基础研究 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-02-15; 11(2): 209-213
在线出版日期: 2003-02-15. doi: 10.11569/wcjd.v11.i2.209
肝细胞生长因子抗肝纤维化作用及对MMP-1, TIMP-1表达的影响
宋刘来, 罗和生, 余保平
宋刘来, 罗和生, 余保平, 武汉大学人民医院消化内科 湖北省武汉市 430060
宋刘来, 男, 1972-10-22生, 安徽省安庆市人, 汉族. 1996年安徽蚌埠医学院本科毕业, 现为武汉大学人民医院硕士研究生, 研究方向为消化系疾病的诊治.
通讯作者: 罗和生, 430060, 湖北省武汉市, 武汉大学人民医院消化内科. luotang@public.wh.hb.cn
收稿日期: 2002-07-12
修回日期: 2002-07-20
接受日期: 2002-07-29
在线出版日期: 2003-02-15

目的: 研究肝细胞生长因子对实验性大鼠肝纤维化的防治作用, 及其对大鼠肝脏基质金属蛋白酶1(MMP-1)抑制因子1(TIMP-1)表达的影响, 探讨HGF抗肝纤维化作用的可能机制.

方法: 将Wistar♀大鼠80只随机分为正常对照组(A组, 16只), 肝纤维化模型组I(B组, 54只), HGF治疗组I(C组, 10只), 采用CCl4复合因素造模, 治疗组于造模同时予HGF 0.5 mg/Kg, ip, qd, 6 wk末造模成功处死C组大鼠, 同时随机处死A组及B组大鼠各6只; 对B组剩下大鼠行二次随机分组为模型对照组II(D组, 12只), HGF治疗组II(E组, 10只), E组于第7周开始给予HGF治疗, 10 wk末处死大鼠. 检测大鼠肝功能, 血清透明质酸(HA), 层粘蛋白(LN), III型前胶原(PcIII), IV型胶原(CIV); 免疫组化法检测肝组织MMP-1, TIMP-1的表达.

结果: C组与B组比较, ALT, AST, HA, LN, CIV, PcIII均显著降低(P<0.01), MMP-1活性升高(0.25±0.02, vs 0.22±0.05, P<0.05); TIMP-1活性明显降低(0.34±0.05, vs 0.45±0.05, P<0.01). E组与D组相较, MMP-1活性变化无显著性, TIMP-1活性有明显降低(0.31±0.07, vs 0.42±0.06, P<0.01).

结论: 肝细胞生长因子对肝纤维化有明显的防治作用, 并可能通过促进MMP-1的活性或抑制TIMP-1活性而促进肝纤维化降解.

关键词: N/A

引文著录: 宋刘来, 罗和生, 余保平. 肝细胞生长因子抗肝纤维化作用及对MMP-1, TIMP-1表达的影响. 世界华人消化杂志 2003; 11(2): 209-213
Effects of hepatocyte growth factor on fibrosis and hepatic expression of MMP-1 andTIMP-1
Liu-Lai Song, He-Sheng Luo, Bao-Ping Yu
Liu-Lai Song, He-Sheng Luo, Bao-Ping Yu, The Devision of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
Correspondence to: He-Sheng Luo, The Devision of gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China. luotang@public.wh.hb.cn
Received: July 12, 2002
Revised: July 20, 2002
Accepted: July 29, 2002
Published online: February 15, 2003

AIM: To investigate the effect of hepatocyte growth factor (HGF) on severity of liver fibrosis and hepatic expressions of MMP-1, TIMP-1 and to explore the mechanism of HGF in preventing liver fibrosis in rats.

METHODS: Eighty male Wistar rats were randomly divided into normal control group (Group A, 16 rats), liver fibrosis model group (Group B, 54 rats) and HGF therapy group (Group C, 10 rats). The liver fibrosis model was induced by administration CCl4 intraperitoneally. Rats in Group C had been administered HGF for six weeks and were sacrificed afterwards. Eight rats from each of group A and B were randomly sacrificed on week 6 simultaneously as that in group C. The remaining rats in-group B were randomly further subdivided into liver fibrosis model group (Group D, 12 rats) and HGF therapy group (Group E, 10 rats), HGF was administered to rats in group E on week. 7.All rats in group D and E were sacrificed on week 10.Liver function and levels of serum hyaluronic acid (HA), mucin (LN), collegen type IV (CIV), procollagen III (PCIII) were tested; the expression of MMP-1 and TIMP-1 were determined by immunohistochemical staining and analyzed by computer.

RESULTS: Compared with Group B, the serum levels of ALT, AST, HA, LN, CIV, PCIII in Group C were significantly reduced (P < 0.01), MMP-1 activity was slightly increased (0.25 ± 0.02, vs 0.22 ± 0.05, P < 0.05), TIMP-1 activity was markedly reduced (0.34 ± 0.05, vs 0.45 ± 005, P < 0.01). TIMP-1 activity in Group E (0.31 ± 0.07) was also markedly reduced in comparison with Group D (0.42 ± 0.06) (P < 0.01).

