肝细胞生长因子对大鼠肾小球系膜细胞表达基质金属蛋白酶-2，-9的影响
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摘要
前言
肾小球细胞外基质(ECM)的成分合成增强、降解被抑制继而造成ECM的过度沉积是引起肾小球硬化的主要原因。肾小球系膜细胞能受许多致病因子的刺激而活化、增殖，并引起ECM过量生成和积聚，与肾小球硬化的发病机理密切相关。基质金属蛋白酶(MMPs)是ECM降解过程中最主要的降解酶之一，MMPs表达降低或活性下降能引起ECM降解减少造成ECM异常沉积，从而导致肾小球硬化和肾间质纤维化。肝细胞生长因子(HGF)是一强的肾营养因子，能通过促进肾上皮细胞合成MMPs以减缓肾小管间质纤维化的进展，但HGF是否能通过调节肾小球系膜细胞的MMPs表达从而改善肾小球ECM的异常代谢目前尚未见到相关报道，因此我们拟通过研究HGF对大鼠系膜细胞(RMC)表达MMP-2，-9的影响，并观察系膜基质的主要成分纤维连接蛋白(FN)的变化以探讨HGF对ECM代谢可能的作用。
材料与方法
1．对体外培养RMC应用不同浓度的人重组HGF(0，5，10，25，50，75ng／ml)孵育24小时，用MTT法检测HGF对RMC增殖的影响，选取对RMC增殖无影响的浓度范围进行下一步试验。
2．对体外培养RMC应用不影响其增殖的不同浓度的人重组HGF(0，5，10，25ng／ml)孵育24小时，收集细胞加入细胞裂解液以提取细胞胞浆总蛋白，用紫外分光光度法测定蛋白浓度，用细胞裂解液调整样品蛋白浓度(5mg／ml)。
3．应用明胶酶谱法(Zymography)检测RMC胞浆总蛋白中MMP-2，-9的活性：取总量为50μg蛋白标本，上样于含1g／L白明胶的8％SDS-聚丙烯酰胺凝胶上电泳，电泳结束后在37℃显影缓冲液下孵育18小时，应用考马斯亮蓝法染色、脱色，扫描凝胶并作计算机信号吸光度分析。
4．应用免疫印迹法(Western blotting)检测RMC胞浆总蛋白中MMP-2，-9，FN的蛋白表达水平：取总量为50μg蛋白标本，上样于6％～8％SDS-
聚丙烯酞胺凝胶上电泳，电泳结束后在4℃、80V电压的条件下转移2一4小
时至PVDF膜，5%脱脂奶粉封闭后，分别加入抗大鼠MMP一，一9、FN多克
隆抗体以及相应的二抗，ECL试剂或碱性磷酸酶显色剂显色，扫描图像并作
计算机信号吸光度分析。
结·果
1.HGF在浓度为0一25n岁耐时对RMC增殖无影响(p>0.05);而
HGF在浓度)50n扩耐时对RMC有明显的促进增殖的作用(p<0.05)。
2.RMC在基础水平只有微弱的MMP一2蛋白表达水平，且未检测到其
活性，HGF在对RMC无增殖影响的浓度范围内对MMP一2的蛋白表达水平
并无影响，但在HGF浓度为10一25n扩nil时可检测到其微弱的活性表达。
3.HGF在对RMC无增殖影响的浓度范围内对MMP一9的活性的影响呈
浓度依赖性地增加，和对照组(on岁rril)相比在HGF浓度为25n群血时增加
MMP一9活性57%(p<0.05)，但只轻度增加其蛋白表达水平，与对照组相比
增加15%(p<0.05)。
4.HGF在对RMC无增殖影响的浓度范围内明显减少FN的蛋白表达
水平，与对照组(On岁耐)相比在HGF浓度为25n扩nil时减少42%(p<0.
05)。
结论
1.HGF对RMC表达MMP一，一的活性及MMP一的蛋白表达水平有不
同程度的促进作用。
2.HGF能下调RMC的FN蛋白表达水平。
3.提示HGF可能具有潜在的抗纤维化的能力，其可能机制是通过促
进MMPs的表达来激活基质降解途径，以减少ECM成分的积聚，从而达到
延缓肾小球硬化的进程。
The hallmark of glomerular sclerosis is the relentless accumulation of extracellular matrix (ECM) , which is believed to result from an imbalance in synthe-sion and degradation. ECM degradation is tightly controlled by a family of zinc-dependent matrix-degrading enzymes known as matrix metalloproteinase (MMPs). Hepatocyte growth factor (HGF) has been shown to reduce netECM accumulation by increasing MMPs expression in renal tubular cells, however whether HGF also modulates the development and progression of glomerular sclerosis by activating matrix degradation pathways in renal mesangial cells is uncertain.
