects of Ganfukang serum on collagen I, III mRNA expression. It shows that Ganfukang serum decreases collagen I, III mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression of collagen I, III mRNA obviously compared with control group P<0.01. Ganfukang serum can decrease collagen I, III mRNA expression significantly compared with model group p<0.01,especially medium dosage serum has the most effective function on inhibition the expression of collagen I mRNA, which has the significant difference compared with low dosage group P<0.01,while there is no significant difference between high dosage group and medium dosage group.
2 Effect of each group serum onα-SMA mRNA expression of HSC in rats
RT-PCR shows that Ganfukang serum decreasesα-SMA mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression ofα-SMA mRNA obviously compared with control group P<0.01. Ganfukang serum can decreaseα-SMA mRNA expression significantly compared with model group p<0.01, especially medium dosage serum has the most effective function on inhibition the expression ofα-SMA mRNA. While there is no significant difference among these three different Ganfukang dosage groups.
3 Protein expression of STAT, ERK and PI3K assay by western blotting
Western blots show the effects of Ganfukang, ERK blocker, PI3K blocker, STAT blocker on PDGF-induced STAT, ERK and PI3K protein levels. The protein levels increased by PDGF. However, The protein levels stimulated by PDGF were significantly inhibited not only by Ganfukang or it’s own blocker, but also by the other two blockers.
4 Comparison of the protein patterns of HSCs
The HSCs were harvested after 12-hour,24-hour and 48-hour treatments of PDGF and Ganfukang respectively. The proteins of control group, PDGF -treated group, and Ganfukang-treated group were extracted and prepared for comparative proteomic analysis. Within the applied pH range (PH 4.0~ 6.0), a protein load of 30μg per 8 cm strip was the optimal quantity for silver-stained gels. Gel scans were analyzed for spot numbers using PDQuest software. After treatments 12 hours, there were 188 spots that the gel of control group contained , 161 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 124 spots. After treatments 24 hours, there were 189 spots that the gel of control group contained,229 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 397 spots. After treatments 48 hours, there were 118 spots that the gel of control group contained,183 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 81 spots. There are many kinds of alterations on protein spots in different time of treatment.
5 Comparison of the protein patterns of liver tissues
For the liver tissues,there were 217 spots that the gel of model groupcontained, the gel of Ganfukang-treated group contained 241 spots.
Conclusions:
1 The anti-fibrosis roles of Ganfukang compound maybe influence the function of PDGF by nonspecific action, thereby inhibit the transcription of I、III collagen、α-SMA mRNA and decrease the production of ECM.
2 The activity of ERK can be inhibited by its inhibitor, while the activity of PI3K and STAT can also be inhibited by ERK inhibitor. So there maybe exist crosstalk among these three pathways. Such crosstalk may contribute to development of liver fibrosis.
3 It was found that there are different protein spots in every different phase by comparing Gel scan among three model groups. The number of protein is changed as the time prolonged. This shows that the pathogenesis of liver fibrosis is involved with many kinds of protein.
4 There is a significant difference at protein level between untreated and PDGF-, Ganfukang- treated cells. It suggests that the differential expression analysis of proteomes may be useful to further study on mechanism of Ganfukang, and search for new targets of anti-liver fibrosis medicine.
肝纤维化是肝脏受到慢性损伤时的一个愈合修复过程,又是所有慢性肝病进展成肝硬化的共同病理基础。有效的治疗肝纤维化可以防止各种慢性肝病向肝硬化发展。近年来,国内外在对肝纤维化启动和进展机制的研究方面取得了一定进展。但是在肝纤维化临床治疗领域,并没有取得明显的进步。许多西药由于抗纤维化作用靶位单一,疗效并不理想,而有些药物毒副作用大于治疗作用,不宜用于临床。我国的中药复方制剂具有多种药效成分协同作用,安全性强的特点,具有显著的优势。中药肝复康是本研究室多年来通过反复实验总结出的抗纤维化经验方,即往的动物实验已多次成功证实了肝复康对肝纤维化组织具有预防及治疗作用。本实验拟在肝星状细胞(hepatic stellate cells,HSC)培养体系中加入血小板衍生生长因子(platelet-derived growth factor,PDGF)及中药血清,应用半定量RT-PCR技术,分别测定肝星状细胞经外源性PDGF处理,再通过不同浓度含药血清干预后,其Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化特点;并以Western blot检测不同方式处理后肝星状细胞STAT、ERK、PI3-K蛋白的不同表达情况。从而进一步揭示中药肝复康对肝星状细胞在肝纤维化中的分子调节机制。
在肝纤维化发病的分子机理方面,尽管目前国内外已从基因和转录水平进行了许多成功的研究。然而基因的功能活性主要靠蛋白质来实现,若能直接从蛋白质整体水平入手来研究将更加全面、真实地揭示肝纤维化的演变过程。但迄今为止,有关中药对肝星状细胞的蛋白质组的干预实验尚无报道。本实验将首次开展中药对肝星状细胞和肝组织蛋白质组的干预实验,可望从蛋白质组学角度进一步揭示肝纤维化的发病机制和中药的药理作用,为新药研制提供理论依据。
方法
一、用RT-PCR方法检测Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化将HSC分为5组:即空白对照组:加10%对照血清的DMEM;模型对照组:加10%对照血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康低剂量组:加10%低剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康中剂量组:加10%中剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康高剂量组:加10%高剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM,分别测定肝星状细胞经外源性PDGF处理,再通过不同浓度含药血清干预后,其Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化特点。
二、Western blot检测不同方式处理后STAT、ERK、PI3-K蛋白的不同表达
将HSC随机分为6组,分别为:对照组:加10%正常对照血清的DMEM;模型组:加10%正常对照血清的DMEM;中药组:加10%中剂量药物血清;ERK阻断剂组:加U0126(ERK阻断剂,终浓度为10μmol/L);PI3K阻断剂组:加LY-294002(PI3K阻断剂,终浓度为10μmol/L);STAT阻断剂组:加Parthenolide(JAK-STAT阻断剂,终浓度为50μmol/L)。以上各组均作用30分钟后,除对照组外,其余各组均加入血小板衍生生长因子(终浓度60ng/ml)。反应10分钟后,提取不同分组HSC总蛋白样品,行Western blot检测STAT、ERK、PI3-K蛋白的不同表达。
三、二维电泳技术检测肝组织及肝星状细胞蛋白质分子的动态表达肝星状细胞2-D电泳:制备正常大鼠肝星状细胞、加入血小板衍生生长因子的肝星状细胞及加入肝复康药物血清和血小板衍生生长因子的肝星状细胞,分别设定为正常对照组、模型组及药物组。分别提取以上各组的总蛋白质并进行等电聚焦电泳,随后进行SDS-PAGE电泳。凝胶染色后比对蛋白质斑点,找出上调和下调的蛋白质。
肝脏组织2-D电泳:将对照组、模型组及药物组大鼠肝脏取出,提取组织总蛋白质,分别进行等电聚焦电泳,随后进行SDS-PAGE电泳。凝胶染色后比对蛋白质斑点,找出上调和下调的蛋白质。统计学处理
多组比较用方差分析,两组间比较用t检验。
结果
一、药物血清对肝星状细胞Ⅰ、Ⅲ型胶原mRNA表达的影响
在空白对照组,HSCⅠ、Ⅲ型胶原mRNA表达呈相对低水平;加入对照血清及PDGF刺激的模型对照组,HSC细胞显示出对外源性PDGF刺激因子的敏感反应性,与空白对照组相比Ⅰ、Ⅲ型胶原mRNA表达明显上调(P<0.01﹚;在药物血清观测组,与模型对照组相比,由PDGF刺激诱导的HSCⅠ、Ⅲ型胶原mRNA表达增加明显受到抑制(P<0.01﹚;不同药物剂量产生的干预效果不同,肝复康各剂量之间比较,以中剂量组Ⅰ型胶原mRNA表达降低最明显,优于低剂量组(P<0.01﹚,和高剂量组相比无显著性差异;中剂量组Ⅲ型胶原mRNA表达降低亦最明显,但三个剂量组之间相比无显著性差异。
二、药物血清对肝星状细胞α-SMA mRNA表达的影响
在空白对照组,HSCα-SMA mRNA表达呈相对低水平;而模型对照组α-SMA mRNA表达呈明显升高(P<0.01﹚;药物血清各组则均具有能明显抑制这种升高的作用(P<0.01﹚,其中以中剂量组干预效果最为明显,但三个剂量组之间相比无显著性差异。
三、肝星状细胞STAT、ERK、PI3K蛋白的表达情况
肝星状细胞STAT、ERK、PI3K蛋白表达在不同干预条件下存在差别,表现为对照组、模型组、中药组、阻断剂组各免疫印迹条带的灰度及面积不同。中药可有效抑制STAT、ERK、PI3K蛋白的表达。此外,在分别应用STAT、ERK、PI3K阻断剂后,除相对应的蛋白质表达明显下调外,其余两种蛋白质的表达也有不同程度的下降。
四、HSC蛋白质的差异展示
(一)培养12小时后蛋白质的差异展示
HSC培养12小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数181个,模型组蛋白质斑点数161个,中药治疗组蛋白质斑点数124个。
(二)培养24小时后蛋白质的差异展示
HSC培养24小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数189个,模型组蛋白质斑点数229个,中药治疗组蛋白质斑点数397个。
(三)培养48小时后蛋白质的差异展示
HSC培养48小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数118个,模型组蛋白质斑点数183个,中药治疗组蛋白质斑点数81个。
(四)不同治疗时间蛋白质的差异展示
随着治疗时间的不断延长,HSC的蛋白质表达在不同时间有了不同变化.有的变
化发生在治疗后24小时,有的则发生在治疗后的48小时。
五、肝组织蛋白质的差异展示
造模4周后,模型组与治疗组肝组织总蛋白质存在着一定差异。具体体现为:蛋白质斑点数不同,模型组217;治疗组241。同时,部分蛋白质在两组中表现出不同的丰度。
结论
一、经肝复康含药血清处理,可下调HSC-T6细胞株Ⅰ、Ⅲ型胶原、α-SMA mRNA表达,不同浓度含药血清作用强度也有所不同。其中以中剂量组干预效果最为明显。中药肝复康对肝纤维化具有明显的抑制作用,其作用机制可能是通过非特异性干扰PDGF功能,抑制Ⅰ、Ⅲ型胶原、α-SMA mRNA的转录,以达到抗纤维化的作用。
二、应用中药肝复康可通过干预HSC主要信号转导途径,从而减弱STAT、ERK、PI3-K的表达,以达到抗肝纤维化的目的。同时,在分别应用STAT、ERK、PI3K阻断剂后,除相对应的蛋白质表达明显下调外,其余两种蛋白质的表达也有不同程度的下降,因此,我们猜测在肝星状细胞内Ras/ERK、PI3K和JAK/STAT途径之间存在着相互作用,既cross-talk。
