抗纤软肝颗粒对大鼠肝星状细胞核仁非组蛋白的影响
|
摘要
目的：
从蛋白质的调控水平研究抗纤软肝颗粒(KXRG)对肝星状细胞(HSC)增殖的调控机制,进一步探讨KXRG抗肝纤维化的作用机制,从而进一步阐明中药抗肝纤维化的药理学机制和作用靶点,为研制新型抗肝纤维化药物提供理论基础。
方法：
采用活化的肝星状细胞系(HSC-T6)培养传代,然后分别加入中药KXRG(1.25mg/ml、2.5mg/ml、5mg/ml)及其大鼠含药血清(5%、10%、20%)进行培养,并设空白对照组。72小时后,各组取部分细胞用流式细胞仪检测HSC各周期DNA含量,并用电子显微镜观察其超微结构变化,研究KXRG对HSC细胞周期的影响；同时,另部分细胞进行细胞核蛋白提取,利用免疫蛋白印迹法(Western blot)观察用药后HSC核仁非组蛋白(B23、C23)活性水平的变化,探讨KXRG在蛋白质表达水平对活化HSC的调控作用。
结果：
1.KXRG及其含药血清对活化的HSC增殖的影响MTT的结果显示KXRG及其含药血清各浓度组均能抑制活化的HSC增殖,且含药血清组明显优于药物组(P<0.01)；各药物组及含药血清组均优于其对照组(P<0.01,P<0.05),且2.5mg/ml药物组分别与1.25mg/ml、5mg/ml药物组比较有差异(P<0.01,P<0.05)；10%含药血清组分别与5%、20%含药血清组比较有明显差异(P<0.01)。
2. KXRG及其含药血清对活化的HSC细胞周期的影响应用流式细胞仪测得各组细胞在不同细胞周期的DNA含量,结果显示不同浓度含药血清组及药物组作用于细胞72h后,随浓度的增加G0/G1期细胞含量逐渐增加,均与对照组比较有差异(P<0.05,P<0.01),而s期和G2/M期细胞含量则逐渐减少。表明KXRG能阻止细胞由G0/G1期进入S期,但对于G2/M期影响不大,从而说明KXRG主要作用于G1/S关卡,即细胞周期限制点,而对M期的限制点无作用。
3. KXRG及其含药血清对活化的HSC超微结构的影响电镜下正常HSC细胞胞膜完整,胞质细胞器结构清晰,核膜完整,核仁清晰,染色质均匀分布于胞核中；经KXRG预处理后,细胞体积缩小,胞膜表面凹凸不平,细胞质电子密度加深,细胞核收缩,核仁松散,并逐渐分散到细胞核膜周围,并可见细胞内空泡形成。
4. KXRG及其含药血清对核仁非组蛋白B23、C23活性水平的影响Western blot的结果显示KXRG可降低HSC核仁中B23、C23蛋白活性的表达,且随药物及含药血清浓度的增加而降低。
结论：
1、KXRG可以抑制活化的HSC增殖；阻滞其细胞周期的正常进行,使其滞留于G0/G1期；并对其超微结构有明显影响。
2、KXRG能降低HSC核仁非组蛋白B23、C23活性水平的表达。
3、本研究结果提示：KXRG可能通过降低HSC核仁非组蛋白B23、C23活性水平的表达,调控细胞周期,抑制活化HSC的增殖,从而达到抗肝纤维化的作用,这可能是KXRG抗肝纤维化的机制。
Objective
From the regulation of protein level studies in the liver of KXRG (anti-fiber soft granule) on hepatic stellate cells (HSC) proliferation control mechanism, to further explore the role of anti-hepatic fibrosis KXRG mechanism so as to further clarify the anti-Chinese medicine pharmacological mechanism of liver fibrosis and the role of the target for the development of new anti-hepatic fibrosis drugs to provide theoretical basis.
Methods
Use of activated hepatic stellate cells (HSC-T6) culture passage, and then were added to traditional Chinese medicine KXRG (1.25mg/ml,2.5mg/ml,5mg/ml) and the rats were drug-containing serum (5%,10%,20%) were cultured, and established a control group. 72 hours later, each group a portion of HSC cells observed by flow cytometry DNA content in each cycle, and used electron microscopy to observe the changes of ultrastructure of the study KXRG of HSC cycle; the same time, another part of the cells for nuclear protein extraction, using Western blot method (Western blot) observed after treatment of non-histone proteins HSC nucleolus (B23, C23) activity level changes, explore KXRG level of protein expression in HSC activation in vitro.
Results
1. KXRG and its drug-containing serum on activated HSC proliferation in MTT results showed that KXRG containing various concentrations of groups and their serum concentrations can inhibit the proliferation of activated HSC, and the drug-containing serum group was significantly better than the drug group (P< 0.01); the drug group and drug-containing serum group was superior to the control group (P<0.01, P<0.05), and 2.5mg/ml treatment group separately with 1.25mg/ml,5mg/ml drug group is a difference (P <0.01, P<0.05); 10% respectively containing serum and 5%,20% drug-containing serum group significant difference (P<0.01).
