摘要
胞外蛋白酶抑制剂基因expi编码的蛋白与蛋白酶活性的有关,并参与细胞的迁移和凋亡活动。本实验室的大鼠再生肝Rat Genome 230 2.0芯片检测结果表明,expi在大鼠2/3肝切除后6h起始表达,6-72h持续表达上调,并在30h达到表达高峰,是对照的33倍。但目前还没有人研究其在肝再生中的功能,而作为重要生命活动的肝再生也涉及蛋白质降解,细胞迁移和凋亡等生理生化活动,了解该基因在肝再生中的功能,有助于揭示肝再生分子机制。为此,本文挑选出基因expi,以Higgins等建立的大鼠2/3肝切除模型做为研究对象,利用大鼠尾静脉液压转基因技术,通过基因添加和RNA干涉的方法,研究expi在大鼠肝再生中的作用。
根据expi的基因序列设计引物并克隆该基因,同时设计其两条RNA干涉片段,然后进行载体的构建。将上述测序正确的基因及其干涉片段连接到不同的载体上,构建三套载体:一套是表达载体pEGFP-N1-expi,用于基因添加实验;另一套是干涉载体,包括pGenesil-1.0-E6和pGenesil-1.0-E147,用于干涉目的基因的实验;最后一套是检验载体,包括pGenesil-1.0-E6-expi,pGenesil-1.0-E147-expi和pGenesil-1.0-HK-expi,用于检验设计的两条干涉片段是否对靶基因产生干涉作用。
将构建的三条检验载体分别进行尾静脉液压转基因操作,通过观察转干涉检验载体的绿色荧光蛋白阳性细胞率(转染率)相较对照检验载体是否有明显减少,来判断设计的干涉片段是否对靶位点具有干涉作用。将表达载体和干涉载体分别进行液压转基因操作,分别在转基因后6、12、24、36、48、60、72和84h取材观察其绿色荧光蛋白阳性细胞率、通过统计分析得出各载体在大鼠肝脏组织中表达高峰的时间点,结合expi芯片检测结果,得出2/3肝切除后的最佳转基因时间。根据以上结果,利用2/3肝切除手术制备的大鼠肝再生模型和液压转基因技术展开expi对大鼠肝再生过程影响的研究。将制备好的转基因后的再生肝材料分别于2/3肝切除后72、120和168h取出,用荧光显微技术、统计学方法和常规组织学技术对死亡率、表达动力学、肝指数和组织形态结构等方面结果进行观察与分析。
测序和电泳检测结果显示:基因expi克隆成功,其表达载体、干涉载体和检验载体构建成功。荧光观察结果表明,转检验载体pGenesil-1.0-E6-expi和pGenesil-1.0-E147-expi的绿色荧光蛋白阳性细胞率明显低于其对照载体pGenesil-1.0-HK-expi,这说明设计的两干涉片段对靶位点均具有干涉作用。转表达载体pEGFP-N1-expi和干涉载体pGenesil- 1.0-E6与pGenesil-1.0-E147后,统计分析阳性细胞率得出各载体在大鼠肝脏组织中表达高峰的时间点是转基因后12h,结合expi芯片检测结果,得出最佳转基因时间点是2/3肝切除后18h。
肝指数检测结果表明,在进行expi基因添加时,肝指数高于对照;进行expi基因干涉时,肝指数则低于对照。总之,本文成功克隆了expi基因,并成功构建了其表达载体、干涉载体和检验载体,根据上述体内检测结果初步推测expi对大鼠肝再生有一定的影响,这为肝再生分子机理研究提供了资料。
Proteinase inhibitor gene expi has the protease activity, and involved in cell migration and apoptosis. According to the expression profile of rat regenerating liver detected with rat genome 230 2.0 chip, expi was observed to begin to express at 6h after 2/3 hepatectomy and increased consistently at 6-72h, reaching peak (33 times higher than control) at 30h. Liver regeneration, an important life process, is also involved in protein degradation, cell migration, and apoptosis and so on. Based on the above description, the study on the function of this gene in liver regeneration will be helpful in revealing the molecular mechanism of liver regeneration. However, at present, the function of this gene on liver regeneration has not been researched yet. Therefore, this paper employed the technique of hydrodynamics-based transgene to explore the influence of expi in the process of rat liver regeneration by means of adding (or interfering) this gene.
Based on the nucleotide sequence of Expi, primers were designed to amplify this gene. At the same time, the two RNA interference fragments of this gene were designed. Then, 3 sets of the recombinant vectors were established by ligation with the above right fragments, respectively, which includes the expression vector pEGFP-N1-expi for gene addition assay, the interference vectors pGenesil-1.0-E6 and pGenesil-1.0-E147 for gene inference test, and the test vectors pGenesil-1.0-E6-expi, pGenesil-1.0-E147 -expi and pGenesil-1.0-HK-expi for testing whether the two interference fragments have interference effect on the target gene or not.
The constructed test vectors were transgened based on hydrodynamics. At 6, 12, 24, 36, 48, 60, 72 and 84h after transgene, the interference effect of interference fragments on the target site were determined by observing whether the GFP-positive cell rates (that’s transfection rate) were decreased more obviously than control. The optimum time for transgene after partial hepatectomy was obtained by statistically measuring the peak time of the hydrodynamics expression of different vectors (including expression vectors and interference vectors) in rat livers, at the same time, expi expression in rat regenerating livers from the chip data must to be considered. Finally, the transgenic-liver samples at 72, 120, and 168h after partial hepatectomy were taken to analyze the mortality, expression kinetics, index of liver regeneration, and morphology structure, etc. by using fluorescence microscopy techniques, statistical methods and conventional histological technique.
DNA sequencing and electrophoresis showed: the cloned expi sequence is right; the vector was successfully constructed; the two interference fragments exhibit the more pronounced interference effect. The best time for transgene was 18 hours after partial hepatectomy.