三、在对三个不同时间段的模型组肝星状细胞蛋白质的凝胶图谱比较发现:每一时间段都有与肝纤维化相关的蛋白质表达,并且随着时间的不断延长,蛋白质的数量也有所变化。这些现象提示了肝纤维化的发生与多种蛋白质的作用有关。
四、中药肝复康治疗肝纤维化是通过调节多个蛋白质表达产生的。随着中药治疗时间的不断延长,肝星状细胞中药治疗组的蛋白质斑点也有了一定的变化。体现出中药复方多成分、多环节、多层次、多靶点的综合药理作用。
Background and objective:
Liver fibrosis is characterized by an accumulated deposition of extracellular matrix materials (ECMs) and the resulting reconstruction of liver parenchyma, companying the impairment of normal liver function. A clinical and experimental study has found that liver cells, hepatic stellate cells (HSC), kupffer cells and sinus endothelial cells all take part in the formation of hepatic fibrosis, in which HSC plays a very important role. Activation of HSC is commonly regarded as the major link of hepatic fibrosis and the main resource of synthesis of ECM. The main characteristic of activation of HSC is excessive proliferation of HSC. In addition,α-SMA is regarded as a marker of activation of HSC Activation of HSC can lead to excessive synthesis of collagen. HSC proliferation is stimulated by growth factors derived from hepatocytes, Kupffer cells, infiltrating macrophages, platelets, and activated HSCs themselves in paracrine and autocrine manners. In particular, PDGF(platelet-derived growth factor,PDGF), the most potent mitogen for HSCs, stimulates their DNA synthesis by activating extracellular signal-regulated kinas(ERK), phophatidylinositol 3-kinase (PI3K) and signal transducer and activator of transcription(STAT) pathways. Hence, PDGF play a very important role in proliferation, activation of HSC and synthesis of ECM. Traditional Chinese medicine has shown its own advantage in treating some difficult diseases. Developed by our department compound Ganfukang has been used as the traditional Chinese medicine for treating hepatic fibrosis. Clinical observations in our affiliated hospital have proved its effective. However, further study of its detailed anti-hepatic fibrosis mechanism is still needed. On the basis of theabove mentioned theory and research developments, our study with the cell culture as technical platform, was to observe the influence of serum collected from rats perfused with Ganfukang on activation and proliferation of HSC in vitro, and using transcription-polymerase chain reaction (RT-PCR) and image analysis technology to observe its influence on the expressions ofα-SMA, collagens I, III, using western blotting to observe its influence on the expressions of protein ERK, PI3K and STAT, particularly through disturbing platelet-derived growth factor dependent signaling pathways and cross-talk among these pathways.
With the rapid development of proteomic methods, it is possible to find and identify the direct sites of Chinese medicine by using comparative proteomic methods. Two-dimensional gel electrophoresis (2-DE) is currently the most common analytical technique available for the study of protein expression patterns. Our study is to observe the influence of Chinese medicine on liver tissues and HSC by using 2-DE. In this way, we can supply the theory basement for drug development.
Materials and methods:
1 Cell culture and grouping for RT-PCR
According to intervening factors, HSCs were divided into 5 groups, i.e. control group, model group, high dosage serum group, medium dosage serum group and low dosage serum group. Upper culture medium was removed after cultivated for 24 h. Control group serum:10% normal SD rat serum +DMEM, model group serum: 10% normal SD rat serum +DMEM+PDGF(final concentration 60ng/ml), high dosage group serum: 10% high dosage serum +DMEM+PDGF(final concentration 60ng/ml), medium dosage group serum: 10% medium dosage serum + DMEM + PDGF(final concentration 60ng/ml) and low dosage group serum: 10% low dosage serum +DMEM+PDGF(final concentration 60ng/ml) were accordingly added. Total RNA was extracted from HSCs. Expression of mRNA was determined by reverse transcription polymerase chain reaction
2 Cell culture and grouping for western blotting
HSC cells were randomly assorted for six groups, those were control group(10% normal SD rat serum +DMEM), model group(10% normal SD rat serum +DMEM), Chinese herb-treated (with 10%medium dosage serum )group, ERK blocker-treated (U0126, final concentration 10μmol/L)group, PI3K blocker-treated (LY-294002, final concentration 10μmol/L )group and STAT blocker-treated (Parthenolide, final concentration 50μmol/L )group, After the treatments of PDGF (10% normal SD rat serum +DMEM+PDGF:final concentration 60ng/ml),and Ganfukang (10% medium dosage serum + DMEM + PDGF:final concentration 60ng/ml) at defined concentration and time(12h, 24h,48h respectively). Cells of control, PDGF-treated or drug-treated, were harvested. Immunoblotting was used to determine the expressions of STAT, ERK and PI3K.
3 Two-dimensional gel electrophoresis
HSCs were assorted for three groups, those were control group, model group, Chinese herb-treated group; Live tissues were divided into 3 groups. Those were control group, model group and Chinese herb-treated group. All protein from those groups was extracted. The method of O’Farrell, with modification, was followed . For the first-dimension, isoelectric focusing was performed by using a vertical mini-IEF system (Jim-X Company) and using analytical SDS-PAGE carried out the second-dimension separation.
Statistical analysis
Values reported in the figures represent means±SD of three or more independent samples. The results were analyzed by the unpaired Student’s t-test. Statistical significance was set at P<0.05.
Results
1 Effect of each group serum on collagen I, III mRNA expression of HSC in rats
By using RT-PCR, we investigated effects of Ganfukang serum on collagen I, III mRNA expression. It shows that Ganfukang serum decreases collagen I, III mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression of collagen I, III mRNA obviously compared with control group P<0.01. Ganfukang serum can decrease collagen I, III mRNA expression significantly compared with model group p<0.01,especially medium dosage serum has the most effective function on inhibition the expression of collagen I mRNA, which has the significant difference compared with low dosage group P<0.01,while there is no significant difference between high dosage group and medium dosage group.
2 Effect of each group serum onα-SMA mRNA expression of HSC in rats
RT-PCR shows that Ganfukang serum decreasesα-SMA mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression ofα-SMA mRNA obviously compared with control group P<0.01. Ganfukang serum can decreaseα-SMA mRNA expression significantly compared with model group p<0.01, especially medium dosage serum has the most effective function on inhibition the expression ofα-SMA mRNA. While there is no significant difference among these three different Ganfukang dosage groups.
3 Protein expression of STAT, ERK and PI3K assay by western blotting
Western blots show the effects of Ganfukang, ERK blocker, PI3K blocker, STAT blocker on PDGF-induced STAT, ERK and PI3K protein levels. The protein levels increased by PDGF. However, The protein levels stimulated by PDGF were significantly inhibited not only by Ganfukang or it’s own blocker, but also by the other two blockers.