2. KXRG and its drug-containing serum on the activation of HSC cell cycle measured by flow cytometry in each group of cells in different cell cycle DNA content showed that serum containing different concentrations of group and drug group acting on the cells, after 72h with the increase in the concentration of content of G0/G1 phase cells increased gradually with the control group is a difference (P<0.05, P<0.01), while the S phase and G2/M phase cell content was gradually reduced. That KXRG can prevent cells from G0/G1 phase to S phase, but G2/M phase had little impact which can show KXRG a major role in the G1/S checkpoint, that is, the cell cycle restriction point, while the M phase restriction point, no effect
3. KXRG and its drug-containing serum on the activation of HSC electron microscope, the ultrastructure of the normal HSC cell membrane integrity and organelle structure, clear cytoplasm, nuclear membrane integrity, a clear nucleolus, chromatin evenly distributed in nucleus; The KXRG pretreatment, the cell volume reduced, the membrane surface, uneven, cytoplasmic electron density deepening contraction in the nucleus, nucleolus loose, and gradually dispersed around the nuclear membrane, and can see the formation of intracellular vacuoles.
4. KXRG and its drug-containing serum on the non-histone proteins nucleolar B23, C23 activity levels Western blot results showed that KXRG can reduce the HSC nucleolar B23, C23 expression of protein activity, and with the KXRG and its drug-containing serum concentration increases.
Conclusion:
1. KXRG can inhibit the proliferation of activated HSC; block the cell cycle of normal to stay in the G0/G1 phase; and had a significant effect on their ultrastructure.
2. KXRG can reduce the HSC nucleolar non-histone proteins B23, C23 activity level expression.
3. The study results suggest that:KXRG possible by reducing the non-histone proteins HSC nucleolar B23, C23 activity level of the expression of cell cycle regulation, inhibit the proliferation of activated HSC to achieve the anti-hepatic fibrosis, which may be KXRG anti-liver fibrosis mechanisms.
从蛋白质的调控水平研究抗纤软肝颗粒(KXRG)对肝星状细胞(HSC)增殖的调控机制,进一步探讨KXRG抗肝纤维化的作用机制,从而进一步阐明中药抗肝纤维化的药理学机制和作用靶点,为研制新型抗肝纤维化药物提供理论基础。
方法：
采用活化的肝星状细胞系(HSC-T6)培养传代,然后分别加入中药KXRG(1.25mg/ml、2.5mg/ml、5mg/ml)及其大鼠含药血清(5%、10%、20%)进行培养,并设空白对照组。72小时后,各组取部分细胞用流式细胞仪检测HSC各周期DNA含量,并用电子显微镜观察其超微结构变化,研究KXRG对HSC细胞周期的影响；同时,另部分细胞进行细胞核蛋白提取,利用免疫蛋白印迹法(Western blot)观察用药后HSC核仁非组蛋白(B23、C23)活性水平的变化,探讨KXRG在蛋白质表达水平对活化HSC的调控作用。
结果：
1.KXRG及其含药血清对活化的HSC增殖的影响MTT的结果显示KXRG及其含药血清各浓度组均能抑制活化的HSC增殖,且含药血清组明显优于药物组(P<0.01)；各药物组及含药血清组均优于其对照组(P<0.01,P<0.05),且2.5mg/ml药物组分别与1.25mg/ml、5mg/ml药物组比较有差异(P<0.01,P<0.05)；10%含药血清组分别与5%、20%含药血清组比较有明显差异(P<0.01)。
2. KXRG及其含药血清对活化的HSC细胞周期的影响应用流式细胞仪测得各组细胞在不同细胞周期的DNA含量,结果显示不同浓度含药血清组及药物组作用于细胞72h后,随浓度的增加G0/G1期细胞含量逐渐增加,均与对照组比较有差异(P<0.05,P<0.01),而s期和G2/M期细胞含量则逐渐减少。表明KXRG能阻止细胞由G0/G1期进入S期,但对于G2/M期影响不大,从而说明KXRG主要作用于G1/S关卡,即细胞周期限制点,而对M期的限制点无作用。
3. KXRG及其含药血清对活化的HSC超微结构的影响电镜下正常HSC细胞胞膜完整,胞质细胞器结构清晰,核膜完整,核仁清晰,染色质均匀分布于胞核中；经KXRG预处理后,细胞体积缩小,胞膜表面凹凸不平,细胞质电子密度加深,细胞核收缩,核仁松散,并逐渐分散到细胞核膜周围,并可见细胞内空泡形成。
4. KXRG及其含药血清对核仁非组蛋白B23、C23活性水平的影响Western blot的结果显示KXRG可降低HSC核仁中B23、C23蛋白活性的表达,且随药物及含药血清浓度的增加而降低。
结论：
1、KXRG可以抑制活化的HSC增殖；阻滞其细胞周期的正常进行,使其滞留于G0/G1期；并对其超微结构有明显影响。
2、KXRG能降低HSC核仁非组蛋白B23、C23活性水平的表达。
3、本研究结果提示：KXRG可能通过降低HSC核仁非组蛋白B23、C23活性水平的表达,调控细胞周期,抑制活化HSC的增殖,从而达到抗肝纤维化的作用,这可能是KXRG抗肝纤维化的机制。
Objective
From the regulation of protein level studies in the liver of KXRG (anti-fiber soft granule) on hepatic stellate cells (HSC) proliferation control mechanism, to further explore the role of anti-hepatic fibrosis KXRG mechanism so as to further clarify the anti-Chinese medicine pharmacological mechanism of liver fibrosis and the role of the target for the development of new anti-hepatic fibrosis drugs to provide theoretical basis.
Methods
Use of activated hepatic stellate cells (HSC-T6) culture passage, and then were added to traditional Chinese medicine KXRG (1.25mg/ml,2.5mg/ml,5mg/ml) and the rats were drug-containing serum (5%,10%,20%) were cultured, and established a control group. 72 hours later, each group a portion of HSC cells observed by flow cytometry DNA content in each cycle, and used electron microscopy to observe the changes of ultrastructure of the study KXRG of HSC cycle; the same time, another part of the cells for nuclear protein extraction, using Western blot method (Western blot) observed after treatment of non-histone proteins HSC nucleolus (B23, C23) activity level changes, explore KXRG level of protein expression in HSC activation in vitro.
