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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 李严严等 / 肝星型细胞分离方法和功能研究进展 July 25, 2014, 30(7): Chin J Biotech, All rights reserved 综述 1059 李严严 1,2, 姜颖 ,.., 2014, 30(7): Li YY, Jiang Y. Update on isolation and functional research of hepatic stellate cells. Chin J Biotech, 2014, 30(7): 摘要 : 肝星型细胞 (Hepatic stellate cells, HSCs), 又叫储脂细胞 (Fat-storing cells, FSCs) 或脂肪细胞 (lipocytes), 是肝脏固有的非实质细胞类型之一, 存在于狄氏腔内, 以脂滴的形式储存人体维生素 A 总量的 50% 80% 原代 HSCs 分离方法, 目前主要集中于密度梯度离心法结合离心淘洗 HSCs 高侧向角的流式分选法紫外激发的自发荧光或特异性抗体标记的流式细胞术等, 将为 HSCs 生理和病理研究提供坚实的基础近年来,HSCs 的研究蓬勃发展, 合作领域不断拓宽生理状态下,HSCs 处于静息状态, 合成细胞外基质 (Extracellular matrix, ECM) 并维持其稳态, 同时广泛摄取和储存维生素 A, 并具有调节肝细胞再生的功能 ; 而病理状态下,HSCs 在肝损伤和持续性刺激条件下被激活, 增殖活性明显增强, 脂滴减少或消失,ECM 合成也明显增加, 具有收缩性, 同时分泌多种促炎因子和粘附分子, 并向肌成纤维细胞转变, 表明 HSCs 的活化是肝纤维化发生发展过程中的关键环节之一有关 HSCs 的分离和功能研究一直是肝脏细胞学和肝脏病理学研究的热点之一文中我们将系统总结和探讨 HSCs 的分离方法和改进策略, 及其功能研究进展和具有潜在价值的研究方向 : 肝星型细胞, 分离方法, 肝脏生理功能, 肝纤维化, 肝再生, 肝脏蛋白质组学 Received: March 22, 2014; Accepted: June 4, 2014 Supported by: National Natural Science Foundation of China (No ). Corresponding author: Ying Jiang. Tel: ; jiangying304@hotmail.com (No )

2 1060 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 Update on isolation and functional research of hepatic stellate cells Yanyan Li 1,2, and Ying Jiang 1 1 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institution of Radiation Medicine, Beijing , China 2 School of Life Science, Tsinghua University, Beijing , China Abstract: Hepatic stellate cells (HSCs), also called Ito cells or lipocytes, are one of inherent liver nonparenchymal cell types located in the Dissé space between hepatocytes and sinusoidal endothelial cells, and account for up to 50% 80% of vitamin A in the form of lipid drops. The methods of primary HSCs isolation mainly focus on density gradient centrifugation combined with centrifugal elutriation, side scatter-activated cell sorting, UV-excited autofluorescence or antibody-based flow cytometry, etc., and will provide solid foundation for the research on physiological and pathological HSCs function. The research of this vitamin A-storing cells has developed and expanded vigorously. In physiological conditions, HSCs are quiescent and play pivotal roles in the synthesis of extracellular matrix (ECM) to maintain its stability with broad uptake and storage of vitamin A, and also regulate liver regeneration. But in pathological conditions, HSCs are activated by constant stimulations or liver injury, then with activated proliferation, reduced lipid drops, and increased ECM synthesis. Morphology of these cells also changes from the star-shaped stellate cells to that of fibroblasts or myofibroblasts with obvious contractibility and secretion of cytokines and chemokines including a variety of proinflammatory factors and adhesion molecules, suggesting that the activation of HSCs is one of the key events in the development of liver fibrosis. Study on the isolation and function of HSCs is always one of the hot topics for liver biology. In this review, we systematically summarize and discuss the recent advances in our understanding of the isolation methods and improvements of HSCs, and functional research of HSCs biology in health and disease, as well as potential directions. Keywords: Hepatic stellate cells (HSCs), isolation method, liver physiology, liver fibrosis, liver regeneration, liver proteomics (Hepatic stellate cells, HSCs) 1.4% 5% 8% [1] HSCs HSCs [2] HSCs [3] HSCs HSCs HSCs [4] HSCs HSCs HSCs