4 Comparison of the protein patterns of HSCs
The HSCs were harvested after 12-hour,24-hour and 48-hour treatments of PDGF and Ganfukang respectively. The proteins of control group, PDGF -treated group, and Ganfukang-treated group were extracted and prepared for comparative proteomic analysis. Within the applied pH range (PH 4.0~ 6.0), a protein load of 30μg per 8 cm strip was the optimal quantity for silver-stained gels. Gel scans were analyzed for spot numbers using PDQuest software. After treatments 12 hours, there were 188 spots that the gel of control group contained , 161 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 124 spots. After treatments 24 hours, there were 189 spots that the gel of control group contained,229 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 397 spots. After treatments 48 hours, there were 118 spots that the gel of control group contained,183 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 81 spots. There are many kinds of alterations on protein spots in different time of treatment.
5 Comparison of the protein patterns of liver tissues
For the liver tissues,there were 217 spots that the gel of model groupcontained, the gel of Ganfukang-treated group contained 241 spots.
Conclusions:
1 The anti-fibrosis roles of Ganfukang compound maybe influence the function of PDGF by nonspecific action, thereby inhibit the transcription of I、III collagen、α-SMA mRNA and decrease the production of ECM.
2 The activity of ERK can be inhibited by its inhibitor, while the activity of PI3K and STAT can also be inhibited by ERK inhibitor. So there maybe exist crosstalk among these three pathways. Such crosstalk may contribute to development of liver fibrosis.
3 It was found that there are different protein spots in every different phase by comparing Gel scan among three model groups. The number of protein is changed as the time prolonged. This shows that the pathogenesis of liver fibrosis is involved with many kinds of protein.
4 There is a significant difference at protein level between untreated and PDGF-, Ganfukang- treated cells. It suggests that the differential expression analysis of proteomes may be useful to further study on mechanism of Ganfukang, and search for new targets of anti-liver fibrosis medicine.
引文
1 Yoshiji H, Kuriyama S, Yoshiji J, Ikenaka Y, Noguchi R, Nakatani T, Tsujinoue H, Yanase K,Namisaki T, Imazu H, Fukui H. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology 2002;36:850-860.
2 Pinzani M. Novel insights into the biology and physiology of the Ito cell. Pharmacol Ther 1995;66: 387-412.
3 Desmoulier A, Xu, G, Costa AM, Youset IM, Gabbiani G, Tuchweber B. Effect of pentoxifylline on early proliferation and phenotypic modulation of fibrogenic cells in two rat models of liver fibrosis and on cultured hepatic stellate cells. J Hepatol 1999;30:621-631.
4 Burt AD. Pathobiology of hepatic stellate cells. J Gastroenterol 1999; 34:299-304.
5 Cales P. Apoptosis and liver fibrosis: antifibrotic strategies. Biorned Pharmacother 1998;52:259-263.
6 徐婷婷,姜妙娜,贾玉杰,车颖 . 肝复康对实验性大鼠肝纤维化肝组织Ⅰ、Ⅲ型胶原含量的影响.山东中医杂志 2004,23(11):682～684.
7 车颖, 徐婷婷,姜妙娜,贾玉杰 . 肝复康抗大鼠肝纤维化的实验研究 .中国中药杂志,2004,29(6):599～600.
8 张国,王天才,唐望先等. Smad3、 Smad7 基因表达与肝纤维化发病关系研究 .中华消化杂志.2002,22:647-650.
9 Shun-Gen Guo, Wei Zhang, Tao Jiang, Min Dai, Lu-Fen Zhang, Yi-Chun Meng, Li-Yun Zhao, Jian-Zhao Niu. Influence of serum collected from rat perfused with compound Biejiaruangan drug on hepatic stellate cells. World Journal of Gastroenterology. 2004;10 (10): 1487-1494.
10 徐婷婷,姜妙娜,贾玉杰,车颖 .肝复康对实验性肝纤维化时转化生长因子 β及层粘连蛋白表达的影响.临床肝胆病杂志,2005,21(1):30～32.
11 车颖, 徐婷婷,姜妙娜,贾玉杰. 肝复康对实验性肝纤维化大鼠肝细胞的保护作用. 中药材,2004,27(6):428～429.
12 车颖 ,王善菊,徐婷婷,姜妙娜,贾玉杰 . 中药肝复康对实验性肝纤维化大鼠肝功能及血清标志物的影响. 放射免疫学杂志 2004,17(4):283～284.
13 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000;275:2247-2250.
14 Gabele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci 2003; 8:69-77.
15 Faouzi S, Le Bail B, Neaud V, et al·[J]·Hepatology, 1999,30:275-284.
16 周俊英 ,甄真 综述 姚树坤 审校 肝星状细胞凋亡与肝纤维化逆转 国外医学内科学分册2004,31(9):402-406.
17 Francis J Eng, Scott L Friedman. Fibrogenesis I. New in-sight into hepatic stellate cellactivation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000,279 (1): G7-G11.
18 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32(4): 496-501.
19 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000, 31 (1):131-140.
20 Burt AD, Cellular and molecular aspects of hepatic fibrosis[J ]J Pathology, 1993, 170 (2)∶105-109.
21 Wang L , Yamasaki G, Johnso R , et al Induction of B2 platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture [J ] J Clin Invest, 1994, 94 (4)∶1563-1567.
22 王琳芳,杨克恭.医学分子生物学原理.北京:高等教育出版社,2001,188-202.
23 王萍 , 袁农 血小板衍生生长因子与肝纤维化的研究进展 西安医科大学学报 2000,21 (3):293-295.
24 Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptor in normal human liver and during active hepatic fibrogenesis. Am J Pathol, 1996, 148(3): 785-800.
25 孙大业,郭艳林,马力耕,等.细胞信号转导.北京.科学出版社,2001,121-129.
26 Friedman SL. Closing in on the signals of hepatic fibrosis [J ] Grastroenterology, 1997, 112 (4) ∶1406-1409.
27 Gressner AM. Cytokines and cellular crosstalk involved in the activation of fat-storing cells. J Hepatology 1995;22(Suppl.2):28-36.
28 Naoki Uyama, Yasuyuki Shimahara, Hiroaki Okuyama, Norifumi Kawada, Shoko Kamo, Kazuo Ikeda, Yoshio Yamaoka Carbenoxolone inhibits DNA synthesis and collagen gene expression in rat hepatic stellate cells in culture. Journal of Hepatology. 2003;39:749-755.
29 Ohyrama H, Shimahara Y, Kawada N. The hepatic stellate cell in the post-genomic era. Histol Histopathol 2002;17:487-495.
30 Guma FCR, Mello TG, Mermelstein CS, Fortuna VA, Wofchuk ST, Gottfried C, Guaragna RM, Costa ML, Borojevic R. Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype. Biochem Cell Biol 2001;79: 409-417.
31 Suzuki C, Kayano K, Uchida I, Okita K. Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity. J Gastroenterol 2001;36:322-329.
32 Buniatian GH, Gebhardt R, Mecke D, Traub P, Wiesinger H. Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo. Neurochem Int 1999; 35:317-327.
33 Woo SW, Nan JX, Lee SH, Park EJ, Zhao YZ, Sohn DH. Aloeemodin suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture. Pharmacol Toxicol 2002;90: 193-198.
34 Levy MT, McCaughan GW, Marinos G, Gorrell MD. Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection. Liver 2002;22:93-101.
35 Friedman S L, Lalazar A,Wang L.HSC-T6 cells, an immortalized rat hepatic stellate cell line. Hepatology,1997,26(p14):338A
36 Davis BH, Chen A , Beno DWA Raf and mitogen activated protein kinase regulate stellate cell collagen gene expression [ J ] J Biol Chem, 1996, 271 ( 3) ∶11039-11043.
37 PinznjM,Marra F, GallgiukiA ,et al Inhibiton by Pentoxifylline of extracelluar signal-regulated kinase activaton by platelet derived growth factor in hepatic stellate cells[J ]Br J Pharmacol, 1996, 119 (6) ∶1117-1121.
38 Preaux AM , Mallat A , Rosenbaum J , et al Pentoxiffyline lnhibits growth and collagen synthesis of cultured human hepatic myofibrobloast-like cells [J ]Hepatology, 1997, 26 (2)∶315-317.
39 Valius M , Kazlaukas A.Phospholipase C-gamma and phosphatidylinostiol 3-kinase are the downstream mediators of the PDGF receptor′s mitogenic signal [J ] Cell, 1993, 73 (1)∶321-323.
40 Bolen JB, Brugge JS. Leukocyte protein tyrosine kinases: Potential targets for drug discovery. Annu Rev Immunol, 1997, 15: 371-404.
41 Ihle JN. STATs: Signal transducers and activator of transcription. Cell, 1996, 84(3):331-334 42 Masaaki M, Hiroshi M, Akira M, et al. Cloning and characterization of novel cis family genes.Biochem Biophys Res Commun, 1997,239 (3) :439-446.
