摘要
目的:Ⅰ、Ⅲ型胶原在肝细胞外过度沉积是肝纤维化、肝硬化形成的一个重要特征。然而,到目前为止仍没有一种有效地治愈肝纤维化、肝硬化的方法。中性粒细胞胶原酶(MMP-8)能够特异的降解Ⅰ型胶原,把MMP-8基因导入肝细胞将有可能是降解胶原纤维有效的方法。本研究通过构建一种能够表达MMP-8的腺病毒,治疗肝硬化的大鼠,探讨其能否有效地降解胶原,减轻肝脏的纤维化程度。
方法:以质粒pGW1GH-MMP8为模板,应用聚合酶链反应(polymerase chain reaction,PCR)和酶切重组技术克隆了全长的MMP-8基因(pro-MMP8)及其截短的MMP-8基因(tMMP-8)片段,构建了复制缺损性腺病毒载体携带乙肝病毒(HBV)载体表达MMP-8基因的质粒pDC-CH-tMMP8、pDC-C-MMP8,经酶切鉴定正确后进行质粒的大量提取,并用同样的方法制备对照质粒pDC-CH-RFP2,构建的质粒与腺病毒骨架质粒pBHGLox△E1,3Cre共转染293细胞以包装、扩增重组腺病毒。在动物实验中,以3×1011病毒颗粒尾静脉注射治疗硫代乙酰胺诱导的肝硬化大鼠模型,分别于2周、4周后处死动物,0.3%戊巴比妥腹腔注射麻醉,腹主动脉取血,收集血清保存于-70℃,做肝功能、肝纤四项等检查,分离肝组织,部分用10%的福尔马林固定,进行组织病理学检查,其余-70℃保存备用。
结果:成功构建了表达MMP-8基因的腺病毒Ad-CH-tMMP8、Ad-C-MMP8,并运用于治疗肝硬化大鼠的研究。在Ad-CH-tMMP8、Ad-C-MMP8尾静脉注射肝硬化大鼠的体内转染研究中,转染2周后相比于模型组和阴性对照组,肝纤维化程度减轻。HE染色及天狼猩红染色结果显示治疗组肝细胞脂肪变性、肝细胞坏死及炎症细胞浸润均有减轻,可见不同程度的肝细胞再生,胶原纤维条索变细变窄,肝小叶结构恢复,与模型组及阴性对照组相比有统计学意义(P<0.05);羟脯氨酸含量测定结果显示与模型组相比,治疗组肝组织羟脯氨酸含量有所下降(28.97±2.36 VS 17.04±0.61、17.62±1.30),具有统计学上的差异(P<0.05),而Ad-CH-RFP2组较模型组又有所升高(30.89±3.67);血清ALT测定结果显示治疗组、阴性对照组大鼠ALT升高,显著高于正常组和模型组(P<0.05),然而,与正常组、模型组、阴性对照组相比,治疗组血清总胆红素、血清白蛋白没有显著的变化,无统计学差异(P>0.05)。转染4周,治疗组大鼠肝纤维化程度继续减轻。而在Ad-CH-RFP2对照组,纤维化程度同模型组类似,并未减轻。
结论:实验研究结果表明,MMP-8能够有效地降解Ⅰ型胶原,减轻大鼠肝纤维化程度。这一创新,有望把MMP-8基因治疗肝硬化带入临床应用,开辟肝硬化软肝治疗新途径。
Objective:Excess typeⅠ,Ⅲcollagen accumulation is a major feature of liver fibrosis and cirrhosis, reversion of this disease has not been fully accomplished. Neutrophil collagenase(MMP-8) can degrade typeⅠcollagen effectively. Introduction of MMP-8 gene into liver cells could be an advantageous tool to degrade collagen. In this study, we constructed a adenovirus expressing MMP-8 and intended to investigate which can degrade collagens and attenuate established hepatic fibrosis in the rat.
Methods:We cloned full length MMP-8 gene (pro-MMP8) and truncated MMP-8 gene (tMMP-8) from plasmid pGW1GH-MMP8 by polymerase chain reaction(PCR) and enzyme-cutting recombination technique, and constructed the plas- mid pDC-CH-tMMP8, pDC-C-MMP8 which express MMP-8 in adenoviral vector carrying replication-deficient HBV vector , we have also constructed the control plasmid pDC-CH-RFP2. The constructed plasmids were conformed by enzyme-cutting technique and then co-transformed with pBHG1ox△E1,3Cre into 293 cells in order to package and expand recombinant adenovirus. Liver cirrhosis rats induced by TAA for ten weeks were infected once with recombinant adenovirus(3×1011 viral particles per kilogram) by intravenous injection through tail veins. At 2 or 4 weeks after the adenoviral infection, rats were killed. Animals were injected with 0.3% Pentobarbital and killed thirty minutes later. Blood samples were collected by puncture of the abdominal aorta, and serum was stored at -70℃until analysis.Samples of the liver were snap-frozen at -70℃or were fixed in 10%buffered formalin for subsequent histological analysis.
