MMP-1/TIMP-1与糖尿病树鼩早期肝纤维化的关系
摘要
目的 研究MMP-1/TIMP-1与糖尿病树鼩早期肝纤维化的关系。方法 将40只树鼩随机分到正常对照组（8只）和链脲佐菌素（STZ）处理组（32只），静脉注射STZ诱导树鼩糖尿病模型，将成模的树鼩归为糖尿病组（13只）， 未成模的树鼩归为STZ对照组（9只）。于注药前和成模后4、8周分别行血生化检查（ALT、AST）和放免检查（Ⅳ·C、LN、HA）。成模后8周对3组树鼩肝组织活检行常规HE染色、PAS、D-PAS染色、Masson染色和MMP-1/TIMP-1免疫组化染色，电镜观察肝脏超微结构。结果 3组树鼩间ALT、AST、Ⅳ·C、LN、HA无明显差别（P>0.05）。糖尿病组树鼩肝细胞光镜和电镜下胞浆糖原明显增多、核内糖原空洞、脂肪变性、细胞水肿、肝窦纤维增生、线粒体结构模糊，粗面内质网脱粒，滑面内质网扩张。糖尿病组肝组织MMP-1/TIMP-1表达的阳性均较其余两组强，其中用病理图象分析仪测MMP-1平均光密度与其余两组无统计学差异，与Masson染色结果无相关性（P﹥0.05）；TIMP-1平均光密度明显高于两组对照组（P﹤0.01）且与Masson染色结果显著相关性（r=0.676，P﹤0.05）。
结论1. 糖尿病树鼩成模8周在电镜下观察到早期肝纤维化的表现2. 糖尿病树鼩在电镜下观察到早期肝纤维化时，肝功能和Ⅳ·C、HA、LN无明显变化3. 糖尿病树鼩早期肝纤维化中MMP-1表达无明显改变，而TIMP-1表达显著增高，且与胶原纤维表达量增高呈显著相关，提示MMP-1/TIMP-1系统失衡在糖尿病早期肝纤维化的发生、发展中有重要作用。
Objective To study the relationship between MMP-1/TIMP-1 and the early stage of liver fibrosis in diabetic tree shrews. Methods 40 tree shrews were divided randomly into normal control group (n=8) and treatment group (n=32). The treatment group were injected of vein with streptozocin (STZ) to induce diabetes. Eight weeks later , the tree shrews maintained fasting blood glucose (FBG) at level of ≥11.1mmol/Lwere divided into diabetes group (n=13), and those maintained FBG<11.1mmol/Linto STZ control group (n=9). ALT , AST , Type Ⅳ collagen (Ⅳ·C) , Laminin (LN) and Hyaluronic acid (HA) were detected respectively before and after STZ injection 4 and 8 weeks. Liver biopsy was performed after the 8th week of the experiment. All of the liver specimens were observed with light microscope and electron microscope. MMP-1 and TIMP-1were studied by immunohistochemistroy and computer image analysis. Result There was no significant difference in the concentrations of serum ALT , AST , Ⅳ·C , LN and HA among the three groups before and after 4 , 8 weeks. The liver histological damages were observed in diabetes group, including hepatocytes swelling , some having fatty degeneration , glycogen holes in cell nuclear , the collagen of perisinusoidal areas increasing by Masson trichome. According to image analysis , the level of Masson trichome was higher in diabetes group than that of in control group (P<0.05) . And the following changes were observed under electron microscope :
glycogen granules in cytoplasm and nuclear increasing , fatty degeneration , configuration of mitochondrial illegibility , rough endoplasmic reticulum degranulation, smooth endoplasmic reticulum expansion . The early stage of liver fibrosis was observed in portal areas. Increase expression of MMP-1 , TIMP-1 was related to the early stage of liver fibrosis . According to image analysis , the level of TIMP-1 was higher in diabetes group than that of the other groups (P<0.01) and so did of MMP-1 but there was no significant difference among three groups (P>0.05) . There was a positive correlation between the level of TIMP-1 and collagen (r=0.676 ,P<0.05) . The correlation between the level of MMP-1 and collagen was not found . Conclusion 1. There is the early stage of liver fibrosis in diabetic tree shrews with electron microscope . 2. There is no significant change in the concentrations of serum ALT , AST , Ⅳ·C , LN and HA on the early stage of diabetic liver fibrosis . 3. MMP-1/TIMP-1 may play a important role influencing the development of early stage of liver fibrosis in diabetes .