Liver index test results show that if adding expi, liver index was higher than control; if interfering expi, lower than control. In short, this study successfully cloned expi and successfully constructed the expression vector, interference vector and test vector. According to the in vivo assays, it is presumed that expi has effect to a extent on rat liver regeneration, which provides the valuable information for elucidating the molecular mechanism of liver regeneration.
引文
[1]张瑞,郭善禹.肝脏功能重建的细胞来源[J].世界华人消化杂志, 2007,15: 1261-126.
[2] Taub R. Liver regeneration: from myth to mechanism [J]. Nat Rev Mol Cell Biol, 2004, 5: 836-847.
[3] Kirimlioglu V, Kirimlioglu H, Yilmaz S, et al. Effect of fish oil, olive oil, and vitamin E on liver pathology, cell proliferation, and antioxidant defense system in rats subjected to partial hepatectomy [J]. Transplant Proc, 2006, 38: 564-567
[4] Cumano A, Godin I. Ontogeny of the hematopoietic system [J]. Annu Rev Immunol, 2007, 25: 745-785.
[5] Markiewski MM, DeAngelis RA, Lambris JD. Liver inflammation and regeneration: two distinct biological phenomena or parallel pathophysiologic processes? [J]. Mol Immunol, 2006, 43: 45-56.
[6] Higgins GM, Anderson RM. Experimental pathology of the liver: restoration of the liver of the white rat following partial surgical removal [J]. Arch Pathol, 1931,12: 186-202.
[7] Cunshuan Xu, Cuifang Chang, Jinyun Yuan, et al. Identification and characterization of 177 unreported genes associated with liver regeneration [J]. Genomics Proteomics & Bioinformatics, 2004, 2: 109-118.
[8] Fausto N, Campbell JS, Riehle KJ. Liver regeneration [J]. Hepatol, 2006, 43: S45-53.
[9] Cunshuan Xu, Shou-Bing Zhang, Xiao-Guang Chen, et al. Correlation analysis of liver tumor-associated genes with liver regeneration [J]. World J Gastroenterol, 2007, 13: 3323-3332.
[10] Michalopoulos GK, De Frances MC. Liver regeneration [J]. Adv Biochem Eng Biotechnol, 2005, 93: 101-134.
[11] LiFeng Zhao, WeiMin Zhang, CunShun Xu. Expression patterns and action analysis of genes associated with blood coagulation responses during rat liver regeneration [J]. World J Gastroenterol, 2006, 12: 6842-6849.
[12] Fausto N. Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells [J]. Hepatol, 2004, 39: 1477-1487.
[13] Nakamura K, Nonaka H, Saito H, et al. Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice [J]. Hepatol, 2004, 39: 635-644.
[14] Hélène Duval, Sasse-Fanie Mbatchi, et al. Reperfusion stress induced during intermittent selective clamping accelerates rat liver regeneration through JNK pathway. Journal of Hepatology.Volume 52, Issue 4, April 2010, Pages 560-569.
[15] Chiduru Yamamoto, Shintaro Yagi, Tomohide Hori, et al. Significance of Portal Venous VEGF During Liver Regeneration After Hepatectomy. Journal of Surgical Research.Volume 159, Issue 2, April 2010, Pages e37-e43.
[16] Cunshuan Xu, Cui-Fang Chang, Jin-Yun Yuan, et al. Expressed genes in regenerating rat liver after partial hepatectomy [J]. World J Gastroenterol, 2005, 11: 2932-2940.
[17] Lai HS, Chen Y, Lin WH, et al. Quantitative gene expression analysis by cDNA microarray during liver regeneration after partial hepatrctomy in rat [J]. Surg Today, 2005, 35: 396-403.
[18]赵利峰,徐存拴.肝干细胞的生长和分化与大鼠肝再生的相关性分析[J].解剖学报,2008, 39: 526-530.
[19]成令忠,钟翠平,朱继红.部分肝切除后大鼠肝细胞超微结构变化[J].解剖学杂志, 1986, 9: 1-6.
[20] Michalopoulos GK, De Frances MC. Liver regeneration [J]. Science, 1997, 276: 60-66.
[21] Payam Samareh Pahlavan, Robert E. Feldmann Jr., Christos Zavos, et al. Prometheus’Challenge: Molecular, Cellular and Systemic Aspects of Liver Regeneration. Journal of Surgical Research. Volume 134, Issue 2, August 2006, Pages 238-251.
[22]徐存拴,唐子阔. PPAR-γ偶联的信号通路可能参与大鼠肝再生[J].基础医学与临床, 2008, 28(8): 810-815.
[23] Yuchang Li, Zhanqiang Ma, Cushuan Xu. Changes of TNF-α, c-myc, p53, p21, PCNA, Bcl-2, TGF-βrelated with the cell proliferation in rat liver regeneration following short interval successive partial hepatectomy [J]. Developmental & Reproductive Biology, 2002, 11: 253-260.
[24] Reynolds ES. Liver parenchymal cell injury. I. Initial alterations of the cell following poisoning with carbon tetrachlo-ride [J]. J Cell Biol, 1963, 19: 139-157.
[25] Naga Chalasani, Robert J Fontana, Herbert L Bonkovsky, et al. Causes, Clinical Features, and Outcomes From a Prospective Study of Drug-Induced Liver Injury in the United States [J]. Gastroenterology, 2008, 135(6): 1924-1934.
[26] LeSage GD, Benedetti A, Glaser S. Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver [J]. Hepatol, 1999, 29: 307-319.
[27] Dabeva MD, Shafritz DA. Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration [J]. Am J Pathol, 1993, 143: 1606-1620.