Results
1. KXRG and its drug-containing serum on activated HSC proliferation in MTT results showed that KXRG containing various concentrations of groups and their serum concentrations can inhibit the proliferation of activated HSC, and the drug-containing serum group was significantly better than the drug group (P< 0.01); the drug group and drug-containing serum group was superior to the control group (P<0.01, P<0.05), and 2.5mg/ml treatment group separately with 1.25mg/ml,5mg/ml drug group is a difference (P <0.01, P<0.05); 10% respectively containing serum and 5%,20% drug-containing serum group significant difference (P<0.01).
2. KXRG and its drug-containing serum on the activation of HSC cell cycle measured by flow cytometry in each group of cells in different cell cycle DNA content showed that serum containing different concentrations of group and drug group acting on the cells, after 72h with the increase in the concentration of content of G0/G1 phase cells increased gradually with the control group is a difference (P<0.05, P<0.01), while the S phase and G2/M phase cell content was gradually reduced. That KXRG can prevent cells from G0/G1 phase to S phase, but G2/M phase had little impact which can show KXRG a major role in the G1/S checkpoint, that is, the cell cycle restriction point, while the M phase restriction point, no effect
3. KXRG and its drug-containing serum on the activation of HSC electron microscope, the ultrastructure of the normal HSC cell membrane integrity and organelle structure, clear cytoplasm, nuclear membrane integrity, a clear nucleolus, chromatin evenly distributed in nucleus; The KXRG pretreatment, the cell volume reduced, the membrane surface, uneven, cytoplasmic electron density deepening contraction in the nucleus, nucleolus loose, and gradually dispersed around the nuclear membrane, and can see the formation of intracellular vacuoles.
4. KXRG and its drug-containing serum on the non-histone proteins nucleolar B23, C23 activity levels Western blot results showed that KXRG can reduce the HSC nucleolar B23, C23 expression of protein activity, and with the KXRG and its drug-containing serum concentration increases.
Conclusion:
1. KXRG can inhibit the proliferation of activated HSC; block the cell cycle of normal to stay in the G0/G1 phase; and had a significant effect on their ultrastructure.
2. KXRG can reduce the HSC nucleolar non-histone proteins B23, C23 activity level expression.
3. The study results suggest that:KXRG possible by reducing the non-histone proteins HSC nucleolar B23, C23 activity level of the expression of cell cycle regulation, inhibit the proliferation of activated HSC to achieve the anti-hepatic fibrosis, which may be KXRG anti-liver fibrosis mechanisms.
引文
[1]#12
[2]孙艳红,方晓艳,刘红雨,等.中药血清药理学方法研究概[J].时珍国医国药,2002,13(2)：114.
[3]王喜军.中药血清药物化学的研究动态及发展趋势[J].中国中药杂志.2006,31(10)：789-792.
[4]史丙俊,陈德宇.中药血清药理学研究初探[J].泸州医学院学报,2003,26(3)：272-273.
[5]陆慧晶.血清药理学研究中的若干问题探讨[J].基层中药杂志2000,14(3)：51.
[6]李静,殷飞,姚树坤,等.血清药理学研究进展[J].中国中医基础医学杂志,2009,15(3)：234-236.
[7]赵万红,曹永孝,袁泽飞.中药血清学的方法学探讨[J].中药新药与临床药理,2002,13(2)：122.
[8]于海荣,王济兴,陈建双.血清药理学方法研究进展[J].承德医学院学报,2006,23(2)：183-185.
[9]崔玲,晏卫东.中药血清药理研究方法回顾与展望[J].武警医学,2004,15(6)：460-461.
[10]张军平,张伯礼,山本清高.中药药物血清的制作方法探讨[J].天津中医药,2004,21(4)：274-276.
[11]张良,徐立,袁冬萍,等.中药血清药理学方法研究进展[J].南京中医药大学学报,2002,18(4)：254.
[12]张德付.中药血清药理学方法在肿瘤研究中存在的主要问题及对策[J].中医研究,2004,17(5)：13-14.
[13]韩笑,刘建勋,林成仁.血清药理学关键问题的存疑与讨论[J].中药药理与临床,2009,25(2)：122-124.
[14]HautekeeteML, GeertsA. VirchowsArch,1997,430(3):195-207.
[15]陈主初.病理生理学.北京：人民卫生出版社,2006,331-335.
[16]Solis-Herruzo JA, de la Torre P, Munoz-Yague MT. Hepatic stellate cells (HSC):architects of hepatic fibrosis [J]. Rev Esp Enferm Dig,2003,95:438-439.
[17]Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell acti-vation[J]. Med Gen Med,2002,4:27-30.
[18]Giannelli G, Antonaci S. Immunological and molecular aspects of liver fibrosis in chronic hepatitis C virus infection. Histol Histopathol,2005,20:939-944.
[19]Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis,2001,21: 437-451.
[20]Friedman S L.Molecular regulation of hepatic fibrosis,an integrated cellular response to tissue injury.J Biol Chem,2000,275 (4):2247-2250.
[21]陈主初.病理生理学.北京：人民卫生出版社,2006.15-20.
[22]杨玲,朱清静,张赤志.软坚消痰活血化瘀法抗肝纤维化的作用机制概述[J].中医药学刊,2005,23(11)：1969-1971.
[23]高建蓉,张赤志,邵志华,等.鳖甲对肝星状细胞增殖影响的研究[J].实用医学杂志,2007,23(11)：1618-1620.