43 刘 景 生 . 细 胞 信 息 与 调 控 . 北 京 : 北 京 医 科 大 学 中 国 协 和 医 科 大 学 联 合 出版,1998,281-302
44 Marra F,Ghosh Choudhury G,Abboud HE. Interferon-gamma-mediated activation of STAT1 alpha regulates growth factor-induced mitogenesis. J Chin Invest, 1996, 98 (5): 1218-1230.
45 Marra F, Arrighi MC, Fazi M,et al,Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology. 1999 Oct;30(4):951-958.
46 Imanishi Y, Maeda N, Otogawa K,et al,Herb medicine Inchin-ko-to (TJ-135) regulates PDGF-BB-dependent signaling pathways of hepatic stellate cells in primary culture and attenuates development of liver fibrosis induced by thioacetamide administration in rats. J Hepatol. 2004 Aug;41(2):242-50.
47 Darnell JE.. STATS and gene regulation . Science 1999;277:1630-1635.
48 David M, Petricoin E III, Benjamin C, Pine R, Weber MJ, Larner AC. Requirement for MAP kinase (ERK2) activity in interferon alpha-and interferon beta-stimulated gene expression through STAT proteins. Science 1995;269:1721-1723.
49 Rosenbaum J, Blazejewski S. Regulation of Ito cell proliferation by soluble factors. J Hepatol,1995, 22 (2 Suppl):65-70.
50 Marra F, Pinzani M, DeFranco R,et al,Involvement of phosphatidylinositol 3-kinase in the activation of extracellular signal-regulated kinase by PDGF in hepatic stellate cells. FEBS Lett.1995 Dec 4;376(3):141-145.
1 郭传勇,申振宇,钟敏章,等.清肝饮对大鼠肝纤维化模型胶原纤维和网状纤维的影响[J].上海铁道大学学报,2000,21(3):1-4.
2 贲长恩,于世瀛,金永三,刘建昭,郭顺根。肝纤维化及中药逆转作用 中医药通报 2002 年 4 月第 1 卷第 2 期 39-45.
3 谢艳爽 郭顺根 血小板衍生生长因子在肝星状细胞内的信号转导 中国组织化学与细胞化学杂志 2004,13(3):321-326.
4 Ikura Y,Morimoto H, Ogam iM , et al Expression of platelet-derived growth factor and its recep tor in livers of patients with chronic liver disease [ J ] J Castroentrology, 1997, 32 (4)∶496-499.
5 Rosenbaum J, Blazejewski S. Regulation of Ito cell proliferation by soluble factors. J Hepatol,1995, 22 (2 Suppl):65-70.
6 Francis J Eng, Scott L Friedman. Fibrogenesis I. New in-sight into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000,279 (1): G7-G11
7 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32(4): 496-501.
8 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000, 31 (1):131-140.
9 高峰 ,程留芳 中医药抗肝纤维化作用机制的研究进展 现代中西医结合杂志 2005 Feb , 14 (3) 395-398.
10 徐婷婷,姜妙娜,贾玉杰,车颖 .肝复康对实验性肝纤维化时转化生长因子β及层粘连蛋白表达的影响.临床肝胆病杂志,2005,21(1):30～32.
11 车颖, 徐婷婷,姜妙娜,贾玉杰. 肝复康对实验性肝纤维化大鼠肝细胞的保护作用. 中药材,2004,27(6):428～429.
12 车颖 ,王善菊,徐婷婷,姜妙娜,贾玉杰 . 中药肝复康对实验性肝纤维化大鼠肝功能及血清标志物的影响. 放射免疫学杂志 2004,17(4):283～284.
13 张国,王天才,唐望先等. Smad3、 Smad7 基因表达与肝纤维化发病关系研究 .中华消化杂志.2002,22:647-650.
14 徐婷婷,姜妙娜,贾玉杰,车颖. 肝复康对实验性大鼠肝纤维化肝组织Ⅰ、Ⅲ型胶原含量的影响.山东中医杂志 2004,23(11):682～684.
15 车颖, 徐婷婷,姜妙娜,贾玉杰 . 肝复康抗大鼠肝纤维化的实验研究.中国中药杂志,2004,29(6):599～600.
16 Willkins MR, et al. Proteome Research: New Frontiers in Function Genomics[M]. New York: Springer, 1997.
17 ennyL,et al. Proteomics: Capacity versus utility[J].Electrophoresois, 2001,21:1071-1081.
18 Hanghui L,et al. Development of mutichannel devices with an array of electrosray tips for highthrough put mass spectrometry[J].Anal Chem,2000,72:3303-3310.
19 刘莺,刘平。大鼠纤维化肝组织高亲水性蛋白质组二维电泳条件的确定[J]。湖南中医学院学报。2003。2:11-16.
20 刘莺,刘平,徐列明等。扶正化瘀方治疗后大鼠纤维化肝组织高亲水性蛋白质分子差异动态展示。 生命科学研究 383-387.
21 Kristensen D B, Norifumi K, et al. Proteome analysis of rat hepatic stellate cell[J]. Hepatology.2000,32(2):269-277.
22 Norifumi K, Kristensen D B, et al. Characterization of a stellate cell activation-associated protein (STAP)with peroxidase activity found in rat hepatic stellate cell[J].The Journal of BiologicaL Chrmistrm,2001,276:25318-25323
23 Sandra Steiner. Proteomics to display lovastatin-induced protein and pathway regulation in rat liver [J]. Electrophoresois,2000,21(14):2129-2137
1 Li D ,Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells : new insights and prospects for therapy[J ] . Gastroenterol Hepatol , 1999 ,14 (7) :618-633.
2 Gressner AM. Mediators of hepatic fibrogenesis [ J ] . Hepatogastroenterology ,1996 ,43 (7) :92-103.
3 Roulot D ,Sevcsik AM,Coste T , et al . Role of transforming growth factor beta type II receptor in hepatic fibrosis : studies of human chronic hepatitis C and experimental fibrosis in rats [J ]. Hepatology ,1999 ,29 (6) :1730-1738.
4 Mallat A ,Preax AM,Blazejewski S , et al . Growth inhibitory and antifi2brogenic effects of interferon- α and interferon-γ on cultured human Itocells [J] . Hepat -ology ,1993 ,18[ Suppl 1 ] :S45-S53.
5 Rockey DC ,Maher JJ ,Jarnagin WR , et al . Inhibition of rat hepatic lipocyte activation in culture by interferon-γ[ J ] . Hepatology , 1992 , 16(4) :776-778.
6 Gressner AM, Polzar B , Lahme B , et al . Induction of rat liver parenchymal cells apoptosis by hepatic myofibroblasts via transforming growth factor beta[J ] . Hepatology ,1996 ,23 (3) :571-581.
7 Mallat A ,Preaus AM, Blazejewski , et al . Interferon alfa and gamma inhibit proliferation and collagen synthes is of human Ito cells in culture [J ] . Hepatology ,1995 ,21(4) :1003.
8 姜藻 ,尤大钰 . 16-16 二甲基前列腺素 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.
9 Okuno M, Moriuaki H , Imai S , et al . Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF- β in liver stellate cells.[J ] . Hepat -ology ,1997 ,26(4) :913-921.
10 Dumont JM,Maignan MF ,Janin B , et al . Effect of malotiate on chronic liver injury induced by carbon tetrachloride in the rat [J ] . Hepatology ,1986 ,3 (2) :260.
11 袁桃霞 ,张锦生 ,张月娥 ,等 . 大鼠肝 Ito 细胞的体外培养及肝素对其抑制作用的观察[J] . 上海医科大学学报,1996 ,23 (2) :902-905.
12 李文才,张锦生,黄光存,等. 肝素对大鼠肝星状细胞生长、细胞外基质和基质金属蛋白酶基因表达的影响[J] . 中华肝脏病杂志, 2000 ,8(4) :200-202.
13 Bockel M,Bader E ,Brochs DG, et al . Beneficial effects of inhibitor ofpoly-hydroxylase in CCl4-induced fibrosis of the liver in rats[J] . Hepa -tology ,1991 ,13[ Suppl 3 ] :S26-S31.
14 Fujimoto J ,Kaneda Y. Reversing liver cirrhosis : impact of gene therapy for liver cirrhosis[J ] . Gene Ther ,1999 ,6 (3) :305-306.
15 Kato R ,Kamiya S ,Ueki M, et al . The Fibronectin-derived antiadensive peptides suppress the myofibroblastic conversion of rat hepatic stellar cells[J ] . Exp Cell Res ,2001 ,265 (1) :54-63.
16 刘忠 ,沈忠义 ,关凤林 ,等 . 中药肝复康对实验性肝硬化内毒素血症的预防及其抗肝纤维化的研究. 中国中西医结合外科杂志,2001 ,7 (3) :148 :2150-2153.