[28] N. Osman, D. Adawi, S. Ahrné, et al. Endotoxin- and d-galactosamine-induced liver injury improved by the administration of Lactobacillus, Bifidobacterium and blueberry [J]. Digestive and Liver Disease, 2007, 39(9): 849-856.
[29] Aaron J. Pugh, Ashutosh J. Barve, Keith Falkner, et al. Drug-Induced Hepatotoxicity or Drug-Induced Liver Injury [J]. Clinics in Liver Disease, 2009, 13(2): 299-294.
[30] Ketevan Pachkoria, M. Isabel Lucena, Esperanza Crespo, et al. Analysis of IL-10, IL-4 and TNF-αpolymorphisms in drug-induced liver injury (DILI) and its outcome [J]. Journal of Hepatology,2008, 49(1): 107-114.
[31] LeSage GD, Benedetti A, Glaser S. Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver [J]. Hepatology, 1999, 29(2):307-319.
[32] Seh–Hoon Oh, Rafal P. Witek, Si–Hyun Bae, et al.Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration [J].Gastroenterology, 2007, 132(3): 1077-1087.
[33] Liya Pi, Seh-Hoon Oh, Thomas Shupe, et al.Role of Connective Tissue Growth Factor in Oval Cell Response During Liver Regeneration After 2-AAF/PHx in Rats [J].Gastroenterology, 2005, 128(7): 2077-2088.
[34]张杨,韩德五.肝细胞再生调控研究的现状与进展[J].山西医科大学学报, 2006, 37(4): 428-432.
[35] A.R. Opoku, I.M. Ndlovu, S.E. Terblanche, et al. In vivo hepatoprotective effects of Rhoicissus tridentata subsp. cuneifolia, a traditional Zulu medicinal plant, against CCl4-induced acute liver injury in rats [J]. South African Journal of Botany, 2007, 73(3): 372-377.
[36] Keith A. Thatch, Edward Y. Yoo, Kim G. Mendelson, et al. Growth factor attenuation of alpha-Naphtylisocyocyanate (ANIT) induced liver injury: A novel approach to the management and treatment of TPN associated liver disease in short bowel syndrome [J]. Journal of the American College of Surgeons, 2008, 207 (3): s25.
[37] Ishak KG. Inherited metabolic diseases of the liver [J]. Clin Liver Dis, 2002, 6(2): 455-479.
[38] Hou CM, Lee PH, Lai YT, et al. Gene expression profiles in living donors immediately after partial hepatectomy-the initial response of liver regeneration [J]. J Formos Med Assoc, 2007, 106(4): 288-294.
[39] Fausto N.Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells [J]. Hepatology, 2004, 39(6): 1477-1487.
[40] K. Narayana, Maie Al-Bader.Ultrastructural and DNA damaging effects of lead nitrate in the liver [J].Experimental and Toxicologic Pathology, 2009, In Press, Corrected Proof.
[41] L Sun J. Kim, Joo W. Lee, Young S. Jung, et al.Ethanol-induced liver injury and changes in sulfur amino acid metabolomics in glutathione peroxidase and catalase double knockout mice [J].Journal of Hepatology, 2009, 50(6): 1184-1191.
[42]刘志刚,钱叶本.肝再生进程调控的研究[J].医学综述, 2006, 12 (6): 332-336.
[43] Matsuo T, Yamaguchi S, Mitsui S, at al. Control mechanism of the circadian clock for timing of cell division in vivo [J]. Science, 2003, 302(5643):255-259
[44] Schibler U. Circadian rhythms. Liver regeneration clocks on [J]. Science, 2003, 302(5643):234-235
[45] Michael S. Chimenti, Mark J.S. Kelly, Diane L. Barber, at al. Molecular Dynamics Simulations Predict A pH-Dependent Conformational Change in the C-Helix of Cell Cycle Checkpoint Kinase Wee1 [J]. Biophysical Journal, 2010, 98 (3) :232a.
[46] Wang X, Kiyokawa H, Dennewitz MB, Costa RH. The Forkhead Box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver regeneration [J]. Proc Natl Acad Sci U S A, 2002, 99(26):16881-16886.
[47] Weglarz TC, Sandgren EP. Timing of hepatocyte entry into DNA synthesis after partial hepatectomy is cell autonomous [J]. Proc Natl Acad Sci U S A, 2000, 97(23):12595-12600.
[48]徐存拴,杨志利,董华明.核糖体相关基因在大鼠再生肝及肝肿瘤中表达异同的比较[J].解剖学报, 2008, 39(3): 281-287.
[49] Shao-Wei Qin, Li-Feng Zhao, Xiao-Guang Chen, at al. Expression pattern and action analysis of genes associated with the responses to chemical stimuli during rat liver regeneration [J]. World J Gastroenterol, 2006, 12(45): 7285-7291.
[50] Qian-Ji Ning, Shao-Wei Qin, Cun-Shuan Xu. Expression patterns and action analysis of genesassociated with drug-induced liver diseases during rat liver regeneration [J]. World J Gastroenterol, 2006, 12(43): 6966-6972.
[51] Olaf Dransfeld, Thor Gehrmann, Karl K?hrer, et al. Oligonucleotide microarray analysis of differential transporter regulation in the regenerating rat liver [J]. Liver Int, 2005, 25: 1243-1258.
[52] Yuceturk H, Yagmurdur MC, Gur G, et al. Role of heparin on TNF-alpha and IL-6 levels in liver regeneration after partial hepatic resection [J]. Eur Surg Res, 2007, 39: 216-221.
[53] Taub R. Liver regeneration in health and disease [J]. Clin Lab Med, 1996, 16: 341-360.
[54] Alcorn JA, Feitelberg SP, Brenner DA. Transient induction of C-Jun during hepatic regeneration [J]. Hepatol, 1990, 11: 909-915.