[24]邝卫红,李建华.鳖甲煎汤抗大鼠肝纤维化作用[J].中药新药与临床药理,2004,15(5)：316.
[25]薛博瑜,胡荣昕,孙丽霞.中医药治疗肝纤维化临床研究思路探讨[J].中华中医药杂志,2007,22(8)：542.
[26]熊益群,严红梅,张赤志.抗纤软肝冲剂抗大鼠肝纤维化的实验研究[J].中国实验方剂学杂志,2000,6(2)：201.
[27]Liu H, LinD, YatesJR. Multidimensional separation forprotein/ pep-tide analysis in the post-genomic era [J]. Biotechniques, 2002,32 (4):898-902.
[28]Wilkins MR, Pasquali C, Appel RD, etal. From proteins to proteomes:large scale protein identification by two-dimensional electrophoresis and amino acid analysis [J]. Biotechnology,1996,14 (1):61-65.
[29]GerlaiR. Phenomics:fiction or the future[J]. TrendsNeurosci, 2002,25(10):506-509.
[30]Anderson NL,Anderson NG. Proteome and proteomic:new tech-nologies, new concepts, and new words[J]. Electrophoresis, 1998,19(11):1853-1861.
[31]Petricoin EF, Zoon KC, Kohn EC, etal. Clinical proteomics: translating benchside promise into bedside reality[J]. Nature Rev Drug Discov,2002,1 (9):683-695.
[32]陈化洋.蛋白质组学研究进展及展望[J].淮北职业技术学院学报,2008,7(3)：39-40.
[33]王慷慨,邓恭华,肖献忠.核仁应激损伤与热休克蛋白研究.生理科学进展,2003,34(2)：175-177.
[34]HernandezVD, LouvetE. The nucleolus:structure, functions, and associateddiseases. Med Sci,2004,20(1):37-44.
[35]DerenziniM. TheAgNORs. Micron,2000,31:117-120.
[36]周晓岩,王琳,姜云鹏,等.核仁组成区嗜银蛋白和增殖指数在肾母细胞瘤研究中的应用[J].吉林大学学报医学版,2003,29(1)：79-81.
[37]Itahana K, Bhat K P, Jin A, etal. Tumor suppressor ARF degrades B23, a nucleoar protein involved in ribosome biogenesis and cell proliferation[J]. Mol Cell,2003,12(5):1151-1164.
[38]Okuda M. The role of nucleophosmin in centrosome duplication [J]. Oncogene,2002,21 (40):6170-6174.
[39]云径平,等.B23在肝再生和肝细胞增生过程中的表达与移位[J].中华医学杂志,2002,82(14)：974-978.
[40]Gao H, Jin S, Song Y, etal.B23 regulates GADD45a nuclear trans-location andcontributes to GADD45a-induced cell cycle G2/M arrest [J].J Biol Chem,2005,280(12):10988-10996.
[41]Kurki S, Peltonen K, Laiho M. Nucleophosmin, HMD2 and p53: players in UV damage incited nucleolar stress response[J]. Cell Cycle,2004,3(8):976-979.
[42]WeberJ D, Taylor L J, Roussel M F, etal. Nucleolar Arf sequesters Mdm2 and activates p53[J]. Nat Cell Biol,1999,1(1):20-26.
[43]Kurki S,Peltonen K,Latonen L,etal. Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation [J]. Cancer Cell,2004, 5 (5):465-475.
[44]Hayne C, Xiang X, Luo Z. MEK inhibition and phosphorylation of serine 4 on B23 are two coincident events in mitosis [J]. Biochem Biophys Res Commun,2004,321 (3):675-680.
[45]Takemura M, Ohoka F, Perpelescu M, etal. Phosphorylation-Dependent Migration of Retinoblastoma Protein into the Nucleolus Triggered by Binding to Nucleophosmin/B23[J]. Exp Cell Res,2002,276(2),233-241.
[46]Jiang P S, Chang J H, Yung B Y. Different kinases phosphorylate nucleophosmin/B23 at different sites during G2 and M phasesof the cell cycle [J]. Cancer Lett,2000,153 (1-2):151-160.
[47]Tokuyama Y, Horn H F, Kawamura K, etal. Specif ic phosphorylation of nucleophosmin on Thr(199)by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication [J]. J Biol Chem,2001,276 (24):21529-21537.
[48]Lu Y Y, Lam C Y, Yung B Y. Decreased accumulation and dephosphorylation of the mitosis-specific from of nucleo-phosmin/B23 in staurosporne-induced chromosome de-condensation [J]. BiochemJ,1996,317 (Pt 1):321-327.
[49]王慷慨,蒋磊,刘可,等.热休克蛋白70对氧化应激所致核仁素裂解的影响[J].中国动脉硬化杂志,2004,12(4)：373-377.
[50]AndersenJS, LyonCE, FoxAH. Directed proteomic analysis of the human nucleolus. CurrBiol,2002,12(1):1-11.
[51]Daniely Y, Dimitrova DD, Borowiec JA. Stress-dependent nucleolin mobilizetion mediated by p53-nucleolin complex formation. Mol Cell Biol,2002,22 (16):6014-6022.
[52]Yanagida,M., Shimamoto, A., Nishikawa,K. etal. Isolation and proteomic characterization of the major proteins of the Nucleolin-binding ribonucleoprotein complexes[J]. Proteomics 1,2001,1390-1404.
[53]Iliakis G, Krieg T, Guan J,etal. Evidence for an S-phase check-point regulating DNA replication after heat shock[J]. Int J Hyper-thermia,2004,20 (2):240-249.