结论1. 糖尿病树鼩成模8周在电镜下观察到早期肝纤维化的表现2. 糖尿病树鼩在电镜下观察到早期肝纤维化时，肝功能和Ⅳ·C、HA、LN无明显变化3. 糖尿病树鼩早期肝纤维化中MMP-1表达无明显改变，而TIMP-1表达显著增高，且与胶原纤维表达量增高呈显著相关，提示MMP-1/TIMP-1系统失衡在糖尿病早期肝纤维化的发生、发展中有重要作用。
Objective To study the relationship between MMP-1/TIMP-1 and the early stage of liver fibrosis in diabetic tree shrews. Methods 40 tree shrews were divided randomly into normal control group (n=8) and treatment group (n=32). The treatment group were injected of vein with streptozocin (STZ) to induce diabetes. Eight weeks later , the tree shrews maintained fasting blood glucose (FBG) at level of ≥11.1mmol/Lwere divided into diabetes group (n=13), and those maintained FBG<11.1mmol/Linto STZ control group (n=9). ALT , AST , Type Ⅳ collagen (Ⅳ·C) , Laminin (LN) and Hyaluronic acid (HA) were detected respectively before and after STZ injection 4 and 8 weeks. Liver biopsy was performed after the 8th week of the experiment. All of the liver specimens were observed with light microscope and electron microscope. MMP-1 and TIMP-1were studied by immunohistochemistroy and computer image analysis. Result There was no significant difference in the concentrations of serum ALT , AST , Ⅳ·C , LN and HA among the three groups before and after 4 , 8 weeks. The liver histological damages were observed in diabetes group, including hepatocytes swelling , some having fatty degeneration , glycogen holes in cell nuclear , the collagen of perisinusoidal areas increasing by Masson trichome. According to image analysis , the level of Masson trichome was higher in diabetes group than that of in control group (P<0.05) . And the following changes were observed under electron microscope :
glycogen granules in cytoplasm and nuclear increasing , fatty degeneration , configuration of mitochondrial illegibility , rough endoplasmic reticulum degranulation, smooth endoplasmic reticulum expansion . The early stage of liver fibrosis was observed in portal areas. Increase expression of MMP-1 , TIMP-1 was related to the early stage of liver fibrosis . According to image analysis , the level of TIMP-1 was higher in diabetes group than that of the other groups (P<0.01) and so did of MMP-1 but there was no significant difference among three groups (P>0.05) . There was a positive correlation between the level of TIMP-1 and collagen (r=0.676 ,P<0.05) . The correlation between the level of MMP-1 and collagen was not found . Conclusion 1. There is the early stage of liver fibrosis in diabetic tree shrews with electron microscope . 2. There is no significant change in the concentrations of serum ALT , AST , Ⅳ·C , LN and HA on the early stage of diabetic liver fibrosis . 3. MMP-1/TIMP-1 may play a important role influencing the development of early stage of liver fibrosis in diabetes .
引文
1. Van Steenbergen W, Lanckmans S. Liver disturbance in obesity and diabetes mellitus. Int J Obes Relat Metab Disord, 1995, Sep 19(suppl 3): 27-36
2. Sasaki A. Mortality and causes of death in patients with diabetes mellitus in Japan. Diabetes Res Clin Pract, 1994,24(suppl): 299-306
3. Shishko PI, Kovalev PA, Goncharov VG, et al. Comparison of peripheral and portal (via the umbilical vein) IDDM patients. Diabetes, 1992, 41(9): 1042-9