17 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 .浙江中医学院学报,1997 ,21 (1) :45-46.
18 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 . 江 苏 中医,2000 ,21 (11) :48-52.
19 吴荣学,高连相,饶娴宜,等.“抗肝纤1 号”抗肝纤维化的实验研究. 中西医结合肝病杂志,1999 ,9 (2) :33-36.
20 姚 立 , 姚 真 敏 , 余 涛 . 鳖 甲 煎 口 服 液 对 大 鼠 肝 纤 维 化 的 影 响 . 中 药 药 理 与 临床,2002 ,18 (6) :527-530.
21 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 . 中西医结合肝病杂志,2000 ,10 (1) :24-25.
22 张 荣 华 ,周 子 成 ,洪 多 伦 ,等 . 三 七 抗 肝 纤 维 化 的 实 验 研 究 . 第 三 军 医 大 学 学报,2000 ,22 (4) :307-309.
23 马红 ,王宝恩 ,马雪梅 . 复方 861 对大鼠肝星状细胞增殖及胶原合成的影响 . 中国临床药理学与治疗杂志,1998 ,3 (3) :172-175.
24 王暴魁 ,王灵台 ,张填 . 肝纤康对体外培养贮脂细胞增殖及胶原合成的影响 . 中西医结合肝病杂志,1997 ,7 (1) :29-31.
25 马跃荣, Ichiro Shimizu ,Susumulto ,等. 小柴胡汤对大鼠实验性肝纤维化的影响. 沪州医学院学报,1997 ,20 (2) :81-83.
26 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 . 中 药材,2001 ,24 (11) :809-810.
27 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. 中国中医药科技,2000 ,7 (5) :285-286.
28 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 . 临 床 和 实 验 医 学 杂 志 ,2002 ,1 (3) :150-153.
29 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. 中国中西医结合脾胃杂志,1998 ,6(1) :29-31.
30 于世赢 ,责长恩 ,杨美娟 ,等 . 清热利湿方药抗肝纤维化的形态学和免疫组织化学定量研究. 北京中医药大学学报,1997 ,20 (1) :28-33.
31 徐列明,刘平,刘成,等. 桃仁提取物合虫草菌丝抗肝纤维化的实验研究. 中国中医药科技1995 ,2 (1) :16-19.
32 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. 新消化病学杂志,1997 ,5 (7) :417-421.
33 朱冰,刘殿武,房文亮,等. 抗纤l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βlmRNA 的影响. 中国中医基础医学杂志,2003 ,9 (1) :21-23.
34 张斌 ,王灵台 ,陈建杰 . 抗纤复方对大鼠纤维化肝贮脂细胞自分泌转化生长因子β1 的影响. 中国中医药科技,1998 ,5 (5) :280-283.
35 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. 中国中医药科技,2001 ,8(5) :292-293.
36 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. 中华消化杂志,2003 ,23 (3) :165-167.
1 Faouzi S, Le Bail B, Neaud V, et al·[J]·Hepatology, 1999,30:275-284.
2 周俊英 ,甄真 综述 姚树坤 审校 肝星状细胞凋亡与肝纤维化逆转 国外医学内科学分册2004,31(9):402-406.
3 谢艳爽 郭顺根 血小板衍生生长因子在肝星状细胞内的信号转导 中国组织化学与细胞化学杂志 2004,13(3):321-326.
4 Ikura Y,Morimoto H, Ogam iM , et al Expression of platelet-derived growth factor and its recep tor in livers of patients with chronic liver disease [ J ] J Castroentrology, 1997, 32 (4)∶496-499.
5 Gaia R, Maurizio P, Fabio M. et al. Interaction between 4-Hydroxy-2, 3-alkenals and the Platelet-derived Growth Factor-β Receptor. J Biol Chem, 2000, 275 (51): 40561-40567.
6 Rosenbaum J, Blazejewski S. Regulation of Ito cell proliferation by soluble factors. J Hepatol,1995, 22 (2 Suppl):65-70.
7 Francis J Eng, Scott L Friedman. Fibrogenesis I. New in-sight into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000,279 (1): G7-G11
8 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32(4): 496-501.
9 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000, 31 (1):131-140.
10 Burt AD, Cellular and molecular aspects of hepatic fibrosis[J ]J Pathology, 1993, 170 (2)∶105-111.
11 Wang L , Yamasaki G, Johnso R , et al Induction of B2 platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture [J ] J Clin Invest, 1994, 94 (4) ∶1563-1567.
12 王琳芳,杨克恭.医学分子生物学原理.北京:高等教育出版社,2001,188-202.
13 王萍 , 袁农 血小板衍生生长因子与肝纤维化的研究进展 西安医科大学学报 2000,21 (3):293-295.
14 Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptor in normal human liver and during active hepatic fibrogenesis. Am JPathol, 1996, 148(3): 785-800.
15 孙大业,郭艳林,马力耕,等.细胞信号转导.北京.科学出版社,2001,121-129.
16 Friedman SL. Closing in on the signals of hepatic fibrosis [J ] Grastroenterology, 1997, 112 (4) ∶1406-1409.
17 周伟琳, 蔡卫民, 丁永祥 血小板衍生生长因子与肝纤维化 浙江大学学报 ( 医学版) 2000,29(6):283-286.
18 Gressner Am. Mediators of hepatic fibrogenesis [J]. Hepatogastroenterology, 1996, 43 (3)∶92-103.
19 田宇彬 , 刘思良 , 李定国 , 等 . 汉防己甲素对血小板衍生生长因子促细胞增殖效应的阻断作用[J ]. 中华医学杂志, 1997, 77 (1) ∶50-53.
20 Tan EML , Q in H, Kennedy SH, et al. Platelet-derived growth factors-AA and -BB regulate collagen and collagenase gene expression differentially in human fibroblasts[J ]. Biochem J , 1995, 310 (P t 2) ∶585-588.
21 辛绍杰 , 赵景民 , 王林杰 , 等 . 血小板衍生生长因子及其受体与病毒性肝炎肝纤维化的关系[J ]. 中华实验和临床病毒学杂志, 1998, 12 (1) ∶51-53.
22 CarloniV , Romanelli RG, P inzaniM , et al. Facal adhesion kinase and phospholipase CC involvement in adhesion and migration of human hepatic stellate cells [J ].Gastroenterology, 1997, 112 (2) ∶522-531.
23 Yang Z, T ian L , Peng L , et al. Immunohistochemical analysis of growth factor expression and localization in gastric coronary vein of cirrhottic patients[J ]. J Tongji Med Univ, 1996, 16 (4) ∶229-233.
24 Pawso T, Protein modules and signalling networks[J] Nature, 1995, 373(4)∶ 573-575.
25 Marra F, Gentilini A , PinzaniM , et al Phosphatidylinositol 3-k inase is requried for plateled-derived growth factor′s actions on hepatic stellate cells [J] Gastroenter– ology, 1997, 112 (4) ∶1297-1299.
26 Davis BH, Chen A , Beno DWA Raf and mitogen activated protein kinase regulate stellate cell collagen gene expression [J] J Biol Chem, 1996, 271 (3)∶11039-11043.
27 PinznjM,Marra F, GallgiukiA ,et al Inhibiton by Pentoxifylline of extracelluar signal-regulated kinase activaton by platelet derived growth factor in hepatic stellate cells[J ]Br J Pharmacol, 1996, 119 (6) ∶1117-1121.
28 Preaux AM , Mallat A , Rosenbaum J , et al Pentoxiffyline lnhibits growth and collagen synthesis of cultured human hepatic myofibrobloast-like cells [J] Hepatology, 1997, 26 (2)∶315-317.
29 Valius M, Kazlaukas A.Phospholipase C-gamma and phosphatidylinostiol 3-kinase are the downstream mediators of the PDGF receptor′s mitogenic signal [J ] Cell, 1993, 73 (1)∶321-323.
30 Bolen JB, Brugge JS. Leukocyte protein tyrosine kinases: Potential targets for drug discovery. Annu Rev Immunol, 1997, 15: 371-404.
31 Ihle JN. STATs: Signal transducers and activator of transcription. Cell, 1996, 84 (3): 331-334 32 Masaaki M, Hiroshi M, Akira M, et al. Cloning and characterization of novel cis family genes. Biochem Biophys Res Commun, 1997,239 (3) :439-446.
33 刘 景 生 . 细 胞 信 息 与 调 控 . 北 京 : 北 京 医 科 大 学 中 国 协 和 医 科 大 学 联 合 出版,1998,281-302
34 Marra F,Ghosh Choudhury G,Abboud HE. Interferon-gamma-mediated activation of STAT1 alpha regulates growth factor-induced mitogenesis. J Chin Invest, 1996, 98 (5): 1218-1230.
35 DiSario A , Sregliati Baroni G, Bendia E, et al. Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells[J ] Am J Physiol , 1997, 273 (36) ∶G39-G43.