[54]王慷慨,等.核仁素在不同细胞膜表面的表达[J].中国动脉硬化杂志,2007,15(1)：63.
[55]Hovanessian AG, Puvion-Dutilleul F, NisoleS, etal. The cell-surface expressed Nucleolin is associated with the actin cytoskeleton[J].Exp Cell Res,2000,26(2):312-328.
[56]Khurts S, Masutomi K, Delgermaa L,etal. Nucleolin interacts with telomerase[J]. J Biol Chem,2004,279(49):51508-51515.
[57]Joo EJ. Nucleolin:acharan sulfate-binding protein on the surface of cancer cells [J]. Glycobiology,2005,15 (1):129.
[1]康谊,王天才,等.15d-PGJ2对肝星状细胞增殖和凋亡的影响[J].中国药理学通报,2007,23(2)：177-180.
[2]肖永红,刘殿武,等.复方中药血清对四氯化碳刺激的肝星状细胞增殖的影响[J].中国中医基础医学杂志,2006,12(1)：18-19.
[3]龚非力.医学免疫学.第2版.科学出版社,2004,61-83.
[4]Borkham-Kamphorst E, Herrmann J,Stol1 D, Treptau J, Gressner AM, Weiskirchen R. Dominant-negative soluble PDGF-beta receptor inhibits HSC activation and attenuates liver fibrosis.Lab Invest,2004,84:766-777.
[5]冯海娟,陆翠华.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.
[6]Bonner JC.Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev 2004,15:255-273.
[7]Tsukada S, Parsons C J, Rippe R A. Mechanisms of liver fibrosis[J]. Clin Chim Acta,2006,364(1-2):33-60.
[8]黄思敏,等.姜黄素对肝纤维化大鼠肝组织中α-SMA,PDGF-BB和TGF-β表达的影响[J].广东医学,2008,29(6)：929-931.
[9]张晓芸,等.苦参碱对血小板衍生生长因子介导的大鼠肝星状细胞迁移、增殖和凋亡的影响[J].中国慢性病预防与控制,2008,16(4)：334-336.
[10]齐荔红,刘晔,等.大黄素对大鼠肝星状细胞增殖和合成胶原的影响[J].第二军医大学学报,2005,26(10)：1190-1191.
[11]杨玲,张赤志,等.抗纤软肝颗粒对PDGF诱导的HSC增殖的影响[J].中国中西医结合消化杂志,2004,12(2)：80-82.
[12]姚希贤,房红梅,王军民,等.拆方益肝康与药物血清对肝星状细胞PDGF及受体表达的影响[J].世界华人消化杂志,2005,13(12)：1412-1416.
[13]王飞.TGF-β1在肝纤维化中的作用[J].中国实用医药,2008,3(23)：189-191.
[14]徐宁,等.TGF-β1于肝纤维化的关系再探讨[J].重庆医药,2008,37(20)：2356-2357.
[15]戴颖,朱萱.生长因子家族在肝纤维化中的作用[J].中国全科医学,2008,11(6A)：1002-1004.
[16]朱清静,等.剔毒护肝方对HSC表达Ⅰ型胶原及TGF-β 1mRNA的影响[J].中国中医药科技,2002,9(2)：76-77.
[17]姜春萌,刘成海,刘成.扶正化瘀胶囊对肝星状细胞激活的干预[J].中国中西医结合消化杂志,2003,11(5)：280.
[18]石小枫,刘杞,徐曼,等肝心康对肝纤维化大鼠血清学指标及TGF-β1的影响[J].中药药理与临床,2004,20(2)：34.
[19]曾军.肝星状细胞、相关因子与肝纤维化关系的研究进展[J].中华肝胆外科杂志,2005,11(6)：427-429.
[20]主余华,等.CTGF shRNA对肝星状细胞细胞因子及细胞外基质表达的影响[J].山东大学学报医学版,2007,45(6)：577-581.
[21]Kim KH, Park GT,Lim YB,etal. Expressionof connective tissue growth factor, a biomarker in senescenceof human diploid fibroblasts, is up-regulated by a transforming growth fac-tor-beta-mediated signaling pathway[J]. Biochem Biophys Res Commun,2004,318(4):819-825.
[22]Abou-Shady M, Friess H, Zimmermann A, etal. Connective tissue growth factor in human liver cirrhosis [J]. Liver,2000,20: 296-304.
[23]贺松其.鳖甲煎丸对肝纤维化模型大鼠CTGF的影响[J].辽宁中医杂志,2005,32(12)：1334-1335.
[24]主余华,张春清,等.银杏叶提取物对肝星状细胞增殖及转化生长因子和结缔组织生长因子基因表达的影响[J].中国病理生理杂志,2007,23(7)：1316-1321.
[25]谢明,廖晓宏,等.姜黄素对肝星状细胞CTGF表达的影响[J].实用医学杂志,2006,22(15)：1724-1726.
[26]宋仕玲,龚作炯,张全荣,等.中药金三莪方对实验性肝纤维化大鼠CTGF及TGF-β1mRNA表达及定位的影响[J].解放军医学杂志,2004,29(2)：130.
[27]叶平,蒋明德.肝纤维化相关因素的研究现状及进展[J].新医学,2008,39(12)：829-831.
[28]薛冬英,洪嘉禾,等.丹参酚酸B对大鼠HSC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.
[29]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)：22-24.
[30]陈主初.病理生理学.北京：人民卫生出版社,2006,34-53.
[31]李骢,等.中药肝复康对PDGF介导的HSC信号转导的影响及意义[J].中华消化杂志,2006,26(9)：636-638.