Results:We have successfully constructed the recombinant adenovirus expressing MMP-8(Ad-CH-tMMP8, Ad-C-MMP8) and test the activity of MMP-8 in animal exprement, compared with model group and negative control group, the fibrosis was dramatically attenuated at two weeks after the infection, Compared with model group and negative control group,HE staining and bitter acid-sirius red staining showed that hepatocyte steatosis, necrosis and inflammation were significantly mild in treatment group, along with hepatocyte proliferation and recovery of hepatic lobules structure(P<0.05); compared with model group, the content of HYP diminished(28.97±2.36 VS 17.04±0.61, 17.62±1.30; P<0.05);compared with model group and normal group, measurement of serum ALT showed that adenovirus expression in the liver transiently induced a significant increase in serum ALT(P<0.05). However, compared with normal group、model group and negative control group, serum total bilirubin and serum albumin levels did not change significantly in treantment group(P>0.05). After 4 weeks, the proliferative effect almost disappeared, but the hepatic fibrosis remained attenuated and the hydroxyproline content remained diminished. Whereas, the fibrosis in Ad- CH-RFP2-treated rats persisted.
Conclusion:Studying results shows that the MMP-8 gene can degrade typeⅠcollagen effectively and attenuate established fibrosis in rat induced by TAA. It is promising for use in a clinical setting and providing a novel therapy tool for liver fibrosis.
引文
1 Eng FJ, Friedman SL. Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol,2000,279:G7-11
2 Chan HL, Leung NW, Hussain M, et al. Hepatitis B e antigen-negative chronic hepatitis B in Hong Kong. Hepatology,2000,31:763-768
3 Progress towards the comprehensive control of hepatitis B. Proceedings of a roundtable meeting.Windsor, Berkshire, United Kingdom, 25-26 July 1995. Gut 1996,38 Suppl 2: S1-70
4 Iredale JP, Murphy G, Hembry RM, et al. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver. J Clin Invest,1992,90:282-287
5 Murawaki Y, Yamamoto H, Kawasaki H.et al. Serum tissue inhibitor of metalloproteinases in patients with chronic liver disease and with hepatocellular carcinoma. Clin Chim Acta 1993,218:47-58
6 Leyland H, Gentry J, Arthur MJ.et al. The plasminogen activating system in hepatic stellate cells. Hepatology, 1996,24:1172-1178
7 Montasser MF, Younis TA, Fahmy IA, et al. Histopath- ological evidences for the antifibrogenic effect of colchicine in schistosomiasis mansoni in mice. J Egypt Soc Parasitol, 1994,24:155-165
8 Leuschner M, Maier KP, Schlichting J, et al. Oral budesonide and ursodeoxycholic acid for treatment of primary biliary cirrhosis: results of a prospective double-blind trial. Gastroenterology,1999,117:918-925
9 Garcia-Banuelos J, Siller-Lopez F, Miranda A, et al.Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion. Gene Ther,2002,9:127-134
10 Dai WJ, Jiang HC. Advances in gene therapy of liver cirrhosis:a review. World J Gastroenterol 2001,7:1-8
11 Gressner AM, Weiskirchen R, Breitkopf K. et al. Roles of TGF-beta in hepatic fibrosis. Front Biosci, 2002,1:793-807
12 Ueki T, Kaneda Y, Tsutsui H, et al. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med, 1999, 5(2):226-30
13 Matsuno Y, Iwata H, Umeda Y.et al. Hepatocyte growth factor gene transfer into the liver via the portal vein using electroporation attenuates rat liver cirrhosis. Gene Ther, 2003,10(18):1559-66
14 Xia JL, Dai C,Michalopoulos GK.et al. Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation. Am J Pathol,2006,168(5):1500-12
15 Nakamuta M, Morizono S,Tsuruta S.et al. Remote delivery and expression of soluble type II TGF-beta receptor in muscle prevents hepatic fibrosis in rats. Int J Mol Med,2005, 16(1):59-64