36 Wang Z, Estacion M , Mordan LJ. Ca2+ Influx via T type channels modulates PDGF-Induced replication of mouse fibroblasts [J ]Am J Physiol, 1993, 265 (39)∶C1239-C1242.
37 Failli P, Ruocco C, F ranco RD ,et al. The mitogenic effect of p latelet-derived growth factor in human hepatic stellate cells requires calcium influx [J ] Am J Physiol, 1995, 269 (38)∶C1133-C1135.
38 PinzanjM ,Marra F, Carlonj V. Signal transduction in hepatic stellate cells[J ] Liver, 1998, 18 (1) ∶2-5.
39 姚迪 , 李定国 , 徐芹芳 1 血小板衍生生长因子和钙离子拮抗剂对大鼠肝细胞胶原合成及细胞内游离钙浓度的影响[J ] 胃肠病学和肝病学杂志, 1998, 7 (2)∶ 132-134.
40 Levitzki A , Gazit A Tytosine kinase inhibition: an approach to drug development [J]Science, 1995, 26T (5) ∶1782-1786.
2 Pinzani M. Novel insights into the biology and physiology of the Ito cell. Pharmacol Ther 1995;66: 387-412.
3 Desmoulier A, Xu, G, Costa AM, Youset IM, Gabbiani G, Tuchweber B. Effect of pentoxifylline on early proliferation and phenotypic modulation of fibrogenic cells in two rat models of liver fibrosis and on cultured hepatic stellate cells. J Hepatol 1999;30:621-631.
4 Burt AD. Pathobiology of hepatic stellate cells. J Gastroenterol 1999; 34:299-304.
5 Cales P. Apoptosis and liver fibrosis: antifibrotic strategies. Biorned Pharmacother 1998;52:259-263.
6 徐婷婷,姜妙娜,贾玉杰,车颖 . 肝复康对实验性大鼠肝纤维化肝组织Ⅰ、Ⅲ型胶原含量的影响.山东中医杂志 2004,23(11):682～684.
7 车颖, 徐婷婷,姜妙娜,贾玉杰 . 肝复康抗大鼠肝纤维化的实验研究 .中国中药杂志,2004,29(6):599～600.
8 张国,王天才,唐望先等. Smad3、 Smad7 基因表达与肝纤维化发病关系研究 .中华消化杂志.2002,22:647-650.
9 Shun-Gen Guo, Wei Zhang, Tao Jiang, Min Dai, Lu-Fen Zhang, Yi-Chun Meng, Li-Yun Zhao, Jian-Zhao Niu. Influence of serum collected from rat perfused with compound Biejiaruangan drug on hepatic stellate cells. World Journal of Gastroenterology. 2004;10 (10): 1487-1494.
10 徐婷婷,姜妙娜,贾玉杰,车颖 .肝复康对实验性肝纤维化时转化生长因子 β及层粘连蛋白表达的影响.临床肝胆病杂志,2005,21(1):30～32.
11 车颖, 徐婷婷,姜妙娜,贾玉杰. 肝复康对实验性肝纤维化大鼠肝细胞的保护作用. 中药材,2004,27(6):428～429.
12 车颖 ,王善菊,徐婷婷,姜妙娜,贾玉杰 . 中药肝复康对实验性肝纤维化大鼠肝功能及血清标志物的影响. 放射免疫学杂志 2004,17(4):283～284.
13 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000;275:2247-2250.
14 Gabele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci 2003; 8:69-77.
15 Faouzi S, Le Bail B, Neaud V, et al·[J]·Hepatology, 1999,30:275-284.
16 周俊英 ,甄真 综述 姚树坤 审校 肝星状细胞凋亡与肝纤维化逆转 国外医学内科学分册2004,31(9):402-406.
17 Francis J Eng, Scott L Friedman. Fibrogenesis I. New in-sight into hepatic stellate cellactivation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000,279 (1): G7-G11.
18 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32(4): 496-501.
19 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000, 31 (1):131-140.
20 Burt AD, Cellular and molecular aspects of hepatic fibrosis[J ]J Pathology, 1993, 170 (2)∶105-109.
21 Wang L , Yamasaki G, Johnso R , et al Induction of B2 platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture [J ] J Clin Invest, 1994, 94 (4)∶1563-1567.
22 王琳芳,杨克恭.医学分子生物学原理.北京:高等教育出版社,2001,188-202.
23 王萍 , 袁农 血小板衍生生长因子与肝纤维化的研究进展 西安医科大学学报 2000,21 (3):293-295.
24 Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptor in normal human liver and during active hepatic fibrogenesis. Am J Pathol, 1996, 148(3): 785-800.
25 孙大业,郭艳林,马力耕,等.细胞信号转导.北京.科学出版社,2001,121-129.
26 Friedman SL. Closing in on the signals of hepatic fibrosis [J ] Grastroenterology, 1997, 112 (4) ∶1406-1409.
27 Gressner AM. Cytokines and cellular crosstalk involved in the activation of fat-storing cells. J Hepatology 1995;22(Suppl.2):28-36.
28 Naoki Uyama, Yasuyuki Shimahara, Hiroaki Okuyama, Norifumi Kawada, Shoko Kamo, Kazuo Ikeda, Yoshio Yamaoka Carbenoxolone inhibits DNA synthesis and collagen gene expression in rat hepatic stellate cells in culture. Journal of Hepatology. 2003;39:749-755.
29 Ohyrama H, Shimahara Y, Kawada N. The hepatic stellate cell in the post-genomic era. Histol Histopathol 2002;17:487-495.
30 Guma FCR, Mello TG, Mermelstein CS, Fortuna VA, Wofchuk ST, Gottfried C, Guaragna RM, Costa ML, Borojevic R. Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype. Biochem Cell Biol 2001;79: 409-417.
31 Suzuki C, Kayano K, Uchida I, Okita K. Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity. J Gastroenterol 2001;36:322-329.
32 Buniatian GH, Gebhardt R, Mecke D, Traub P, Wiesinger H. Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo. Neurochem Int 1999; 35:317-327.
33 Woo SW, Nan JX, Lee SH, Park EJ, Zhao YZ, Sohn DH. Aloeemodin suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture. Pharmacol Toxicol 2002;90: 193-198.
34 Levy MT, McCaughan GW, Marinos G, Gorrell MD. Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection. Liver 2002;22:93-101.
35 Friedman S L, Lalazar A,Wang L.HSC-T6 cells, an immortalized rat hepatic stellate cell line. Hepatology,1997,26(p14):338A
36 Davis BH, Chen A , Beno DWA Raf and mitogen activated protein kinase regulate stellate cell collagen gene expression [ J ] J Biol Chem, 1996, 271 ( 3) ∶11039-11043.
37 PinznjM,Marra F, GallgiukiA ,et al Inhibiton by Pentoxifylline of extracelluar signal-regulated kinase activaton by platelet derived growth factor in hepatic stellate cells[J ]Br J Pharmacol, 1996, 119 (6) ∶1117-1121.
38 Preaux AM , Mallat A , Rosenbaum J , et al Pentoxiffyline lnhibits growth and collagen synthesis of cultured human hepatic myofibrobloast-like cells [J ]Hepatology, 1997, 26 (2)∶315-317.
39 Valius M , Kazlaukas A.Phospholipase C-gamma and phosphatidylinostiol 3-kinase are the downstream mediators of the PDGF receptor′s mitogenic signal [J ] Cell, 1993, 73 (1)∶321-323.
40 Bolen JB, Brugge JS. Leukocyte protein tyrosine kinases: Potential targets for drug discovery. Annu Rev Immunol, 1997, 15: 371-404.
41 Ihle JN. STATs: Signal transducers and activator of transcription. Cell, 1996, 84(3):331-334 42 Masaaki M, Hiroshi M, Akira M, et al. Cloning and characterization of novel cis family genes.Biochem Biophys Res Commun, 1997,239 (3) :439-446.
43 刘 景 生 . 细 胞 信 息 与 调 控 . 北 京 : 北 京 医 科 大 学 中 国 协 和 医 科 大 学 联 合 出版,1998,281-302
44 Marra F,Ghosh Choudhury G,Abboud HE. Interferon-gamma-mediated activation of STAT1 alpha regulates growth factor-induced mitogenesis. J Chin Invest, 1996, 98 (5): 1218-1230.
45 Marra F, Arrighi MC, Fazi M,et al,Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology. 1999 Oct;30(4):951-958.
46 Imanishi Y, Maeda N, Otogawa K,et al,Herb medicine Inchin-ko-to (TJ-135) regulates PDGF-BB-dependent signaling pathways of hepatic stellate cells in primary culture and attenuates development of liver fibrosis induced by thioacetamide administration in rats. J Hepatol. 2004 Aug;41(2):242-50.