[32]侯丽颖,贺松其,等.保肝宁对瘦素刺激HSC增殖及其JAK2-STAT3信号通路影响的实验研究[J].上海中医药杂志,2007,41(3)：60-62.
[33]侯丽颖,季幸姝,等.瘦素诱导HSC增殖的信号转导通路及保肝宁调控作用的实验研究[J].中医药通报,2008,7(2)：61-62.
[34]陈主初.病理生理学.北京：人民卫生出版社,2006.15-20.
[35]李荣华,聂慧.中药血清调节肝星状细胞增殖凋亡机制实验研究进展[J].辽宁中医杂志,2008,35(3)：468-470.
[36]杨玲,等.β-榄香烯对HSC周期及调亡的影响[J].中华消化杂志,2006,28(8)：555-556.
[37]来丽娜,方步武.青蒿琥酯对大鼠肝星状细胞增殖的影响[J].中药药理与临床,2006,22(3、4)：25-26.
[38]赵宏贤,陈霞,等.粉防已碱对大鼠肝星状细胞增殖及细胞周期的调控[J].现代预防医学,2007,34(23)：4437-39.
[39]赵宏贤,陈霞,等.粉防已碱对大鼠肝星状细胞增殖及细胞周期素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.
[40]韩海啸,李军祥,等.赤芍水提物对肝星状细胞株HSC-T6促凋亡作用的实验研究[J].深圳中西医结合杂志,2007,17(1)：1-4.
[2]孙艳红,方晓艳,刘红雨,等.中药血清药理学方法研究概[J].时珍国医国药,2002,13(2)：114.
[3]王喜军.中药血清药物化学的研究动态及发展趋势[J].中国中药杂志.2006,31(10)：789-792.
[4]史丙俊,陈德宇.中药血清药理学研究初探[J].泸州医学院学报,2003,26(3)：272-273.
[5]陆慧晶.血清药理学研究中的若干问题探讨[J].基层中药杂志2000,14(3)：51.
[6]李静,殷飞,姚树坤,等.血清药理学研究进展[J].中国中医基础医学杂志,2009,15(3)：234-236.
[7]赵万红,曹永孝,袁泽飞.中药血清学的方法学探讨[J].中药新药与临床药理,2002,13(2)：122.
[8]于海荣,王济兴,陈建双.血清药理学方法研究进展[J].承德医学院学报,2006,23(2)：183-185.
[9]崔玲,晏卫东.中药血清药理研究方法回顾与展望[J].武警医学,2004,15(6)：460-461.
[10]张军平,张伯礼,山本清高.中药药物血清的制作方法探讨[J].天津中医药,2004,21(4)：274-276.
[11]张良,徐立,袁冬萍,等.中药血清药理学方法研究进展[J].南京中医药大学学报,2002,18(4)：254.
[12]张德付.中药血清药理学方法在肿瘤研究中存在的主要问题及对策[J].中医研究,2004,17(5)：13-14.
[13]韩笑,刘建勋,林成仁.血清药理学关键问题的存疑与讨论[J].中药药理与临床,2009,25(2)：122-124.
[14]HautekeeteML, GeertsA. VirchowsArch,1997,430(3):195-207.
[15]陈主初.病理生理学.北京：人民卫生出版社,2006,331-335.
[16]Solis-Herruzo JA, de la Torre P, Munoz-Yague MT. Hepatic stellate cells (HSC):architects of hepatic fibrosis [J]. Rev Esp Enferm Dig,2003,95:438-439.
[17]Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell acti-vation[J]. Med Gen Med,2002,4:27-30.
[18]Giannelli G, Antonaci S. Immunological and molecular aspects of liver fibrosis in chronic hepatitis C virus infection. Histol Histopathol,2005,20:939-944.
[19]Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis,2001,21: 437-451.
[20]Friedman S L.Molecular regulation of hepatic fibrosis,an integrated cellular response to tissue injury.J Biol Chem,2000,275 (4):2247-2250.
[21]陈主初.病理生理学.北京：人民卫生出版社,2006.15-20.
[22]杨玲,朱清静,张赤志.软坚消痰活血化瘀法抗肝纤维化的作用机制概述[J].中医药学刊,2005,23(11)：1969-1971.
[23]高建蓉,张赤志,邵志华,等.鳖甲对肝星状细胞增殖影响的研究[J].实用医学杂志,2007,23(11)：1618-1620.
[24]邝卫红,李建华.鳖甲煎汤抗大鼠肝纤维化作用[J].中药新药与临床药理,2004,15(5)：316.
[25]薛博瑜,胡荣昕,孙丽霞.中医药治疗肝纤维化临床研究思路探讨[J].中华中医药杂志,2007,22(8)：542.
[26]熊益群,严红梅,张赤志.抗纤软肝冲剂抗大鼠肝纤维化的实验研究[J].中国实验方剂学杂志,2000,6(2)：201.
[27]Liu H, LinD, YatesJR. Multidimensional separation forprotein/ pep-tide analysis in the post-genomic era [J]. Biotechniques, 2002,32 (4):898-902.
[28]Wilkins MR, Pasquali C, Appel RD, etal. From proteins to proteomes:large scale protein identification by two-dimensional electrophoresis and amino acid analysis [J]. Biotechnology,1996,14 (1):61-65.
[29]GerlaiR. Phenomics:fiction or the future[J]. TrendsNeurosci, 2002,25(10):506-509.
[30]Anderson NL,Anderson NG. Proteome and proteomic:new tech-nologies, new concepts, and new words[J]. Electrophoresis, 1998,19(11):1853-1861.
[31]Petricoin EF, Zoon KC, Kohn EC, etal. Clinical proteomics: translating benchside promise into bedside reality[J]. Nature Rev Drug Discov,2002,1 (9):683-695.