Objective
To study the regulation of expression of MMP -2,-9 and ECM secretion by HGF in rat mesangial cells ( RMC) .
Methods
Cultured RMC were incubated with different concertration recombinant human HGF(0 ~75ng/ml) for 24 hours, cell proliferation was assessed by MTT colorimetric assay, protein levels of MMP-2,-9 and Fibronectin ( FN) in the RMC were measured by western blotting analysis, and MMP-2, -9 activities were measured by gelatin zymography.
Results
Administration 0 ~25ng/ml HGF into culture medium had no effect on proliferation of RMC, in this range, HGF upregulate MMP-9 activities (increased by 57% ,p <0. 05) when compared with control, and its protein secretion by RMC also increased by 15%. MMP-2 was normally expressed, at a very low level, in the cells, and HGF had no effect on its protein expression. Zymogra-phy showed that no degradtive activity of MMP-2 in serum-free RMIP1640 was observed, while we can detect its activity at a low level by HGF (10 ~ 25ng/ ml). Moreover HGF decreases the protein expression of FN by 42% compared with the control.
Conclusion
HGF can stimulate MMP -2,-9 activities and MMP-9 protein secretion in various degree, also increased the degradation of FN, which may inhibite accumulation of ECM and delay progressive of glomerulosclerosis.
肾小球细胞外基质(ECM)的成分合成增强、降解被抑制继而造成ECM的过度沉积是引起肾小球硬化的主要原因。肾小球系膜细胞能受许多致病因子的刺激而活化、增殖，并引起ECM过量生成和积聚，与肾小球硬化的发病机理密切相关。基质金属蛋白酶(MMPs)是ECM降解过程中最主要的降解酶之一，MMPs表达降低或活性下降能引起ECM降解减少造成ECM异常沉积，从而导致肾小球硬化和肾间质纤维化。肝细胞生长因子(HGF)是一强的肾营养因子，能通过促进肾上皮细胞合成MMPs以减缓肾小管间质纤维化的进展，但HGF是否能通过调节肾小球系膜细胞的MMPs表达从而改善肾小球ECM的异常代谢目前尚未见到相关报道，因此我们拟通过研究HGF对大鼠系膜细胞(RMC)表达MMP-2，-9的影响，并观察系膜基质的主要成分纤维连接蛋白(FN)的变化以探讨HGF对ECM代谢可能的作用。
材料与方法
1．对体外培养RMC应用不同浓度的人重组HGF(0，5，10，25，50，75ng／ml)孵育24小时，用MTT法检测HGF对RMC增殖的影响，选取对RMC增殖无影响的浓度范围进行下一步试验。
2．对体外培养RMC应用不影响其增殖的不同浓度的人重组HGF(0，5，10，25ng／ml)孵育24小时，收集细胞加入细胞裂解液以提取细胞胞浆总蛋白，用紫外分光光度法测定蛋白浓度，用细胞裂解液调整样品蛋白浓度(5mg／ml)。
3．应用明胶酶谱法(Zymography)检测RMC胞浆总蛋白中MMP-2，-9的活性：取总量为50μg蛋白标本，上样于含1g／L白明胶的8％SDS-聚丙烯酰胺凝胶上电泳，电泳结束后在37℃显影缓冲液下孵育18小时，应用考马斯亮蓝法染色、脱色，扫描凝胶并作计算机信号吸光度分析。
4．应用免疫印迹法(Western blotting)检测RMC胞浆总蛋白中MMP-2，-9，FN的蛋白表达水平：取总量为50μg蛋白标本，上样于6％～8％SDS-
聚丙烯酞胺凝胶上电泳，电泳结束后在4℃、80V电压的条件下转移2一4小
时至PVDF膜，5%脱脂奶粉封闭后，分别加入抗大鼠MMP一，一9、FN多克
隆抗体以及相应的二抗，ECL试剂或碱性磷酸酶显色剂显色，扫描图像并作
计算机信号吸光度分析。
结·果
1.HGF在浓度为0一25n岁耐时对RMC增殖无影响(p>0.05);而
HGF在浓度)50n扩耐时对RMC有明显的促进增殖的作用(p<0.05)。
2.RMC在基础水平只有微弱的MMP一2蛋白表达水平，且未检测到其
活性，HGF在对RMC无增殖影响的浓度范围内对MMP一2的蛋白表达水平
并无影响，但在HGF浓度为10一25n扩nil时可检测到其微弱的活性表达。
3.HGF在对RMC无增殖影响的浓度范围内对MMP一9的活性的影响呈
浓度依赖性地增加，和对照组(on岁rril)相比在HGF浓度为25n群血时增加
MMP一9活性57%(p<0.05)，但只轻度增加其蛋白表达水平，与对照组相比
增加15%(p<0.05)。
4.HGF在对RMC无增殖影响的浓度范围内明显减少FN的蛋白表达
水平，与对照组(On岁耐)相比在HGF浓度为25n扩nil时减少42%(p<0.