CONCLUSIONS: HGF has obvious effect in preventing development of liver fibrosis; it might facilitate degradation of hepatic fibrosic tissue via increasing the MMP-1 activity and or inhibiting TIMP-1 activity.

Key Words: N/A


0 引言

肝纤维化是慢性肝病发展到肝硬化的必经病理过程. 肝纤维化进程是可以控制, 甚至可以逆转的[1-13]. 肝纤维化发展的机制和抗肝纤维化治疗-直是国内外研究的热点. 近年来肝细胞生长因子(hepatocyte growth factor, HGF)抗肝纤维化作用引起关注. 国外有文献[14-19]报道应用HGF基因治疗已形成肝纤维化的大鼠模型, 可使肝纤维化消散. 但关于HGF抗肝纤维化作用的具体机制, 尤其是HGF对肝纤维化降解系统的影响尚未见相关报道. 我们通过预防性和治疗性给予HGF研究其对实验性大鼠肝纤维化的防治作用, 及对肝组织中MMP-1和TIMP-1表达的影响, 旨在进一步揭示HGF抗肝纤维化作用的机制.

1 材料和方法
1.1 材料

Wistar♂大鼠80只, 体质量120-140 g, 由湖北省动物实验协会提供; HGF制剂获赠于威海赛洛金药业有限公司, 批号: 国药准字20 010 003, 国家一类新药; MMP-1, TIMP-1-抗和二抗均购自北京中山生物技术公司, 为美国山道士公司产品; 透明质酸(HA), 层粘蛋白(LN), III型前胶原(PcIII), IV型胶原(CIV)放免试剂盒购自南京建成生物公司; CCl4分析纯, 郑州试剂二厂生产.

1.2 方法

将实验大鼠随机分为正常对照组(A组, 16只), 肝纤维化模型组I(B组, 54只), HGF治疗组I(C组, 10只), 模型制备方法参照文献[20], B组及C组用1 mL注射器ip 0.025 mL CCl4+0.15 mL花生油, 3次/wk; A组以0.025 mL NS+0.15 mL花生油ip, 3次/wk, 所有大鼠均自由饮水, 进食条杆状动物饲料. C组于造模同时予HGF 0.5 mg/Kg, 0.5 mL, ip, qd. 每周给各组大鼠称重以调整用药量. 2, 4 wk末B组随机处死2-3只大鼠作病理检查以确定造模情况及了解肝纤维化分期. 6 wk末造模成功处死C组大鼠, 同时随机处死A, B组大鼠各8只. 对剩下的B组大鼠行二次随机分组为模型组II(D组, 12只), HGF治疗组II(E组, 10只), E组大鼠于7 wk予HGF治疗, 剂量同前, D组则注射等量生理盐水, 10 wk末处死所有大鼠. (1)血清学检测: 将大鼠血清即时送武汉大学人民医院检验科检验肝功能指标. 血清HA, LN, CIV, PcIII用SN-628放射免疫γ记数器测定. (2)免疫组化检测: 肝组织经中性甲醛固定, 石蜡包埋, 连续切片厚度4-6 mm, 作HE染色, 光镜下作病理学检查, 肝纤维化分期采用Scheuer法. 免疫组织化学法测定肝组织MMP1和TIMP1的表达, 采用链霉菌抗生物素蛋白-过氧化物酶染色法(即S-P法)染色, DAB显色, 操作按试剂盒说明进行. 染色结果以棕黄色为阳性, 应用高清晰彩色病理图像免疫组化测量系统(HPLAS-100)进行图像分析, 经标准灰度校正后, 随机取5个视野, 测定MMP1, TIMP1的平均积分光密度值(IDP).

统计学处理 计量资料用t检验, 计数资料Ridit检验, P<0.05为差异有显著性. (实验中途死亡大鼠均不列入统计)

2 结果

实验中B组先后有7只大鼠死亡, D组死亡3只, E组死亡2只, 其余均存活. 病理学检查, A组: 肝小叶结构完整, 中央静脉窦周围肝细胞索放射状排列, 肝细胞未见坏死, 汇管区清晰可辨, 无明显炎性细胞浸润. B组: 可见多个坏死区, 纤维组织明显增生, 肝小叶结构破坏, 有假小叶形成(图1). C组: 肝小叶结构基本完整, 偶见小的坏死灶, 纤维间隔略有增宽, 汇管区有轻度炎细胞浸润. D组所见基本同B组, E组纤维化情况较D为轻. Scheuer分期(表1)可见C组肝纤维化程度明显低于B组, E组肝纤维化轻于D组.

表1 各组大鼠肝组织肝纤维化Scheuer分期.
分组nS0S1S2S3S4
A880000
B800125
Cb1004510
D900063
Ea800341
图1
图1 肝纤维化模型组(B组)HE切片: 可见若干肝细胞坏死区; 小叶间隔增宽, 有假小叶形成.
2.1 肝功能及肝纤维化指标检测

经HGF治疗的C, E组ALT, AST值较相应的B, D组明显降低(P<0.01). C, E组肝纤维化4项指标较相应模型对照组有显著降低(P<0.01)(表2).