[32]陈化洋.蛋白质组学研究进展及展望[J].淮北职业技术学院学报,2008,7(3)：39-40.
[33]王慷慨,邓恭华,肖献忠.核仁应激损伤与热休克蛋白研究.生理科学进展,2003,34(2)：175-177.
[34]HernandezVD, LouvetE. The nucleolus:structure, functions, and associateddiseases. Med Sci,2004,20(1):37-44.
[35]DerenziniM. TheAgNORs. Micron,2000,31:117-120.
[36]周晓岩,王琳,姜云鹏,等.核仁组成区嗜银蛋白和增殖指数在肾母细胞瘤研究中的应用[J].吉林大学学报医学版,2003,29(1)：79-81.
[37]Itahana K, Bhat K P, Jin A, etal. Tumor suppressor ARF degrades B23, a nucleoar protein involved in ribosome biogenesis and cell proliferation[J]. Mol Cell,2003,12(5):1151-1164.
[38]Okuda M. The role of nucleophosmin in centrosome duplication [J]. Oncogene,2002,21 (40):6170-6174.
[39]云径平,等.B23在肝再生和肝细胞增生过程中的表达与移位[J].中华医学杂志,2002,82(14)：974-978.
[40]Gao H, Jin S, Song Y, etal.B23 regulates GADD45a nuclear trans-location andcontributes to GADD45a-induced cell cycle G2/M arrest [J].J Biol Chem,2005,280(12):10988-10996.
[41]Kurki S, Peltonen K, Laiho M. Nucleophosmin, HMD2 and p53: players in UV damage incited nucleolar stress response[J]. Cell Cycle,2004,3(8):976-979.
[42]WeberJ D, Taylor L J, Roussel M F, etal. Nucleolar Arf sequesters Mdm2 and activates p53[J]. Nat Cell Biol,1999,1(1):20-26.
[43]Kurki S,Peltonen K,Latonen L,etal. Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation [J]. Cancer Cell,2004, 5 (5):465-475.
[44]Hayne C, Xiang X, Luo Z. MEK inhibition and phosphorylation of serine 4 on B23 are two coincident events in mitosis [J]. Biochem Biophys Res Commun,2004,321 (3):675-680.
[45]Takemura M, Ohoka F, Perpelescu M, etal. Phosphorylation-Dependent Migration of Retinoblastoma Protein into the Nucleolus Triggered by Binding to Nucleophosmin/B23[J]. Exp Cell Res,2002,276(2),233-241.
[46]Jiang P S, Chang J H, Yung B Y. Different kinases phosphorylate nucleophosmin/B23 at different sites during G2 and M phasesof the cell cycle [J]. Cancer Lett,2000,153 (1-2):151-160.
[47]Tokuyama Y, Horn H F, Kawamura K, etal. Specif ic phosphorylation of nucleophosmin on Thr(199)by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication [J]. J Biol Chem,2001,276 (24):21529-21537.
[48]Lu Y Y, Lam C Y, Yung B Y. Decreased accumulation and dephosphorylation of the mitosis-specific from of nucleo-phosmin/B23 in staurosporne-induced chromosome de-condensation [J]. BiochemJ,1996,317 (Pt 1):321-327.
[49]王慷慨,蒋磊,刘可,等.热休克蛋白70对氧化应激所致核仁素裂解的影响[J].中国动脉硬化杂志,2004,12(4)：373-377.
[50]AndersenJS, LyonCE, FoxAH. Directed proteomic analysis of the human nucleolus. CurrBiol,2002,12(1):1-11.
[51]Daniely Y, Dimitrova DD, Borowiec JA. Stress-dependent nucleolin mobilizetion mediated by p53-nucleolin complex formation. Mol Cell Biol,2002,22 (16):6014-6022.
[52]Yanagida,M., Shimamoto, A., Nishikawa,K. etal. Isolation and proteomic characterization of the major proteins of the Nucleolin-binding ribonucleoprotein complexes[J]. Proteomics 1,2001,1390-1404.
[53]Iliakis G, Krieg T, Guan J,etal. Evidence for an S-phase check-point regulating DNA replication after heat shock[J]. Int J Hyper-thermia,2004,20 (2):240-249.
[54]王慷慨,等.核仁素在不同细胞膜表面的表达[J].中国动脉硬化杂志,2007,15(1)：63.
[55]Hovanessian AG, Puvion-Dutilleul F, NisoleS, etal. The cell-surface expressed Nucleolin is associated with the actin cytoskeleton[J].Exp Cell Res,2000,26(2):312-328.
[56]Khurts S, Masutomi K, Delgermaa L,etal. Nucleolin interacts with telomerase[J]. J Biol Chem,2004,279(49):51508-51515.
[57]Joo EJ. Nucleolin:acharan sulfate-binding protein on the surface of cancer cells [J]. Glycobiology,2005,15 (1):129.
[1]康谊,王天才,等.15d-PGJ2对肝星状细胞增殖和凋亡的影响[J].中国药理学通报,2007,23(2)：177-180.
[2]肖永红,刘殿武,等.复方中药血清对四氯化碳刺激的肝星状细胞增殖的影响[J].中国中医基础医学杂志,2006,12(1)：18-19.
[3]龚非力.医学免疫学.第2版.科学出版社,2004,61-83.
[4]Borkham-Kamphorst E, Herrmann J,Stol1 D, Treptau J, Gressner AM, Weiskirchen R. Dominant-negative soluble PDGF-beta receptor inhibits HSC activation and attenuates liver fibrosis.Lab Invest,2004,84:766-777.