47 Darnell JE.. STATS and gene regulation . Science 1999;277:1630-1635.
48 David M, Petricoin E III, Benjamin C, Pine R, Weber MJ, Larner AC. Requirement for MAP kinase (ERK2) activity in interferon alpha-and interferon beta-stimulated gene expression through STAT proteins. Science 1995;269:1721-1723.
49 Rosenbaum J, Blazejewski S. Regulation of Ito cell proliferation by soluble factors. J Hepatol,1995, 22 (2 Suppl):65-70.
50 Marra F, Pinzani M, DeFranco R,et al,Involvement of phosphatidylinositol 3-kinase in the activation of extracellular signal-regulated kinase by PDGF in hepatic stellate cells. FEBS Lett.1995 Dec 4;376(3):141-145.
1 郭传勇,申振宇,钟敏章,等.清肝饮对大鼠肝纤维化模型胶原纤维和网状纤维的影响[J].上海铁道大学学报,2000,21(3):1-4.
2 贲长恩,于世瀛,金永三,刘建昭,郭顺根。肝纤维化及中药逆转作用 中医药通报 2002 年 4 月第 1 卷第 2 期 39-45.
3 谢艳爽 郭顺根 血小板衍生生长因子在肝星状细胞内的信号转导 中国组织化学与细胞化学杂志 2004,13(3):321-326.
4 Ikura Y,Morimoto H, Ogam iM , et al Expression of platelet-derived growth factor and its recep tor in livers of patients with chronic liver disease [ J ] J Castroentrology, 1997, 32 (4)∶496-499.
5 Rosenbaum J, Blazejewski S. Regulation of Ito cell proliferation by soluble factors. J Hepatol,1995, 22 (2 Suppl):65-70.
6 Francis J Eng, Scott L Friedman. Fibrogenesis I. New in-sight into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000,279 (1): G7-G11
7 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32(4): 496-501.
8 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000, 31 (1):131-140.
9 高峰 ,程留芳 中医药抗肝纤维化作用机制的研究进展 现代中西医结合杂志 2005 Feb , 14 (3) 395-398.
10 徐婷婷,姜妙娜,贾玉杰,车颖 .肝复康对实验性肝纤维化时转化生长因子β及层粘连蛋白表达的影响.临床肝胆病杂志,2005,21(1):30～32.
11 车颖, 徐婷婷,姜妙娜,贾玉杰. 肝复康对实验性肝纤维化大鼠肝细胞的保护作用. 中药材,2004,27(6):428～429.
12 车颖 ,王善菊,徐婷婷,姜妙娜,贾玉杰 . 中药肝复康对实验性肝纤维化大鼠肝功能及血清标志物的影响. 放射免疫学杂志 2004,17(4):283～284.
13 张国,王天才,唐望先等. Smad3、 Smad7 基因表达与肝纤维化发病关系研究 .中华消化杂志.2002,22:647-650.
14 徐婷婷,姜妙娜,贾玉杰,车颖. 肝复康对实验性大鼠肝纤维化肝组织Ⅰ、Ⅲ型胶原含量的影响.山东中医杂志 2004,23(11):682～684.
15 车颖, 徐婷婷,姜妙娜,贾玉杰 . 肝复康抗大鼠肝纤维化的实验研究.中国中药杂志,2004,29(6):599～600.
16 Willkins MR, et al. Proteome Research: New Frontiers in Function Genomics[M]. New York: Springer, 1997.
17 ennyL,et al. Proteomics: Capacity versus utility[J].Electrophoresois, 2001,21:1071-1081.
18 Hanghui L,et al. Development of mutichannel devices with an array of electrosray tips for highthrough put mass spectrometry[J].Anal Chem,2000,72:3303-3310.
19 刘莺,刘平。大鼠纤维化肝组织高亲水性蛋白质组二维电泳条件的确定[J]。湖南中医学院学报。2003。2:11-16.
20 刘莺,刘平,徐列明等。扶正化瘀方治疗后大鼠纤维化肝组织高亲水性蛋白质分子差异动态展示。 生命科学研究 383-387.
21 Kristensen D B, Norifumi K, et al. Proteome analysis of rat hepatic stellate cell[J]. Hepatology.2000,32(2):269-277.
22 Norifumi K, Kristensen D B, et al. Characterization of a stellate cell activation-associated protein (STAP)with peroxidase activity found in rat hepatic stellate cell[J].The Journal of BiologicaL Chrmistrm,2001,276:25318-25323
23 Sandra Steiner. Proteomics to display lovastatin-induced protein and pathway regulation in rat liver [J]. Electrophoresois,2000,21(14):2129-2137
1 Li D ,Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells : new insights and prospects for therapy[J ] . Gastroenterol Hepatol , 1999 ,14 (7) :618-633.
2 Gressner AM. Mediators of hepatic fibrogenesis [ J ] . Hepatogastroenterology ,1996 ,43 (7) :92-103.
3 Roulot D ,Sevcsik AM,Coste T , et al . Role of transforming growth factor beta type II receptor in hepatic fibrosis : studies of human chronic hepatitis C and experimental fibrosis in rats [J ]. Hepatology ,1999 ,29 (6) :1730-1738.
4 Mallat A ,Preax AM,Blazejewski S , et al . Growth inhibitory and antifi2brogenic effects of interferon- α and interferon-γ on cultured human Itocells [J] . Hepat -ology ,1993 ,18[ Suppl 1 ] :S45-S53.
5 Rockey DC ,Maher JJ ,Jarnagin WR , et al . Inhibition of rat hepatic lipocyte activation in culture by interferon-γ[ J ] . Hepatology , 1992 , 16(4) :776-778.
6 Gressner AM, Polzar B , Lahme B , et al . Induction of rat liver parenchymal cells apoptosis by hepatic myofibroblasts via transforming growth factor beta[J ] . Hepatology ,1996 ,23 (3) :571-581.
7 Mallat A ,Preaus AM, Blazejewski , et al . Interferon alfa and gamma inhibit proliferation and collagen synthes is of human Ito cells in culture [J ] . Hepatology ,1995 ,21(4) :1003.
8 姜藻 ,尤大钰 . 16-16 二甲基前列腺素 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.
9 Okuno M, Moriuaki H , Imai S , et al . Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF- β in liver stellate cells.[J ] . Hepat -ology ,1997 ,26(4) :913-921.
10 Dumont JM,Maignan MF ,Janin B , et al . Effect of malotiate on chronic liver injury induced by carbon tetrachloride in the rat [J ] . Hepatology ,1986 ,3 (2) :260.
11 袁桃霞 ,张锦生 ,张月娥 ,等 . 大鼠肝 Ito 细胞的体外培养及肝素对其抑制作用的观察[J] . 上海医科大学学报,1996 ,23 (2) :902-905.
12 李文才,张锦生,黄光存,等. 肝素对大鼠肝星状细胞生长、细胞外基质和基质金属蛋白酶基因表达的影响[J] . 中华肝脏病杂志, 2000 ,8(4) :200-202.
13 Bockel M,Bader E ,Brochs DG, et al . Beneficial effects of inhibitor ofpoly-hydroxylase in CCl4-induced fibrosis of the liver in rats[J] . Hepa -tology ,1991 ,13[ Suppl 3 ] :S26-S31.
14 Fujimoto J ,Kaneda Y. Reversing liver cirrhosis : impact of gene therapy for liver cirrhosis[J ] . Gene Ther ,1999 ,6 (3) :305-306.
15 Kato R ,Kamiya S ,Ueki M, et al . The Fibronectin-derived antiadensive peptides suppress the myofibroblastic conversion of rat hepatic stellar cells[J ] . Exp Cell Res ,2001 ,265 (1) :54-63.
16 刘忠 ,沈忠义 ,关凤林 ,等 . 中药肝复康对实验性肝硬化内毒素血症的预防及其抗肝纤维化的研究. 中国中西医结合外科杂志,2001 ,7 (3) :148 :2150-2153.
17 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 .浙江中医学院学报,1997 ,21 (1) :45-46.
18 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 . 江 苏 中医,2000 ,21 (11) :48-52.
19 吴荣学,高连相,饶娴宜,等.“抗肝纤1 号”抗肝纤维化的实验研究. 中西医结合肝病杂志,1999 ,9 (2) :33-36.
20 姚 立 , 姚 真 敏 , 余 涛 . 鳖 甲 煎 口 服 液 对 大 鼠 肝 纤 维 化 的 影 响 . 中 药 药 理 与 临床,2002 ,18 (6) :527-530.
21 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 . 中西医结合肝病杂志,2000 ,10 (1) :24-25.
22 张 荣 华 ,周 子 成 ,洪 多 伦 ,等 . 三 七 抗 肝 纤 维 化 的 实 验 研 究 . 第 三 军 医 大 学 学报,2000 ,22 (4) :307-309.
23 马红 ,王宝恩 ,马雪梅 . 复方 861 对大鼠肝星状细胞增殖及胶原合成的影响 . 中国临床药理学与治疗杂志,1998 ,3 (3) :172-175.