3 李严严等 / 肝星型细胞分离方法和功能研究进展 1061 [5] HSCs 1 HSCs 分离方法进展 1982 HSCs [6] HSCs HSCs [7] HSCs 1.1 密度梯度离心法结合离心淘洗法 HSCs HSCs HSCs [2] Seglen Stractan [8] Nycodenz [9] Percoll TM[10] OptiPrep TM[11] HSCs HSCs g/ml HSCs Desmin Vimentin N-CAM GFAP HSCs [12] HSCs [2] HSCs HSCs HSCs A HSCs [2] HSCs HSCs [13] HSCs HSCs 1.2 结合 HSCs 细胞形态特性的流式细胞术分选法 HSCs HSCs HSCs cjb@im.ac.cn

4 1062 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 ( Desmin GFAP ) HSCs HSCs 50% 70% 96% [12] 1.3 自发荧光的流式细胞术分选法 HSCs A HSCs A HSCs 355 nm HSCs HSCs HSCs [14] HSCs HSCs 1.4 抗体标记的流式细胞术分选法 HSCs HSCs HSCs [15] GFAP HSCs Desmin HSCs HSCs CD3 (T ) CD19 (B ) F4/80 CD146 HSCs HSCs 3 HSCs [3] HSCs HSCs HSCs HSCs ( ) HSCs HSCs HSCs HSCs

5 李严严等 / 肝星型细胞分离方法和功能研究进展 1063 HSCs HSCs HSCs HSCs 2 HSCs 功能研究进展 2.1 HSCs 生理功能研究进展 HSCs HSCs ECM A A HSCs 1 HSCs A HSCs A HSCs 50% 80% A A [16] A [17] A A A (Cellular retinol-binding protein, CRBP) A A A RBP ( RBP4) CRBP 3 ( RBP1 RBP2 RBP3) A A [18] A HSCs HSCs RBP mrna (Western blotting) HSCs RBP 3 H -RBP [19] [20] RBP HSCs HSCs HSCs A HSCs A HSCs [21-22] HSCs [23] TGF-β [24] TGF-β HSCs [25] HSCs HSCs HSCs [26] [27] TGF-ɑ EGF HSCs HSCs HSCs cjb@im.ac.cn

6 1064 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 HGF [28] PDGF HSCs [29] HSCs PDGF PDGFR HSCs afgf [30] PDGFR [31] HSCs PDGFR Ras ERK/MAPK PI3K STAT-1 HSCs PDGF PDGFR HSCs HSCs ECM [32] ECM [33] ECM HSCs HSCs HSCs HSCs ECM ECM 图 1 HSCs 的生理和病理功能 [34-35] Fig. 1 Physiological and pathological functions of HSCs (From Atzori et al [34], Yin et al [35] ).

李严严等 / 肝星型细胞分离方法和功能研究进展 1065 2.2 HSCs 病理功能研究进展 HSCs A HSCs HSCs HSCs HSCs HSCs 2.2.1 HSCs [36] HSCs HSCs HSCs [37] HSCs A HSCs 2 HSCs A HSCs 图 2 HSCs 活化过程和效应以及逆转方向示意图 [5] Fig.

7 李严严等 / 肝星型细胞分离方法和功能研究进展 HSCs 病理功能研究进展 HSCs A HSCs HSCs HSCs HSCs HSCs HSCs [36] HSCs HSCs HSCs [37] HSCs A HSCs 2 HSCs A HSCs 图 2 HSCs 活化过程和效应以及逆转方向示意图 [5] Fig. 2 Diagram of HSCs activation, effect and reversion (From Friedman [5] ). cjb@im.ac.cn

8 1066 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 HSCs A A HSCs HSCs HSCs PDGF PDGFR PDGF HSCs PDGFR 3- HSCs TGF-β [38] HSCs [39] HSCs ET-1 [40] ET-1 HSCs ETBR ETAR HSCs ET-1 NO HSCs ET-1 HSCs HSCs HSCs NO [41] ET-1 NO HSCs HSCs Ⅰ ECM MMP Ⅳ TIMP-1 ECM [4] HSCs TGF-β1 [42] TGF-β1 (TGFRⅠ TGFRⅡ) Smad TGF-β1 [43] HSCs TIMP-1 [44] MMPs TIMPs MMPs HSCs [34] HSCs HSCs HSCs A HSCs TGF-β [45] PDGF TGF-β EGF HSCs HSCs Fas [46]