16 Hung KS, Lee TH, Chou WY.et al. Interleukin-10 gene therapy reverses thioacetamide-induced liver fibrosis in mice. Biochem Biophys Res Commun, 2005,14,336 (1):324-31
17 Iimuro Y, Nishio T, Morimoto T, et al. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat. Gastroenterology,2003,124(2):445-58
18 Protzer U, Nassal M, Chiang PW, et al. Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection. Proc Natl Acad Sci USA, 1999, 96(19):10818-23
19 Siller-Lopez F, Garcia-Banuelos J, Hasty KA, et al. Truncat- ed active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen. J Hepatol,2000,33(5):758 -63
1 Mulligan RC.The basic science of gene therapy.science, 1993,260:936-932
2 Miller DG, Adam MA, Miller AD.Gene thansfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.Mol Cell Biol.1990, 10:4239-4242
3 Friedman T, Yee JK.Pseudotyped retroviral vecters for studies of human gene therapy.Nature Med,1995, 1:275-277
4 Siller-Lopez F, Sandoval A, Salgado S, et al.Treatment with human metalloproteinase-8 gene delivery ameliorates exper- imental rat liver cirrhosis. Gastroenterology, 2004,126(4): 1122-1133
5 孙殿兴, 胡大荣, 邬光惠.抗乙型肝炎病毒分子治疗新策略.中华肝脏病杂志,2001,9(4):315-316
1 张杰, 陈积圣, 王坤等.硫代乙酰胺诱导肝硬化模型方法的改进,中华实验外科杂志 1999 年 11 月第 16 卷第 6 期
2 Dashti H, Jeppsson B, Haggerstrand I, et al. Thioacetamide and carbon tetrachloride induced liver cirrhosis. Eur Surg Res,1989,21:83
3 Muller D, Zimmerman ST, Schiller F. Drug metabolism in rat liver injured by Thioacetamide. Arch Toxicol,(suppl)5, 1982,368
4 Fitzhugh OG, Nilsson A.Liver tumours in rats fed thiourea or Thioacetamide. Science, 1948,108 :626
5 Muller A, Machnik F, Zimmermann T.et al. Thioacetamide induced cirrhosis-like liver lesions in rats-usefulness and reliability of this animal model. Exp Pathol,1988;34: 229-236
6 Oren R, Dotan I, Papa M.et al. Inhibition of experimentallyinduced cirrhosis in rats by hypothyroidism. Hepatology, 1996, 24:419-423
1 Michalopoulos GK, Defrances MC, Liver regeneration. Science,1997,276(5309):60-66
2 Grappone C, Pinzani M, Parola M.et al. Expression of platelet-derived growth factor in newly formed cholang- iocytes during experimental biliary fibrosis in rats. J Hepatol, 1999,31(1):100-109
3 Iimuro Y, Nishio T, Morimoto T, et al. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat. Gastroenterology,2003, 124(2):445-58
4 Siller-Lopez F, Garcia-Banuelos J, Hasty KA.et al.Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen. J Hepatol,2000, 33(5):758-63
5 Yasuda M, Shimizu I, Shiba M,et al. Suppressive effects of estradiol on dimethylnitrosamine-induced fibrosis of the liv- er in rats. Hepatology,1999, 29:719-727
6 Ueno H,Sakamoto T,Nakamura T, et al. soluble transforming growth factor betareceptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats. HumGene Ther,2000,11:33-42
7 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, 117:1198-1204
8 Qi Z, Atsuchi N, Ooshima A, et al. Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci US A,1999, 96:2345-2349
9 Masunaga H, Fujise N, Shiota A, et al. Preventive effects of the deleted formof hepatocyte growth factor against various liver injuries. Eur J Pharmacol,1998,342:267-279
10 Murata T, Arii S, Nakamura T, et al. Inhibitory effect of Y-27632, a ROCK inhibitor, on progression of rat liver fibrosis in association with inactivation of hepatic stellate cells. J Hepatol,2001,35:474-481
11 Tada S, Iwamoto H, Nakamuta M, et al. A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats.J Hepatol,2001,34:529-536
12 Ramadori G, Knittel T, Saile B. Fibrosis and altered matrix synthesis. Digestion,1998,59(4):372-375
13 Iredale JP. A cut above the rest? MMP-8 and liver fibrosis gene therapy.Gastroenterology, 2004,126(4):1199-1201
14 Salgado S, Garcia J, Vera J.et al. Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy. Mol Ther,2000,2:545-551