05)。
结论
1.HGF对RMC表达MMP一，一的活性及MMP一的蛋白表达水平有不
同程度的促进作用。
2.HGF能下调RMC的FN蛋白表达水平。
3.提示HGF可能具有潜在的抗纤维化的能力，其可能机制是通过促
进MMPs的表达来激活基质降解途径，以减少ECM成分的积聚，从而达到
延缓肾小球硬化的进程。
The hallmark of glomerular sclerosis is the relentless accumulation of extracellular matrix (ECM) , which is believed to result from an imbalance in synthe-sion and degradation. ECM degradation is tightly controlled by a family of zinc-dependent matrix-degrading enzymes known as matrix metalloproteinase (MMPs). Hepatocyte growth factor (HGF) has been shown to reduce netECM accumulation by increasing MMPs expression in renal tubular cells, however whether HGF also modulates the development and progression of glomerular sclerosis by activating matrix degradation pathways in renal mesangial cells is uncertain.
Objective
To study the regulation of expression of MMP -2,-9 and ECM secretion by HGF in rat mesangial cells ( RMC) .
Methods
Cultured RMC were incubated with different concertration recombinant human HGF(0 ~75ng/ml) for 24 hours, cell proliferation was assessed by MTT colorimetric assay, protein levels of MMP-2,-9 and Fibronectin ( FN) in the RMC were measured by western blotting analysis, and MMP-2, -9 activities were measured by gelatin zymography.
Results
Administration 0 ~25ng/ml HGF into culture medium had no effect on proliferation of RMC, in this range, HGF upregulate MMP-9 activities (increased by 57% ,p <0. 05) when compared with control, and its protein secretion by RMC also increased by 15%. MMP-2 was normally expressed, at a very low level, in the cells, and HGF had no effect on its protein expression. Zymogra-phy showed that no degradtive activity of MMP-2 in serum-free RMIP1640 was observed, while we can detect its activity at a low level by HGF (10 ~ 25ng/ ml). Moreover HGF decreases the protein expression of FN by 42% compared with the control.
Conclusion
HGF can stimulate MMP -2,-9 activities and MMP-9 protein secretion in various degree, also increased the degradation of FN, which may inhibite accumulation of ECM and delay progressive of glomerulosclerosis.