9 李严严等 / 肝星型细胞分离方法和功能研究进展 1067 HSCs [47] HSCs [48] 1 HSCs 2 HSCs 3 HSCs 4 HSCs ECM HSCs HSCs ECM HSCs HSCs 1) A HSCs 2) 3) 4) 5) 6) HSCs 7) HSCs HSCs ECM [49] HSCs [50] HSCs 2AAF/PH [51] L- [52] HSCs CCl 4 Foxf1+/- HSCs [53] HSCs HSCs HSCs HSCs 2AAF/PH HSCs HGF [54] HSCs HGF P75NTR HSCs P75NTR HSCs (Plg-/-) P75NTR Plg HGF [55] P75NTR HSCs Rho HSCs P75NTR Rho HSCs HSCs HGF HSCs [56] ECM cjb@im.ac.cn

10 1068 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 ECM ECM HSCs ECM TGF-β HSCs TGF-β [57] HSCs HGF-TGF-β1 HSCs HGF TGF-β1 HSCs TGF-β1 HSCs B (5-HT2B) TGF-β1 PH BDL CCl 4 5-HT2B [58] HSCs HSCs HSCs [59] [60] / [61] [62] HSCs [63] HSC HSC HSCs [64] HSC [35] HCC 2.3 HSCs 蛋白质组学研究进展 HSC HSC HSC HSC HSC HSC HSC 2001 Kristensen [65] HSC HSC HSC STAP

11 李严严等 / 肝星型细胞分离方法和功能研究进展 1069 HSC HSC STAP HSC STAP HSC HSC Schwabe [66] CCl 4 HSC HSC CCl 4 HSC HSC HSC HSC HSC HSC HSC HSC [67] HSC HSC HSC Coulouarn [68] HSC HSC VEGFA MMP9 HSC HSC 3 展望 HSCs HSCs HSCs HSCs HSCs ( ) HSCs HSCs HSCs cjb@im.ac.cn

12 1070 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 HSCs HSCs REFERENCES [1] Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol, 1977, 72(2): [2] Weiskirchen R, Gressner AM. Isolation and culture of hepatic stellate cells. Methods Mol Med, 2005, 117: [3] Tacke F, Weiskirchen R. Update on hepatic stellate cells: pathogenic role in liver fibrosis and novel isolation techniques. Expert Rev Gastroenterol Hepatol, 2012, 6(1): [4] Senoo H, Yoshikawa K, Morii M, et al. Hepatic stellate cell (vitamin A-storing cell) and its relative-past, present and future. Cell Biol Int, 2010, 34(12): [5] Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008, 88(1): [6] Knook DL, Seffelaar AM, de Leeuw AM. Fat-storing cells of the rat liver. Their isolation and purification. Exp Cell Res, 1982, 139(2): [7] Gridelli B, Vizzini G, Pietrosi G, et al. Efficient human fetal liver cell isolation protocol based on vascular perfusion for liver cell-based therapy and case report on cell transplantation. Liver Transplantation, 2012, 18(2): [8] Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem, 1987, 161(1): [9] Schäfer S, Zerbe O, Gressner AM. The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology, 1987, 7(4): [10] Blomhoff R, Berg T. Isolation and cultivation of rat liver stellate cells. Methods Enzymol, 1990, 190: [11] Sicklick JK, Li YX, Choi SS, et al. Role for Hedgehog signaling in hepatic stellate cell activation and viability. Laboratory Investigation, 2005, 85(11): [12] Geerts A, Niki T, Hellemans K, et al. Purification of rat hepatic stellate cells by side scatter-activated cell sorting. Hepatology, 1998, 27(2): [13] Yata Y, Enosawa S, Suzuki S, et al. An improved method for the purification of stellate cells from rat liver with dichloromethylene diphosphate (CL2MDP). Methods Cell Sci, 1999, 21(1): [14] Ichikawa S, Mucida D, Tyznik AJ, et al. Hepatic stellate cells function as regulatory bystanders. J Immunol, 2011, 186(10): [15] Cassiman D, Libbrecht L, Desmet V, et al. Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers. J Hepatol, 2002, 36(2): [16] Blomhoff R, Blomhoff HK. Overview of retinoid metabolism and function. J Neurobiol, 2006, 66(7): [17] Blomhoff R, Green MH, Berg T, et al. Transport and storage of vitamin A. Science, 1990, 250(4979): [18] Blomhoff R, Helgerud P, Rasmussen M, et al. In vivo uptake of chylomicron [3H]retinyl ester by rat liver: evidence for retinol transfer from parenchymal to nonparenchymal cells. Proc Natl Acad Sci USA, 1982, 79(23): [19] Blomhoff R, Norum KR, Berg T. Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells. J Biol Chem, 1985, 260(25): [20] Andersen KB, Nilsson A, Blomhoff HK, et al. Direct mobilization of retinol from hepatic perisinusoidal stellate cells to plasma. J Biol Chem, 1992, 267(2): [21] Davis BH, Coll D, Beno DW. Retinoic acid suppresses the response to platelet-derived growth factor in human hepatic Ito-cell like myofibroblasts: a post-receptor mechanism independent of raf/fos/jun/egr activation. Biochem J, 1993, 294: [22] Okuno M, Moriwaki H, Imai S, et al. Retinoids exacerbate rat liver fibrosis by inducing the