15 Hecht N, Pappo O, Shouval D.et al.Hyper-IL-6 gene therapy reverses fulminant hepatic failure. Mol Ther,2001,3: 683- 687
16 Nakatani T, Kuriyama S, Tominaga K.et al. Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers. Gut,2000,47:563- 570
17 Garc?′a-Ban?uelos J, Siller-Lo′pez F, Miranda A.et al. Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors.Evidence of cirrhosis reversion. Gene Ther,2002,9:127-134
18 Carmeliet P, Moons L, Lijnen R. et al. Urokinasegenerated plasmin activates matrix metalloproteinases during aneury- sm formation. Nat Genet,1997,17:439-444
19 Salgado S, Garcia J, Vera J.et al. Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy.Mol Ther,2000,2:545-551
20 Ueki T,Kaneda Y,Tsutsui H.et al. Hepatocyte growth factor gene therapy of liver cirrhosis in rats.Nat Med,1999,5:226- 230
21 Bickel M, Baringhaus KH, Gerl M.et sl.Selective inhibition of hepatic collagen accumulation in experimental liver fibro- sis in rats by a new prolyl 4-hydroxylase inhibitor.Hepatolo- gy,1998,28:404-411
22 Sakaida I, Hironaka K, Uchida K.et al.Fibrosis accelerates the development of enzyme-altered lesions in the rat liver.Hepatology,1998,28:1247-1252
1 Gressner AM. Transdifferentiation of hepatic stellate cells (Ito cells)to myofibroblasts: a key event in hepatic fibrogenesis. Kidney IntSuppl,1996,54:39-45
2 Eng FJ, Friedman SL. Fibrogenesis: I. New insights into hepaticstellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol,2000,279:G7-11
3 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000,275:2247-2250
4 Owen CA, Hu Z, Lopez-O-tin C, et al. Membrane-bound matrix metalloproteinase-8 on activated polymorphonuclear cells is a potent,tissue inhibitor of metalloproteinase resistant collagenase and serp inase. J Immunol,2004,172: 7791-78031
5 Wojtowicz-Praga SM, Dickson RB, et al. Matrix metallo- proteinase inhibitors. Invest New Drugs,1997,15(1): 61-75
6 Suzuki K, Enghild JJ, Morodomi T,et al.Mechanisms of activation of tissue procollagenase by matrix metalloprotein- ase 3 (stromelysin).Biochemistry,1990,6;29(44):10261-70
7 Hozumi A, Nishimura Y, et al. Induction of MMP-9 in normal human bronchial epithelial cells by TNF-alpha via NF-kappa B-mediated pathway.Am J Physiol Lung Cell Molphysiol,2001,6:1444
8 Tsukada S, Parsons CJ, Rippe RA. Mechanisms of liver fibrosis. Clin Chim Acta,2006,364(1-2):33-60
9 Brenner DA, Waterboer T,et al.New aspects of hepatic fibrosis. J Hepatol,2000,32:32-38
10 Perez-Tamayo R, Montfort I, et al. Collagenolytic activity in experimental cirrhosis of the liver.Exp Mol Pathol,1987,47:300-308
11 Benyon RC, Arthur MJP. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis,2001, 21:373-384
12 Emonard H, Grimaud JA. Matrix metalloproteinases. A review.Cell Mol Biol,1990,36:131-153
13 Holmbeck K, Bianco P, et al. MT1-MMP-deficient mice develop dwarfism,osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell,1999,99: 81-92
14 Ohuchi E, Imai K, et al. Membrane type 1 matrix metalloproteinase digests interstitital collagenas and other extracellular matrix macromolecules. J Biol Chem,1997, 272:2446-2451
15 Pardo A, Selman M. MMP-1: the elder of the family. Int J Biochem Cell Biol, 2005,37(2):283-288
16 Siller-Lopez F, Sandoval A, et al.Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis. Gastroenterology, 2004,126(4):1122-1133
17 Iimuro Y, Nishio T, et al. Delivery of matrix metallo- proteinase-1 attenuates established liver fibrosis in the rat. Gastroenterology,2003,124(2):445-458
18 Salgado S, Garcia J, et al.Liver Cirrhosis Is Reverted by Urokinase-Type Plasminogen Activator Gene Therapy. Molecular Therapy,2000,2(6)545-551
19 Friedman SL. Liver fibrosis—from bench to bedside. JHepatol,2003,38(Suppl 1):S38-53
20 Jansen PL. Non-alcoholic steatohepatitis.Eur J Gastroenterol Hepatol,2004,16:1079-1085