24 王暴魁 ,王灵台 ,张填 . 肝纤康对体外培养贮脂细胞增殖及胶原合成的影响 . 中西医结合肝病杂志,1997 ,7 (1) :29-31.
25 马跃荣, Ichiro Shimizu ,Susumulto ,等. 小柴胡汤对大鼠实验性肝纤维化的影响. 沪州医学院学报,1997 ,20 (2) :81-83.
26 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 . 中 药材,2001 ,24 (11) :809-810.
27 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. 中国中医药科技,2000 ,7 (5) :285-286.
28 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 . 临 床 和 实 验 医 学 杂 志 ,2002 ,1 (3) :150-153.
29 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. 中国中西医结合脾胃杂志,1998 ,6(1) :29-31.
30 于世赢 ,责长恩 ,杨美娟 ,等 . 清热利湿方药抗肝纤维化的形态学和免疫组织化学定量研究. 北京中医药大学学报,1997 ,20 (1) :28-33.
31 徐列明,刘平,刘成,等. 桃仁提取物合虫草菌丝抗肝纤维化的实验研究. 中国中医药科技1995 ,2 (1) :16-19.
32 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. 新消化病学杂志,1997 ,5 (7) :417-421.
33 朱冰,刘殿武,房文亮,等. 抗纤l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βlmRNA 的影响. 中国中医基础医学杂志,2003 ,9 (1) :21-23.
34 张斌 ,王灵台 ,陈建杰 . 抗纤复方对大鼠纤维化肝贮脂细胞自分泌转化生长因子β1 的影响. 中国中医药科技,1998 ,5 (5) :280-283.
35 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. 中国中医药科技,2001 ,8(5) :292-293.
36 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. 中华消化杂志,2003 ,23 (3) :165-167.
1 Faouzi S, Le Bail B, Neaud V, et al·[J]·Hepatology, 1999,30:275-284.
2 周俊英 ,甄真 综述 姚树坤 审校 肝星状细胞凋亡与肝纤维化逆转 国外医学内科学分册2004,31(9):402-406.
3 谢艳爽 郭顺根 血小板衍生生长因子在肝星状细胞内的信号转导 中国组织化学与细胞化学杂志 2004,13(3):321-326.
4 Ikura Y,Morimoto H, Ogam iM , et al Expression of platelet-derived growth factor and its recep tor in livers of patients with chronic liver disease [ J ] J Castroentrology, 1997, 32 (4)∶496-499.
5 Gaia R, Maurizio P, Fabio M. et al. Interaction between 4-Hydroxy-2, 3-alkenals and the Platelet-derived Growth Factor-β Receptor. J Biol Chem, 2000, 275 (51): 40561-40567.
6 Rosenbaum J, Blazejewski S. Regulation of Ito cell proliferation by soluble factors. J Hepatol,1995, 22 (2 Suppl):65-70.
7 Francis J Eng, Scott L Friedman. Fibrogenesis I. New in-sight into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000,279 (1): G7-G11
8 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32(4): 496-501.
9 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000, 31 (1):131-140.
10 Burt AD, Cellular and molecular aspects of hepatic fibrosis[J ]J Pathology, 1993, 170 (2)∶105-111.
11 Wang L , Yamasaki G, Johnso R , et al Induction of B2 platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture [J ] J Clin Invest, 1994, 94 (4) ∶1563-1567.
12 王琳芳,杨克恭.医学分子生物学原理.北京:高等教育出版社,2001,188-202.
13 王萍 , 袁农 血小板衍生生长因子与肝纤维化的研究进展 西安医科大学学报 2000,21 (3):293-295.
14 Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptor in normal human liver and during active hepatic fibrogenesis. Am JPathol, 1996, 148(3): 785-800.
15 孙大业,郭艳林,马力耕,等.细胞信号转导.北京.科学出版社,2001,121-129.
16 Friedman SL. Closing in on the signals of hepatic fibrosis [J ] Grastroenterology, 1997, 112 (4) ∶1406-1409.
17 周伟琳, 蔡卫民, 丁永祥 血小板衍生生长因子与肝纤维化 浙江大学学报 ( 医学版) 2000,29(6):283-286.
18 Gressner Am. Mediators of hepatic fibrogenesis [J]. Hepatogastroenterology, 1996, 43 (3)∶92-103.
19 田宇彬 , 刘思良 , 李定国 , 等 . 汉防己甲素对血小板衍生生长因子促细胞增殖效应的阻断作用[J ]. 中华医学杂志, 1997, 77 (1) ∶50-53.
20 Tan EML , Q in H, Kennedy SH, et al. Platelet-derived growth factors-AA and -BB regulate collagen and collagenase gene expression differentially in human fibroblasts[J ]. Biochem J , 1995, 310 (P t 2) ∶585-588.
21 辛绍杰 , 赵景民 , 王林杰 , 等 . 血小板衍生生长因子及其受体与病毒性肝炎肝纤维化的关系[J ]. 中华实验和临床病毒学杂志, 1998, 12 (1) ∶51-53.
22 CarloniV , Romanelli RG, P inzaniM , et al. Facal adhesion kinase and phospholipase CC involvement in adhesion and migration of human hepatic stellate cells [J ].Gastroenterology, 1997, 112 (2) ∶522-531.
23 Yang Z, T ian L , Peng L , et al. Immunohistochemical analysis of growth factor expression and localization in gastric coronary vein of cirrhottic patients[J ]. J Tongji Med Univ, 1996, 16 (4) ∶229-233.
24 Pawso T, Protein modules and signalling networks[J] Nature, 1995, 373(4)∶ 573-575.
25 Marra F, Gentilini A , PinzaniM , et al Phosphatidylinositol 3-k inase is requried for plateled-derived growth factor′s actions on hepatic stellate cells [J] Gastroenter– ology, 1997, 112 (4) ∶1297-1299.
26 Davis BH, Chen A , Beno DWA Raf and mitogen activated protein kinase regulate stellate cell collagen gene expression [J] J Biol Chem, 1996, 271 (3)∶11039-11043.
27 PinznjM,Marra F, GallgiukiA ,et al Inhibiton by Pentoxifylline of extracelluar signal-regulated kinase activaton by platelet derived growth factor in hepatic stellate cells[J ]Br J Pharmacol, 1996, 119 (6) ∶1117-1121.
28 Preaux AM , Mallat A , Rosenbaum J , et al Pentoxiffyline lnhibits growth and collagen synthesis of cultured human hepatic myofibrobloast-like cells [J] Hepatology, 1997, 26 (2)∶315-317.
29 Valius M, Kazlaukas A.Phospholipase C-gamma and phosphatidylinostiol 3-kinase are the downstream mediators of the PDGF receptor′s mitogenic signal [J ] Cell, 1993, 73 (1)∶321-323.
30 Bolen JB, Brugge JS. Leukocyte protein tyrosine kinases: Potential targets for drug discovery. Annu Rev Immunol, 1997, 15: 371-404.
31 Ihle JN. STATs: Signal transducers and activator of transcription. Cell, 1996, 84 (3): 331-334 32 Masaaki M, Hiroshi M, Akira M, et al. Cloning and characterization of novel cis family genes. Biochem Biophys Res Commun, 1997,239 (3) :439-446.
33 刘 景 生 . 细 胞 信 息 与 调 控 . 北 京 : 北 京 医 科 大 学 中 国 协 和 医 科 大 学 联 合 出版,1998,281-302
34 Marra F,Ghosh Choudhury G,Abboud HE. Interferon-gamma-mediated activation of STAT1 alpha regulates growth factor-induced mitogenesis. J Chin Invest, 1996, 98 (5): 1218-1230.
35 DiSario A , Sregliati Baroni G, Bendia E, et al. Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells[J ] Am J Physiol , 1997, 273 (36) ∶G39-G43.
36 Wang Z, Estacion M , Mordan LJ. Ca2+ Influx via T type channels modulates PDGF-Induced replication of mouse fibroblasts [J ]Am J Physiol, 1993, 265 (39)∶C1239-C1242.
37 Failli P, Ruocco C, F ranco RD ,et al. The mitogenic effect of p latelet-derived growth factor in human hepatic stellate cells requires calcium influx [J ] Am J Physiol, 1995, 269 (38)∶C1133-C1135.
38 PinzanjM ,Marra F, Carlonj V. Signal transduction in hepatic stellate cells[J ] Liver, 1998, 18 (1) ∶2-5.
39 姚迪 , 李定国 , 徐芹芳 1 血小板衍生生长因子和钙离子拮抗剂对大鼠肝细胞胶原合成及细胞内游离钙浓度的影响[J ] 胃肠病学和肝病学杂志, 1998, 7 (2)∶ 132-134.
40 Levitzki A , Gazit A Tytosine kinase inhibition: an approach to drug development [J]Science, 1995, 26T (5) ∶1782-1786.