引文
1．王海燕，刘平，张鸣和等．肾小球疾病进展与硬化机理．肾脏病学(第二版)，1995，514-23
2. Su SanV,Mclennan,Elizabeth Fisher,et al. Effects of glucose on matrix metalloproteinase and plasmin activities in mesangial cells:possible role in diabetic nephropathy. Kidney Int, 2000,58(1) :s81-7
3. Suzuki D, Miyazaki M ,Jinde K, et al. In situ hybridization studies of matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1 and type Ⅳ collagen in diabetic nephropathy. Kidney Int, 1997,52( 1 ):111-19
4. Balkovetz DF. Evdence that hepatocyte growth factor abrogates contact inhibition of mitosis in Madin-Darby canine kidney cell monolayers [J]. J Biol Chem, 1997,272 (6) : 3471-77
5. Liu Y. Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms[J]. Am J Physiol,1999, 277 (4Pt2) : F624-33
6. Liu Y, Rajur K, Tolbert E, et al. Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. Kidney Int,2000,58(5) :2028-43
7．梅长林，张黎明，陈蕾．大鼠系膜细胞系的建立及鉴定．肾脏病与透析肾移植杂志，1996，6：90-3
8．孙田美，梅长林，张黎明等．洛伐他汀对系膜细胞增殖及细胞基质分泌的影响．肾脏病与透析肾移植杂志，2001，10(5)：426-28
9. Liu Y, Tolbert EM, Lin L, et al. Up-regulation of hepatocyte growth factor receptor : An amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure. Kidney Int, 1999,55 (2) :442-53
10. Kitamura M, Burton S, English J, et al. Transfer of a mutated gene encoding active transforming growth factor-1 suppresses mitogenesis and IL-1 response in the glomerulurs. Kidney Int, 1995,48(6) :1747-57
11. Lelongt B, BengattaS, Delauche M, et al. Matrix eatalloproteinase 9 protects mice fron anti-glomerular basement membrane nephritis through its fibrinolytic activeity. J Exp Med,2001,193 (7) :793～820
12. Mc Miilan JI, Riordan JW, Couser WG, et al. Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy. J Clin Invest,1996,97(4):1094 -110
13. Mizuno S, Kurosawa T, Matsumoto K, et al. Hepatocyte growth factor prevents renal fibrosis and dysfunction in mouse model of chronic renal diseases. J Clin Invest, 1998,101 (9) : 1827-34
14. Kallincos NC ,Pollard AN, Couper JJ, et al. Evdence for a fuctional hepatocyte growth factor recptor in human mesangial cells [J]. Regulatory Peptides, 1998,74 (2-3):137-42
15．史跃先，黎磊石，周虹等．纤维连接蛋白在IgA肾病肾组织内的分布及意义．中华内科杂志，1997，6：390-93
2. Su SanV,Mclennan,Elizabeth Fisher,et al. Effects of glucose on matrix metalloproteinase and plasmin activities in mesangial cells:possible role in diabetic nephropathy. Kidney Int, 2000,58(1) :s81-7
3. Suzuki D, Miyazaki M ,Jinde K, et al. In situ hybridization studies of matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1 and type Ⅳ collagen in diabetic nephropathy. Kidney Int, 1997,52( 1 ):111-19
4. Balkovetz DF. Evdence that hepatocyte growth factor abrogates contact inhibition of mitosis in Madin-Darby canine kidney cell monolayers [J]. J Biol Chem, 1997,272 (6) : 3471-77
5. Liu Y. Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms[J]. Am J Physiol,1999, 277 (4Pt2) : F624-33
6. Liu Y, Rajur K, Tolbert E, et al. Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. Kidney Int,2000,58(5) :2028-43
7．梅长林，张黎明，陈蕾．大鼠系膜细胞系的建立及鉴定．肾脏病与透析肾移植杂志，1996，6：90-3
8．孙田美，梅长林，张黎明等．洛伐他汀对系膜细胞增殖及细胞基质分泌的影响．肾脏病与透析肾移植杂志，2001，10(5)：426-28
9. Liu Y, Tolbert EM, Lin L, et al. Up-regulation of hepatocyte growth factor receptor : An amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure. Kidney Int, 1999,55 (2) :442-53
10. Kitamura M, Burton S, English J, et al. Transfer of a mutated gene encoding active transforming growth factor-1 suppresses mitogenesis and IL-1 response in the glomerulurs. Kidney Int, 1995,48(6) :1747-57
11. Lelongt B, BengattaS, Delauche M, et al. Matrix eatalloproteinase 9 protects mice fron anti-glomerular basement membrane nephritis through its fibrinolytic activeity. J Exp Med,2001,193 (7) :793～820
12. Mc Miilan JI, Riordan JW, Couser WG, et al. Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy. J Clin Invest,1996,97(4):1094 -110
13. Mizuno S, Kurosawa T, Matsumoto K, et al. Hepatocyte growth factor prevents renal fibrosis and dysfunction in mouse model of chronic renal diseases. J Clin Invest, 1998,101 (9) : 1827-34
14. Kallincos NC ,Pollard AN, Couper JJ, et al. Evdence for a fuctional hepatocyte growth factor recptor in human mesangial cells [J]. Regulatory Peptides, 1998,74 (2-3):137-42
15．史跃先，黎磊石，周虹等．纤维连接蛋白在IgA肾病肾组织内的分布及意义．中华内科杂志，1997，6：390-93