[55] ZoéD. Burke, Karen R. Reed, Toby J. Phesse, et al. Liver Zonation Occurs Through aβ-Catenin–Dependent, c-Myc–Independent Mechanism [J]. Gastroenterology, 2009, 136(7): 2316-2324.
[56] Morello D, FitzGerald, M.J, Babinet C, et al. C-myc, c-fos and c-jun regulation in the regeneration livers of normal and H2K/c-myc transgenic mice [J]. Mol Cell Biol, 1990, 10: 3185-3193.
[57]李玉昌,徐存拴,张云汉.应用抑制性消减杂交技术克隆大鼠肝再生过程中特异表达基因[J].遗传, 2002, 24: 152-154.
[58] Haber BA, Mohn KL, Diamond RH, et al. Induction patterns of 70 gene during nine days after hepatectomy define the temporal course of liver regeneration [J]. Clin Invest, 1993, 91: 1319-1326.
[59]徐存拴,张守兵杨志利等.蛋白质代谢、折叠、运输、定位、装配相关基因在大鼠肝再生中表达变化[J].分子细胞生物学报,2008, 23(2): 107-119.
[60] Schoen JM, Lautt WW. Nitric oxide potentiates C-Fos mRNA expression after 2/3 partial hepatectomy [J]. Proc West Pharmacol Soc, 2002, 45: 47-48.
[61] Frank Hilberg, Adriano Aguzzi, Norma Howells, et al. C-jun is essential for normal mouse development and hepatogenesis [J]. Nature ,1993, 365: 179-181.
[62] Moser MJ, Gong Y, Zhang MN, et al. The effects of colectomy on immediate-early proto-oncogene expression and hepatic regeneration in the rat [J]. Dig Dis Sci, 2006, 51: 1179-1182.
[63] Job C. Tharappel, Brett T. Spear, Howard P. Glauert. Effect of phenobarbital on hepatic cell proliferation and apoptosis in mice deficient in the p50 subunit of NF-κB [J]. Toxicology and Applied Pharmacology, 2008, 226(3): 338-344.
[64] Frank G?tschel, Claudia Kern, Simona Lang, et al. Inhibition of GSK3 differentially modulates NF-κB, CREB, AP-1 andβ-catenin signaling in hepatocytes, but fails to promote TNF-α-induced apoptosis [J]. Experimental Cell Research, 2008, 314(6): 1351-1366.
[65] David E. Klick, Jeff D. Shadley, Ronald N. Hines. Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding proteinβ(C/EBPβ) liver inhibitory and liver activating proteins [J]. Biochemical Pharmacology, 2008, 76(2): 268-278.
[66] FitzGerald MJ, EM Webber, JR Donovan, et al. Rapid DNA binding by nuclear factor kappa B in hepatocytes at the start of liver regeneration [J]. Cell Growth Differ,1995, 6: 417-427.
[67] Yamada Y, Fausto N. Deficient liver regeneration after carbon tetrachloride injury in mice lacking type1 but not type 2 tumor necrosis factor receptor [J]. Am J Pathol, 1998, 152: 1577-1589.
[68] Ekihiro Seki, Yuichi Kondo, Yuji Iimuro, et al. Demonstration of cooperative contribution of MET- and EGFR-mediated STAT3 phosphorylation to liver regeneration by exogenous suppressor of cytokine signalings [J]. Journal of Hepatology, 2008, 48(2): 237-245.
[69] Chia Hung Chou, Pei-Jer Chen, Yung-Ming Jeng, et al. Synergistic Effect of Radiation and Interleukin-6 on Hepatitis B Virus Reactivation in Liver Through STAT3 Signaling Pathway [J]. International Journal of Radiation Oncology Biology Physics, 2009, 75(5): 1545-1552.
[70] Takuji Takahashi, Shinji Togo, Takafumi Kumamoto, et al. Transfection of NF-κB Decoy Oligodeoxynucleotides into Macrophages Reduces Murine Fatal Liver Failure After Excessive Hepatectomy [J]. Journal of Surgical Research, 2009, 154(2): 179-186.
[71] Kirillova I, Chaisson M, Fausto N. Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation [J]. Cell Growth Differ, 1999, 10: 819-828.
[72] Wen-Huang Peng, Yun-Chen Tien, Chih-Yang Huang, et al.Fraxinus rhynchophylla ethanol extract attenuates carbon tetrachloride-induced liver fibrosis in rats via down-regulating the expressions of uPA, MMP-2, MMP-9 and TIMP-1 [J]. Journal of Ethnopharmacology, 2010, 127(3): 606-613.
[73] Araceli Arellano-Plancarte, Judith Hernandez-Aranda, Kevin J. Catt, et al. Angiotensin-induced EGF receptor transactivation inhibits insulin signaling in C9 hepatic cells [J]. Biochemical Pharmacology, 2010, 79(5): 733-745.
[74] Matthew Hennig, Michele T. Yip-Schneider, Patrick Klein, et al. Ethanol-TGFα-MEK Signaling Promotes Growth of Human Hepatocellular Carcinoma [J]. Journal of Surgical Research, 2009, 154(2): 187-195.
[75] Kiyoshi Migita, Yumi Maeda, Seigo Abiru, et al. Immunosuppressant FK506 inhibits matrix metalloproteinase-9 induction in TNF-α-stimulated human hepatic stellate cells [J]. Life Sciences ,2006, 78(21): 2510-2515.
[76] Cruise JL, Houck KA, Michalopoulos GK. Induction of DNA synthesis in cultured rat hepatocytes through stimulation of alpha 1 adrenoreceptor by norepinephrine [J]. Science, 1985, 227: 749-751.
[77] Tomoharu Shimizu, Takao Suzuki, Huang-Ping Yu, et al. The role of estrogen receptor subtypes on hepatic neutrophil accumulation following trauma-hemorrhage: Direct modulation of CINC-1 production by Kupffer cells [J]. Cytokine, 2008, 43(1): 88-92.