4. Hans Olov Adami, Wong Ho Chow, Olof Nyren, et al. Excess risk of liver cancer in patients with diabetes mellitus. J Natl Cancer Inst, 1996,88(20):1472
5. 沈稚舟，吴松华，邵福源等. 糖尿病慢性并发症. 上海医科大学出版社，1999年1月第1版： 314-22
6. Mura waki Y, Koda M, Yamada S. Kawasaki H, Shima H, Burkhardt H. Serum collagenase activity in patients with chronic liver fibrosis. J hepatol, 1993; 18:328-334
7. Arthur MJP. Matrix degradation in liver: a role in injury and repair. Hepatology. 1997; 26:1069-1071
8.Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression
of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94:2493-2503
9. Nakamura T, Fukui M, Ebihara I, et al. Abnormal gene expression of matrix metalloproteinase and its inhibitor in glomeruli from diabetic rats [J]. Ren Physiol Biochem. 1994,17 :316-25
10. De La Paz MA, Itoh Y, Toth CA, et al. Matrix metalloproteinases and their inhibitors in human vitreous. Invest Ophthalmol Vis Sci 1998; 39: 1256-1260
11. 冼苏，黄松，苏建家等. 链脲佐菌素诱导树鼩糖尿病动物模型的研究. 广西医科大学学报. 2000;17(6):491-495
12. Aarthur MJP. Fibrosis and altered matrix degradation. Digestion, 1998; 59: 376-80
13. Wang Y, Gao Y, Yang JZ. Study on the expression of interstitial collagenase in experimental liver fibrosis. Shijie Huaren Xiaohua Zazhi, 1999; 7: 1004-6
14. Herbst H, Wege T, Milani S, et al. Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis. Am J Pathol, 1997, 150: 1647
15. 成军，主编：细胞外基质的分子生物学与临床疾病。第1版。
北京：北京医科大学出版社。1999，149-62
16. 冼苏, 王乃尊, 韦敏怡等. 糖尿病性肝肿大临床及病理研究. 广西医科大学学报, 1996;13(3): 7
17. 刘新民主编. 内分泌代谢疾病鉴别诊断学. 科学出版社，1992 年2月第一版： 553
18. 韦敏怡. 糖尿病性肝病的病理研究. 广西医科大学学报, 1995;12(4):492-95
19. 秦映芬，冼苏，韦敏怡. 糖尿病患者肝脏的超微结构研究。临床肝胆病杂志，2000.16(4):241
20. 刘景章，赵子粼，高毅等。基质金属蛋白酶在肝纤维化组织中的表达。广东医学，2001；22（5）：374-75
21. 黄宇琦，高毅，杨继震等。大鼠肝纤维化基质金属蛋白酶及其抑制因子的表达。世界华人消化杂志，1999；7（9）：795-96
22. Iredale JP, Murphy G, Hembry RM, et al. Human hepatic lipo- cytes synthesize tissue inhibitor of metalloproteinase-1(TIMP-1):
Implications for regulation of matrix degradation in liver. J Clin Invest, 1992, 90:282
23. Benyon RC, Irendale JP, Goddard S, et al. Expression of tissue inhibitor of metalloproteinase 1 and 2 is increased in fibrosis human liver [J]. Gasrtroenterology, 1996,110(3); 821-31
Iredale JP, Benyon RC, Arthur MJP, et al. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver
24. injury and fibrosis. Hepatology, 1996; 24:176-84
25. Gao Y, Huang YQ, Wang Y, et al. The detection of tissue inhibitor of metalloproteinase-1 in experimental hepatic fibrosis by RT-PCR. Shijie Huaren Xiaohua Zazhi, 1999; 7:993
26. 朱妍，施法兴等。糖尿病患者血清肿瘤坏死因子的变化。江苏医药，1997；23（9）：614-16
27. 刘润华，刘聪等。糖尿病患者血清肿瘤坏死因子α的水平及临床意义。中国实用内科杂志，1997；17（5）：288-89
28. 梁真, TNF-α与树鼩糖尿病性肝脏病变关系的研究.(硕士论文)
29. Mclennan S, Fisher E, Martell S, et al. Effects of glucose on matrix metalloproteinase and plasmin activities in mesangial cells: possible role in diabetic nephropathy. Kidney Int 2000；58(suppl 77): 581
30. 哈迪，糖尿病树鼩早期肝损害中TGF-β1的意义. (硕士论文)
31. Wakisaka M, Spiro MJ, Spiro RG, et al. Sythesis of type Ⅵ collagen by cultured glomerular cells and comprison of its regulation by glucose and other facters with that of type Ⅴ collagen. Diabetes 1994；43:95
2. Sasaki A. Mortality and causes of death in patients with diabetes mellitus in Japan. Diabetes Res Clin Pract, 1994,24(suppl): 299-306