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14 1072 ISSN CN /Q Chin J Biotech July 25, 2014 Vol.30 No.7 [47] Zhu J, Wu J, Frizell E, et al. Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis. Gastroenterology, 1999, 117(5): [48] Schuppan D, Kim YO. Evolving therapies for liver fibrosis. J Clin Invest, 2013, 123(5): [49] Roskams T. Relationships among stellate cell activation, progenitor cells, and hepatic regeneration. Clin Liver Dis, 2008, 12(4): [50] Yang L, Y Jung, A Omenetti, et al. Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers. Stem Cells, 2008, 26(8): [51] Shen K, Chang W, Gao X, et al. Depletion of activated hepatic stellate cell correlates with severe liver damage and abnormal liver regeneration in acetaminophen-induced liver injury. Acta Biochim Biophys Sin, 2011, 43(4): [52] Pintilie DG, Shupe TD, Oh S, et al. Hepatic stellate cells involvement in progenitor-mediated liver regeneration. Lab Invest, 2010, 90(8): [53] Kalinichenko VV, Bhattacharyya D, Zhou Y, et al. Foxf1+/ mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury. Hepatology, 2003, 37(1): [54] Chen L, Zhang W, Zhou Q, et al. HSCs play a distinct role in different phases of oval cell-mediated liver regeneration. Cell Biochem Funct, 2012, 30(7): [55] Passino MA, Adams RA, Sikorski SL, et al. Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75ntr. Science, 2007, 315(5820): [56] Wells RG. Cellular sources of extracellular matrix in hepatic fibrosis. Clin Liver Dis, 2008, 12(4): [57] Karkampouna S, Ten DP, Dooley S, et al. TGF-beta signaling in liver regeneration. Curr Pharm Des, 2012, 18(27): [58] Ebrahimkhani MR, Oakley F, Murphy LB, et al. Stimulating healthy tissue regeneration by targeting the 5-HT(2)B receptor in chronic liver disease, Nat Med, 2011, 17(12): [59] Beis D, Stainier DYR. In vivo cell biology: following the zebrafish trend. Trends Cell Biol, 2006, 16(2): [60] Chu J, Sadler KC. New school in liver development: lessons from zebrafish. Hepatology, 2009, 50(5): [61] Curado S, Anderson RM, Jungblut B, et al. Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev Dyn, 2007, 236(4): [62] Curado S, Ober EA, Walsh S, et al. The mitochondrial import gene tomm22 is specifically required for hepatocyte survival and provides a liver regeneration model. Dis Model Mech, 2010, 3(7/8): [63] Yin C, Evason KJ, Maher JJ, et al. The bhlh transcription factor Hand2 marks hepatic stellate cells in zebrafish: Analysis of stellate cell entry into the developing liver. Hepatology, 2012, 56(5): [64] Amann T, Bataille F, Spruss T, et al. Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma. Cancer Sci, 2009, 100(4): [65] Kristensen DR, Kawada N, Imamura K, et al. Proteome analysis of rat hepatic stellate cells. Hepatology, 2000, 32(2): [66] De Minicis S, Seki E, Uchinami H, et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology, 2007, 132(5): [67] Natalia N. A systems biology approach for understanding the collagen regulatory network in alcoholic liver disease. Liver International, 2012, 32(2): [68] Coulouarn C, Corlu A, Glaise D, et al. Hepatocyte stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma. Cancer Res, 2012, 72(10): ( 本文责编郝丽芳 )

Microsoft Word - 360145.doc

Microsoft Word - 360145.doc 项目批准号 81273814 归口管理部门申请代码收件日期 H2716 360145 y 国家自然科学基金委员会资助项目计划书资助类别 : 面上项目亚类说明 : 非连续资助类项目附注说明 : 项目名称 : 益气养肺法拮抗 bfgf 对肺癌微环境介导化疗