[78]宁黔冀,李恩中,翟心慧等.外源性雌二醇对大鼠再生肝细胞转录活性的调节[J].解剖学报, 2004, 35: 413-416.
[79] Richard G. Ruddell, Derek A. Mann, Grant A. Ramm. The function of serotonin within the liver [J]. J Hepatol, 2008, 48(4): 666-675.
[80] Ciecierski R, Wi?niewski M, Paczek L. Liver regeneration [J]. Pol Merkur Lekarski, 2005, 18: 473-477.
[81] Donna Beer Stolz, Wendy M Mars, Bryon E. Petersen, et al. Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat [J]. Cancer Res, 1999, 59: 3954-3960.
[82] Natarajan A, Wagner B, Sibilia M. The EGF receptor is required for efficient liver regeneration [J]. Proc Natl Acad Sci U S A, 2007, 104: 17081-17086.
[83]张文学,孙卫华,裴云飞等.血管内皮生长因子(VEGF)与肝再生[J].河南师范大学学报(自然科学版), 2004, 32: 65-68.
[84] Earp HS, Dawson TL, Li X, et al. Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research [J]. Breast Cancer Res Treat, 1995, 35: 115-132.
[85]张文学,赵良真,赵艳红等.血管内皮生长因子在大鼠肝再生过程中的表达变化[J].解剖学报, 2006, 34: 402-406.
[86] Fausto N. Liver regeneration [J]. Hepatol, 2000, 32(1 Suppl):19-31.
[87] Akcali KC, Dalgic A, Ucar A, et al. Expression of bcl-2 gene family during resection induced liver regeneration: comparison between hepatectomized and sham groups [J]. World J Gastroenterol, 2004, 10: 279-283.
[88] Holger Willenbring, Amar Deep Sharma, Arndt Vogel, et al. Loss of p21 Permits Carcinogenesis from Chronically Damaged Liver and Kidney Epithelial Cells despite Unchecked Apoptosis [J]. Cancer Cell, 2008, 14(1): 59-67.
[89] Starkel P, L Lambotteb, C Sempouxc, et al. After portal branch ligation in the rat, cellular proliferation is associated with selective induction of c-Ha-rss, p53, cyclin E, and cdk2 [J]. Gut, 2001, 49: 119-130.
[90] Nakano K, Chijiiwa K, Tanaka M. Lower activity of CCAAT/enhance-binding protein and expression of cyclin E, but not cydin D1, activation protein-1 and p2I(WAF1), after partial hepatectomy in obstructive jaundice [J]. Biochem Biophys Res Commun, 2001, 280: 640-645.
[91] Masson S, Daveau M, Hiron M, et al. Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats [J]. Liver, 1999, 19: 312-317.
[92] Hunter T, Pines J. Cyclins and cancer II: Cyelin D and CDK inhibitors come of age [J]. Cell, 1994, 79: 573-582.
[93] Malgorzata Borowiak, Alistair N. Garratt, Torsten Wüstefeld, et al. Met provides essential signals for liver regeneration [J]. Proc Natl Acad Sci U S A, 2004, 101: 10608-10613.
[94] Manuel A. Fernández, Cecilia Albor, Mercedes Ingelmo-Torres, et al. Caveolin-1 is essential for liver regeneration [J]. Science, 2006, 313: 1628-1632.
[95] Raquel García-Olivas, Senén Vilaró, Manuel Reina, et al. PDGF-stimulated cell proliferation and migration of human arterial smooth muscle cells: Colocalization of PDGF isoforms with glycosaminoglycans [J]. The International Journal of Biochemistry & Cell Biology, 2007, 39(10): 1915-1929.
[96] Biglarnia, Markus Kamler, Eva M. Niehues, et al. VEGF is Important for Early Liver Regeneration After Partial Hepatectomy [J]. Journal of Surgical Research, 2007, 138(2): 291-299.
[97] Hashimoto, Masaharu Sakamoto, Ryukichi Kumashiro, et al. Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma [J]. Journal of Hepatology, 2004, 40(5): 799-807.
[98] Chundong Yu, Fen Wang, Chengliu Jin, et al. Role of fibroblast growth factor type 1 and 2 in carbon tetrachloride-induced hepatic injury and fibrogenesis [J]. Am J Pathol, 2003, 163: 1653-1662.
[99] Shimizu H, Mitsuhashi N, Ohtsuka M, et al. Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats [J]. World J Gastroenterol, 2005, 11: 7254-7260.
[100] Papastefanou VP, Bozas E, Mykoniatis MG, et al. VEGF isoforms and receptors expression throughout acute acetaminophen-induced liver injury and regeneration [J]. Arch Toxicol, 2007, 81: 729-741.
[101] LeCouter J, Moritz DR, Li B,et al. Angiogenesis-independent endothelial protection of liver: Role of VEGFR-1 [J]. Science, 2003, 299: 890-893.
[102] Herrera B, Alvarez AM, Beltrán J, et al. Resistance to TGF-beta-induced apoptosis in regenerating hepatocytes [J]. J Cell Physiol, 2004, 201: 385-392.
[103] Reddy JK, Azarnoff DL, Sirtori CR. Hepatic peroxisome proliferation: Induction by BR-931, a hypo- lipidemic analog of WY-14,643 [J]. Arch Int Pharmacodyn Ther, 1978; 234: 4-14
[104] Sakamoto T, Liu Z, Murase N, et al. Mitosis and apoptosis in the liver of interleukin-6-deficientmice after partial hepatectomy [J]. Hepatol, 1999, 29: 403-411.
[105] Starzl TE, Fung J, Tzakis A, et al. Baboon-to-human liver transplantation [J]. Lancet, 1993, 341: 65-71.