3. Shishko PI, Kovalev PA, Goncharov VG, et al. Comparison of peripheral and portal (via the umbilical vein) IDDM patients. Diabetes, 1992, 41(9): 1042-9
4. Hans Olov Adami, Wong Ho Chow, Olof Nyren, et al. Excess risk of liver cancer in patients with diabetes mellitus. J Natl Cancer Inst, 1996,88(20):1472
5. 沈稚舟，吴松华，邵福源等. 糖尿病慢性并发症. 上海医科大学出版社，1999年1月第1版： 314-22
6. Mura waki Y, Koda M, Yamada S. Kawasaki H, Shima H, Burkhardt H. Serum collagenase activity in patients with chronic liver fibrosis. J hepatol, 1993; 18:328-334
7. Arthur MJP. Matrix degradation in liver: a role in injury and repair. Hepatology. 1997; 26:1069-1071
8.Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression
of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94:2493-2503
9. Nakamura T, Fukui M, Ebihara I, et al. Abnormal gene expression of matrix metalloproteinase and its inhibitor in glomeruli from diabetic rats [J]. Ren Physiol Biochem. 1994,17 :316-25
10. De La Paz MA, Itoh Y, Toth CA, et al. Matrix metalloproteinases and their inhibitors in human vitreous. Invest Ophthalmol Vis Sci 1998; 39: 1256-1260
11. 冼苏，黄松，苏建家等. 链脲佐菌素诱导树鼩糖尿病动物模型的研究. 广西医科大学学报. 2000;17(6):491-495
12. Aarthur MJP. Fibrosis and altered matrix degradation. Digestion, 1998; 59: 376-80
13. Wang Y, Gao Y, Yang JZ. Study on the expression of interstitial collagenase in experimental liver fibrosis. Shijie Huaren Xiaohua Zazhi, 1999; 7: 1004-6
14. Herbst H, Wege T, Milani S, et al. Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis. Am J Pathol, 1997, 150: 1647
15. 成军，主编：细胞外基质的分子生物学与临床疾病。第1版。
北京：北京医科大学出版社。1999，149-62
16. 冼苏, 王乃尊, 韦敏怡等. 糖尿病性肝肿大临床及病理研究. 广西医科大学学报, 1996;13(3): 7
17. 刘新民主编. 内分泌代谢疾病鉴别诊断学. 科学出版社，1992 年2月第一版： 553
18. 韦敏怡. 糖尿病性肝病的病理研究. 广西医科大学学报, 1995;12(4):492-95
19. 秦映芬，冼苏，韦敏怡. 糖尿病患者肝脏的超微结构研究。临床肝胆病杂志，2000.16(4):241
20. 刘景章，赵子粼，高毅等。基质金属蛋白酶在肝纤维化组织中的表达。广东医学，2001；22（5）：374-75
21. 黄宇琦，高毅，杨继震等。大鼠肝纤维化基质金属蛋白酶及其抑制因子的表达。世界华人消化杂志，1999；7（9）：795-96
22. Iredale JP, Murphy G, Hembry RM, et al. Human hepatic lipo- cytes synthesize tissue inhibitor of metalloproteinase-1(TIMP-1):
Implications for regulation of matrix degradation in liver. J Clin Invest, 1992, 90:282
23. Benyon RC, Irendale JP, Goddard S, et al. Expression of tissue inhibitor of metalloproteinase 1 and 2 is increased in fibrosis human liver [J]. Gasrtroenterology, 1996,110(3); 821-31
Iredale JP, Benyon RC, Arthur MJP, et al. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver
24. injury and fibrosis. Hepatology, 1996; 24:176-84
25. Gao Y, Huang YQ, Wang Y, et al. The detection of tissue inhibitor of metalloproteinase-1 in experimental hepatic fibrosis by RT-PCR. Shijie Huaren Xiaohua Zazhi, 1999; 7:993
26. 朱妍，施法兴等。糖尿病患者血清肿瘤坏死因子的变化。江苏医药，1997；23（9）：614-16
27. 刘润华，刘聪等。糖尿病患者血清肿瘤坏死因子α的水平及临床意义。中国实用内科杂志，1997；17（5）：288-89
28. 梁真, TNF-α与树鼩糖尿病性肝脏病变关系的研究.(硕士论文)
29. Mclennan S, Fisher E, Martell S, et al. Effects of glucose on matrix metalloproteinase and plasmin activities in mesangial cells: possible role in diabetic nephropathy. Kidney Int 2000；58(suppl 77): 581
30. 哈迪，糖尿病树鼩早期肝损害中TGF-β1的意义. (硕士论文)
31. Wakisaka M, Spiro MJ, Spiro RG, et al. Sythesis of type Ⅵ collagen by cultured glomerular cells and comprison of its regulation by glucose and other facters with that of type Ⅴ collagen. Diabetes 1994；43:95