表2 各组大鼠肝功能肝纤维化指标变化及肝组织MMP-1,TIMP-1的表达(mean±SD).
分组n肝功能指标
肝纤维化指标(mg/L)
免疫组化检测(IDP)
AST(nkat/L)ALT(nkat/L)ALb(g/L)TP(g/L)HALNPCIIICIVMMP-1TIMP-1
A81 380±256693±16729.3±1.657.9±2.783±21103±1896±1442±110.07±0.030.13±0.04
B83 220±6741 869±17927.4±0.957.1±1.1332±47167±11279±32138±200.22±0.050.45±0.05b
C101 396±174b920±203b27.8±1.357.4±1.8125±31b109±23b144±24b56±18b0.25±0.02a0.34±0.05
D92 942±5421 235±11026.9±1.257.2±0.7362±58171±18275±43153±290.18±0.030.42±0.06
E81 963±398b977±193b27.2±2.457.6±2.3289±37b135±20b177±30b98±23b0.18±0.040.31±0.07b
2.2 免疫组化检查结果

MMP1和TIMP1在A组(正常对照组)仅有微弱表达, 其余各组表达比A组均有不同程度增强(图2-7). HPLAS-1 000图像分析结果(表2)示C组MMP1较B组升高(P<0.05), E组与D组差异无显著性; TIMP1活性C组明显低于B组, E组明显低于D组(P<0.01).

图2
图2 正常肝组织(A组)TIMP-1表达活性很微弱.
图3
图3 B组MMP-1表达,经HPLAS分析, 其活性略低于C组(P<0. 05).
图4
图4 C组MMP-1表达.
图5
图5 C组TIMP-1表达经分析其活性低于B组(P<0. 01).
图6
图6 D组TIMP-1表达, 经分析其活性明显高于E组(P<0. 01).
图7
图7 E组TIMP-1表达. 以上切片均为×200.
3 讨论

肝细胞生长因子(HGF)最初是从血浆和血小板中分离纯化出来并被认为具有刺激肝细胞再生活性的物质 . 以后的研究发现HGF是具有多种生物学活性的细胞因子. HGF有刺激肝细胞增生的活性, 并可抑制受损肝细胞的凋亡, 故有-定的抗肝损伤和预防肝纤维化作用. 但HGF抗肝纤维化作用可能是多环节的. 沈敏 et al[21]报道HGF能显著抑制从大鼠肝脏星状细胞建株的肌成纤维样细胞增生及细胞外基质(ECM)的产生. Ueki et al[14,16]应用HGF基因治疗用二甲基亚硝胺制造的肝纤维化大鼠, 发现他可使已形成的肝纤维化完全消散, 提示HGF抗肝纤维化作用可能还与促进细胞外基质的降解有关. 我们的研究表明HGF预防性给药组(C组)和治疗性给药组(E组), 肝功能及肝纤维化指标较相应对照组均有明显改善(见表1, 2). 此外从实验结果还可以看出, HGF可能促进MMP1的活性, 并明显抑制TIMP1活性(表2). MMP1, TIMP1在ECM的降解中起重要作用[22-33], TIMP-1活性的超常表达或MMP-1活性受抑制被认为是肝纤维化进展的重要因素, 因此HGF对MMP-1和TIMP-1的作用可能是其抗肝纤维化的机制之一. 总之HGF抗肝纤维化的作用可能有以下几方面: 促进肝细胞再生, 减少肝细胞凋亡, 从而减少肝细胞的坏死与损伤, 减轻炎性刺激; 抑制肝星状细胞(HSC)的活化与增生, 抑制细胞外基质的产生; 通过促进基质蛋白酶活性或降低其抑制因子活性表达而促进细胞外基质的降解.

肝纤维化是慢性肝病的共同病理基础, 他持续发展的中心环节在于肝细胞损伤或坏死引起的炎性反应不断刺激肝组织非实质细胞(主要是HSC)[34-39], 导致细胞外基质大量合成与沉积, 并超过其降解速度. 由于肝纤维化是肝硬变的必经病理过程, 多年来-直是研究的热点. 目前对肝纤维化机制的研究已由病理组织学发展到细胞学, 分子学乃至基因水平. 从理论上说, 任何可以作用于肝纤维化发生发展的某-环节的药物都可能有抗肝纤维化作用. 但迄今为止, 人们尚未发现理想的抗肝纤维化药物. 已报道的多种抗肝纤维化药物或由于疗效不佳,或由于毒副作用大, 其临床应用效果均不甚满意.

HGF有着较为肯定的抗肝纤维化作用, 对肝功能有-定保护作用, 在实验或临床应用中, 尚未发现有明显的毒副反应, 因此进一步探讨其抗肝纤维化作用机制, 研究新的疗法(如基因治疗), 仍具有潜在的科研和临床价值.

编辑: N/A

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