[106] A.Mather, X.M.Chen, S.McGinn, et al. High glucose induced endothelial cell growth inhibition is associated with an increase in TGFβ1 secretion and inhibition of Ras prenylation via suppression of the mevalonate pathway [J]. Biochemistry & Cell Biology, 2009, 41(3): 561-569.
[107] Kiba T, Saito S, Numata K, et al. Fas(APO-1/CD95)mRNA is down-regulated in liver regeneration after hepateetomy in rats [J]. Gastroenteml, 2000, 35: 34-38.
[108]李红蕾,陈晓光,张富春等.细胞外基质相关基因在大鼠肝再生中表达模式分析[J].遗传,2008, 30: 333-340.
[109] Mónika Gallai a, Anna Sebestyéna, Péter Nagya, et al. Proteoglycan gene expression in rat liver after partial hepatectomy [J]. Biochem Biophys Res Commun, 1996, 228: 690-694.
[110] K.Lenhard Rudolph, ChristianTrautwein, Stefan Kubicka, et al. Differential regulation of extracellular matrix synthesis during liver regeneration after partial hepatectomy in rats [J]. Hepatol, 1999, 30: 1159-1166.
[111] Gkretsi V, Bowen WC, Yang Y, et al. Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation [J]. Biochem Biophys Res Commun, 2007, 353: 638-643.
[112]赵利峰,徐存拴,王林嵩等.调控肝再生的基因和生长因子的研究进展[J].解剖学报, 2004, 35: 334-336.
[113] Lalor PF, Lai WK, Curbishley SM, et a1. Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo [J]. World J Gastroenterol, 2006,12: 5429-5439.
[114] Michalopoulos GK. Liver regeneration [J]. J Cell Physiol, 2007, 213: 286-300.
[115] Khan Z, Michalopoulos GK, Stolz DB. Peroxisomal localization of hypoxia-inducible factors and hypoxia-inducible factor regulatory hydroxylases in primary rat hepatocytes exposed to hypoxia-reoxygenation [J]. Am J Pathol, 2006, 169: 1251-1269.
[116] S.Marubashi, M.Sakon, H.Nagano, et a1. Effect of portal hemodynamics on liver regeneration studied in a novel portohepatic shunt rat model [J]. Surgery, 2004, 136: 1028-1037.
[117] Nelsen CJ, Rickheim DG, Tucker MM, at al. Amino acids regulate hepatocyte proliferation through modulation of cyclin D1 expression [J]. J Biol Chem, 2003, 278(28):25853- 25858.
[118] Avruch J, Lin Y, Long X, at al. Recent advances in the regulation of the TOR pathway by insulin and nutrients [J]. Curr Opin Clin Nutr Metab Care, 2005, 8(1):67-72.
[119] Volarevic S, Stewart MJ, Ledermann B, at al. Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6 [J]. Science, 2000, 288(5473):2045-2047.
[120] Goggin MM, Nelsen CJ, Kimball SR, at al. Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver [J]. Hepatology, 2004, 40(3):537-544.
[121] Sven A. Lang, Christina Hackl, Christian Moser. Implication of RICTOR in the mTOR inhibitor-mediated induction of insulin-like growth factor-I receptor (IGF-IR) and human epidermal growth factor receptor-2 (Her2) expression in gastrointestinal cancer cells [J]. Molecular Cell Research, 2010, 1803(4): 435-442.
[122] Bordereaux D, Fichelson S, Tambourin P, at al. Alternative splicing of the EVI1 zinc finger gene generates mRNAs which differ by number of zinc finger motifs [J]. Oncogene, 1990, 5:925-927.
[123] Mosthaf L, Grako K., Dull T J, at al. Functionally distinct insulin receptors generated by tissue-specific alternative splicing [J]. EMBOJ, 1990, 9:2409-2413.
[124] LaRosa, G.J, Gudas, L. Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the Homeobox [J]. Mol. Cell. Biol, 1988, 8:3906-3917.
[125] Hayzer, D.J, Iynedjian, P.B. Alternative splicing of glucokinase mRNA in rat liver [J]. Biochem, 1990, 270: 261-263
[126] Caras I.W, Davitz M.A., Rhee L., at al. Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins [J]. Nature, 1987, 325:545-549.
[127] Bond, R.W, Wyborski R.J, GottIieb, D.I. Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase [J]. Proc. natI.Acad. Sci. USA , 1990, 87:8771-8775.
[128] T.N. Dear, R.F. Kefford. The WDNM1 gene product is a novel member of the‘four-disulphide core’family of proteins [J]. Biochem. Biophys. Res. Commun., 1991,176:247-254.
[129] B. Morrison, P. Leder. Neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2 initiated tumors [J]. Oncogene, 1994, 9:3417-3426.
[130] Weinberg R.A. Positive and negative controls on cell growth [J].Biochem, 1989, 28: 8263-8269.
[131] Schirrmacher V. Cancer metastasis: Experimental ap-proaches, theoretical concepts and impacts for treatment strategies [J]. Adv. Cancer Res. , 1985, 43: l-73.
[132] DearT.N, Ramshaw I.A. and Kefford R.F. rential expression of a novel gene, WDNM1, in nonmetastatic rat mammary adenocarcinoma cells [J]. Cancer Res., 1988,48:5203-5209.
[133] T.N. Dear, D.A. McDonald , R.F. Kefford. Transcriptional down-regulation of a rat gene, WDNM2, inmetastatic DMBA-8 cells [J]. Cancer Res, 1989, 49:5323-5328.
[134] P.S. Steeg, Search for metastasis suppressor genes [J]. Invasion Metastasis, 1989, 9:351-359.