[5]冯海娟,陆翠华.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.
[6]Bonner JC.Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev 2004,15:255-273.
[7]Tsukada S, Parsons C J, Rippe R A. Mechanisms of liver fibrosis[J]. Clin Chim Acta,2006,364(1-2):33-60.
[8]黄思敏,等.姜黄素对肝纤维化大鼠肝组织中α-SMA,PDGF-BB和TGF-β表达的影响[J].广东医学,2008,29(6)：929-931.
[9]张晓芸,等.苦参碱对血小板衍生生长因子介导的大鼠肝星状细胞迁移、增殖和凋亡的影响[J].中国慢性病预防与控制,2008,16(4)：334-336.
[10]齐荔红,刘晔,等.大黄素对大鼠肝星状细胞增殖和合成胶原的影响[J].第二军医大学学报,2005,26(10)：1190-1191.
[11]杨玲,张赤志,等.抗纤软肝颗粒对PDGF诱导的HSC增殖的影响[J].中国中西医结合消化杂志,2004,12(2)：80-82.
[12]姚希贤,房红梅,王军民,等.拆方益肝康与药物血清对肝星状细胞PDGF及受体表达的影响[J].世界华人消化杂志,2005,13(12)：1412-1416.
[13]王飞.TGF-β1在肝纤维化中的作用[J].中国实用医药,2008,3(23)：189-191.
[14]徐宁,等.TGF-β1于肝纤维化的关系再探讨[J].重庆医药,2008,37(20)：2356-2357.
[15]戴颖,朱萱.生长因子家族在肝纤维化中的作用[J].中国全科医学,2008,11(6A)：1002-1004.
[16]朱清静,等.剔毒护肝方对HSC表达Ⅰ型胶原及TGF-β 1mRNA的影响[J].中国中医药科技,2002,9(2)：76-77.
[17]姜春萌,刘成海,刘成.扶正化瘀胶囊对肝星状细胞激活的干预[J].中国中西医结合消化杂志,2003,11(5)：280.
[18]石小枫,刘杞,徐曼,等肝心康对肝纤维化大鼠血清学指标及TGF-β1的影响[J].中药药理与临床,2004,20(2)：34.
[19]曾军.肝星状细胞、相关因子与肝纤维化关系的研究进展[J].中华肝胆外科杂志,2005,11(6)：427-429.
[20]主余华,等.CTGF shRNA对肝星状细胞细胞因子及细胞外基质表达的影响[J].山东大学学报医学版,2007,45(6)：577-581.
[21]Kim KH, Park GT,Lim YB,etal. Expressionof connective tissue growth factor, a biomarker in senescenceof human diploid fibroblasts, is up-regulated by a transforming growth fac-tor-beta-mediated signaling pathway[J]. Biochem Biophys Res Commun,2004,318(4):819-825.
[22]Abou-Shady M, Friess H, Zimmermann A, etal. Connective tissue growth factor in human liver cirrhosis [J]. Liver,2000,20: 296-304.
[23]贺松其.鳖甲煎丸对肝纤维化模型大鼠CTGF的影响[J].辽宁中医杂志,2005,32(12)：1334-1335.
[24]主余华,张春清,等.银杏叶提取物对肝星状细胞增殖及转化生长因子和结缔组织生长因子基因表达的影响[J].中国病理生理杂志,2007,23(7)：1316-1321.
[25]谢明,廖晓宏,等.姜黄素对肝星状细胞CTGF表达的影响[J].实用医学杂志,2006,22(15)：1724-1726.
[26]宋仕玲,龚作炯,张全荣,等.中药金三莪方对实验性肝纤维化大鼠CTGF及TGF-β1mRNA表达及定位的影响[J].解放军医学杂志,2004,29(2)：130.
[27]叶平,蒋明德.肝纤维化相关因素的研究现状及进展[J].新医学,2008,39(12)：829-831.
[28]薛冬英,洪嘉禾,等.丹参酚酸B对大鼠HSC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.
[29]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)：22-24.
[30]陈主初.病理生理学.北京：人民卫生出版社,2006,34-53.
[31]李骢,等.中药肝复康对PDGF介导的HSC信号转导的影响及意义[J].中华消化杂志,2006,26(9)：636-638.
[32]侯丽颖,贺松其,等.保肝宁对瘦素刺激HSC增殖及其JAK2-STAT3信号通路影响的实验研究[J].上海中医药杂志,2007,41(3)：60-62.
[33]侯丽颖,季幸姝,等.瘦素诱导HSC增殖的信号转导通路及保肝宁调控作用的实验研究[J].中医药通报,2008,7(2)：61-62.
[34]陈主初.病理生理学.北京：人民卫生出版社,2006.15-20.
[35]李荣华,聂慧.中药血清调节肝星状细胞增殖凋亡机制实验研究进展[J].辽宁中医杂志,2008,35(3)：468-470.
[36]杨玲,等.β-榄香烯对HSC周期及调亡的影响[J].中华消化杂志,2006,28(8)：555-556.
[37]来丽娜,方步武.青蒿琥酯对大鼠肝星状细胞增殖的影响[J].中药药理与临床,2006,22(3、4)：25-26.
[38]赵宏贤,陈霞,等.粉防已碱对大鼠肝星状细胞增殖及细胞周期的调控[J].现代预防医学,2007,34(23)：4437-39.
[39]赵宏贤,陈霞,等.粉防已碱对大鼠肝星状细胞增殖及细胞周期素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.
[40]韩海啸,李军祥,等.赤芍水提物对肝星状细胞株HSC-T6促凋亡作用的实验研究[J].深圳中西医结合杂志,2007,17(1)：1-4.