[135] Nicolson G.L. Cancer metastasis: tumor cell and host organ properties important in metastasis to specific secondary sites [J].Biochim. Biophys. Acta, 1988, 948:175-224.
[136] Matrisian L.M, Bowden G.T, Krieg P, at al. The mRNA coding for the secreted protease transin is expressed more abundantly in malignant than in benign tumors [J]. Proc. natl. Acad. Sci. USA, 1986, 83:9413-9417.
[137] Ostrwoski L.E, Finch J, Krieg P, at al. Expression pattern of a gene for a secreted metalloproteinase during late stages of tumor progression. [J]. Mol. Carcinog., 1988, 1:13-19.
[138] Sappino A.P, Busso N, Belin D, at al. Increase of urokinase-type plasminogen activator gene expression in human lung and breast carcinomas. [J]. Cancer Res., 1987, 47:4043-4046.
[139] Denhardt D.T, Greenberg A.H, Egan S.E, at al. Cysteine proteinase cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts. [J]. Oncogene, 1987, 2:55-59.
[140] Bonfil R.D, Reddel R.R, Ura H, at al. Invasive and metastatic potential of a v-Ha-ras-transformed human bronchial epithelial cell line [J]. Cancer Inst. , 1989, 81:587-594.
[141] Khokha R, Waterhouse. P, Yagel S, at al. ntisense RNA-induced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells [J]. Science , 1989,243:947-950.
[142] T.N. Dear, R.F. Kefford. The WDNMl gene product is a novel member of the‘four disulphide core’family of proteins [J]. BIOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1991, 176:247-254.
[143] Ramshaw LA., Badenoch-Jones P. Studies on rat mammary adenocarcinomas: a model for metastasis. [J]. Cancer Metastasis Rev, 1985, 4:195-208.
[144] Grant A., Ramshaw I.A., Badenoch-Jones P, at al. Purification and characterization of an inhibitor of plasminogen activator released by rat mammary adenocarcinoma cells [J]. Eur. J. Biochem., 1986, 154:635-641.
[145] B. Choi, K. Myung, M. Lee, at al. Cloning of involution-induced cDNAs from mouse mammary gland [J]. Mol. Cells, 1996, 6:311-315.
[146] Dae Jun Jung, Jin Jong Bong, Myunggi Baik. Extracellular proteinase inhibitor-accelerated apoptosis is associated with B cell activating factor in mammary epithelial cells [J]. Exp Cell Res, 2004, 292(1):115-22.
[147] P. Schneider, F. MacKay, V. Steiner, at al. a novel ligand of the tumor necrosis factor family, stimulates B cell growth [J]. Exp. Med, 1999,189:1747-1756.
[148] M. Karpusas, T.G. Cachero, F. Qian, at al. Crystal structure of extracellular human BAFF, a TNF family member that stimulates B lymphocytes [J]. Mol. Biol, 2002, 315:1145-1154.
[149] F. Mackay and C.R. Mackay. The role of BAFF in B cell maturation, T cell activation and autoimmunity [J]. Trends Immunol, 2002, 23:113-115.
[150] Y. Laabi, A. Strasser, Immunology: Lymphocyte survival—Ignorance is Blys [J]. Science, 2000,289:883-884.
[151] A. Mukhopadhyay, J. Ni, Y. Zhai, at al. Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-B, and c-Jun NH2-terminal kinase [J]. J. Biol. Chem. 1999, 274 :15978-15981.
[152] Y. Laabi, A. Egle, A. Strasser. TNF cytokine family: more BAFF-ling complexities [J]. Curr. Biol. 2001, 11:1013-1016.
[153] Hampton R.Y. ER-associated degradation in protein quality control and cellular regulation [J]. Curr Opin Cell Biol, 2002, 14: 476-482.
[154] Gomes AV, Zong C, Ping P. Protein degradation by the 26S proteasome system in the normal and stressed myocardium [J]. Antioxid Redox Signal, 2006, 8(9-10): 1677-1691.
[155] Otlewski J, Jelen F, Zakrzewska M, at al. The many faces of protease-protein inhibitor interaction [J]. EMBO J, 2005, 24: 1303-1310.
[156] Bordusa F. Substrate mimetics in protease catalysis: characteristics, kinetics, and synthetic utility [J]. Curr Protein Pept Sci, 2002, 3: 159-180.
[157] Klaudia Brix, Anna Dunkhorst, Kristina Mayer, at al. Cysteine cathepsins: Cellular roadmap to different functions [J]. Biochimie, 2008, 90(2): 194-207
[158] Barrett AJ, Rawlings ND. Evolutionary lines of cysteine peptidases [J]. Biol Chem, 2001, 382(5):727-733.
[159] Barrett AJ, Rawlings ND. Evolutionary lines of cysteine peptidases [J]. Biol Chem, 2001, 382: 727-733.
[160] Dickinson DP. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease [J]. Crit Rev Oral Biol Med, 2002, 13: 238-275.
[161] Ascenzi P, Salvati L, Bolognesi M, et al. Inhibition of cysteine protease activity by NO-donors [J]. Curr Protein Pept Sci, 2001, 2(2):137-153
[162] Dickinson DP. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease [J]. Crit Rev Oral Biol Med, 2002, 13(3):238-275
[163] Boris Turk, Veronika Stoka. Protease signalling in cell death: caspases versus cysteine cathepsins [J]. FEBS Letters, 2007, 581(15): 2761-2767
[164] Siewinski M, Saleh Y, Ziolkowski P. Cysteine peptidases in health and diseases [J]. Folia Med Cracov, 2003, 44(1-2):169-178.
[165]李春蕊,刘文励,孙汉英.焦点粘附物及其在细胞迁移中的作用[J].国外医学?生理、病理科学与临床分册. 2002, 22 (6): 583-596.
[166]王玉珍,姜慧卿,扈彩霞. Rho信号转导通路与细胞迁移[J].医学综述. 2006, 12 (11): 651-653.
[167] Dan Du, Esben Pedersen, Zhipeng Wang, at al. Cdc42 is crucial for the maturation of primordial cell junctions in keratinocytes independent of Rac1 [J]. Experimental Cell Research, 2009, 315 (8): 1480-1489.
[168] Hee-YoungYang, Ju Kim, Kyung-Yeol Lee, at al. Rac/ROS-related protein kinase C and phosphatidylinositol-3-kinase signaling are involved in a negative regulating cascade in B cellactivation by antibody-mediated cross-linking of MHC class II molecules [J]. Molecular Immunology, 2010, 47 (4): 706-712
[169] Schmitz AA, Govek EE, B?ttner B, at al. Rho GTPases: signaling, migration, and invasion [J]. Exp Cell Res. 2000, 261 (1): 1-12.
[170]芦曙辉,王玉琦.细胞迁移机制的研究进展[J].中国临床医学. 2003, 10 (6): 801-803.
[171] M JuliaGarcía-Fuster, Alfredo Ramos-Miguel, Antonio Miralles, at al. Opioid receptor agonists enhance the phosphorylation state of Fas-associated death domain (FADD) protein in the rat brain: Functional interactions with casein kinase Iα, Gαi proteins, and ERK1/2 signaling[J]. Neuropharmacology, 2008,55(5): 886-899.
[172] Zha J,Weiler S,Oh KJ,et al. Posttranslational N-myristoy-lation of Bid as a molecular switch for targeting mitochondria and apoptosis[J].Science,2000,290(5497):1761-1765.
[173] Ali. M. Sharifi, Habib Eslami, Bagher Larijani, et al. Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells[J]. Neuroscience Letters, 2009, 459(2)47-51
[174] Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes [J]. Cell, 2003, 114:181-190.
[175] Guy C. Brown, Vilmante Borutaite. Regulation of apoptosis by the redox state of cytochrome c [J]. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008, 1777(7-8): 877-881.
[176] Boehning D, van Roswm DB, Patterson RL, et al. A peptide inhibitor of rytochrome c/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and ex-trinsic cell death pathways [J]. Proc Natl Acad Scl U S, 2005, 102:1466-1471.
[177] Ueli S. Liver regeneration clocks on [J]. Science, 2003, 302: 234-235.
[178] Sanchez A, Factor VM, Schroeder IS, at al. Activation of NF-kappaB and STAT3 in rat oval cells during 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration [J]. Hepatology, 2004, 39(2): 376-385.
[179] Yasukatsu Takushi, Masayuki Shiraishi, Eiji Nozato, at al. Expression of Anti-Apoptotic Protein, Bcl-2, in Liver Regeneration After a Partial Hepatectomy [J]. Surgical Research, 2006, 134(1): 93-101.
[180] Maria Cecília M. Coelho, Uenis Tannuri, Ana Cristina A. Tannuri, at al. Expression of interleukin 6 and apoptosis-related genes in suckling and weaning rat models of hepatectomy and liver regeneration [J]. Pediatric Surgery, 2007, 42(4): 613-619.
[181] Ketsia B. Pierre, Christopher M. Jones, Janene M. Pierce, at al. NFAT4 Deficiency Results in Incomplete Liver Regeneration Following Partial Hepatectomy [J]. Surgical Research, 2009, 154(2): 226-233.
[182] Maren Ilowski, Christine Putz, Thomas S. Weiss, at al. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner [J]. Biochemical and Biophysical Research Communications, 2010, 394(4): 915-920.
[183] Hughes RD, Evans LW. Activin A and follistatin in acute liver failure [J]. Eur J Gastroenterol Hepatol, 2003, 15(2): 127-131.
[184] Date M, Matsuzaki K, Matsushita M, et al. Differential regulation of activin A for hepatocyte growth and fibronectin synthesis in rat liver injury [J]. J Hepatol, 2000, 32(2): 251-260.
[185]徐存拴,邢雪琨,谢来峰等.液压转基因技术应用于大鼠肝脏转基因研究[J].解剖学报,2009,40 (1): 103-107.
[186] Wolff JA,Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo [J]. Science, 1990, 247 (4949 pt 1):1465-1468.
[187] Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA [J]. Gene Ther, 1999, 6 (7):1258-1266.
[188] Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA [J]. Hum Gene Ther, 1999, 10 (10):1735-1737.
[189]徐存拴,邢雪琨,杨献光等.液压转基因技术应用于大鼠再生肝转基因实验[J].解剖学报, 2009,40 (4): 599-603.
[190] Chen ZY, Yant SR, Lie CY, et al. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver [J]. Mol Ther, 2001, 3 (3):403-410.
[191] Miao CH, Thompson AR, Loeb K, et al. Long-term and therapeutic-level hepatic gene expression of human factor FIX after naked plasmid transfer [J]. Mol Ther, 2001, 3 (6):947-957.
[192] Ge Y, Powell S, Van Roey M, et al. Factors influencing the development of an anti-factorⅨ(FⅨ) immune response following administration of adeno-associated virus-FⅨ[J]. Blood, 2001, 97 (12):3733-3737.
[193] Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against virus [J]. Nature, 2001, 411 (6839):834-841.
[194] Qin L, Ding Y, Pahud DR, et al. Promoter attenuation in gene therapy: interferon-γand tumor necrosis factor-αinhibit transgene expression [J]. Hum Gene Ther, 1997, 8 (17):2019-2029.
[195] Harms JS, Splitter GA. Interferon-γinhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian HNCI promoter [J]. Hum Gene Ther, 1995, 6 (10):1291-1297.
[196] Clark AJ, Harold G, Yull F. Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice [J]. Nucleic Acids Res, 1997, 25 (5):1009-1014.