明胶酶A、B在MRL/Lpr自发性狼疮小鼠中的表达以及水蛭调控的机理研究
摘要
目的：本研究旨在探讨 MRL/1pr自发性狠疮小鼠肾脏明胶酶A、
B(MMP-2与MMP-9)的表达变化，明确水蛭的调节作用及机理。
方法：8周龄狠疮小鼠60只，随机分为治疗组与对照组。治疗
组经饲料服用生水蛭粉3g/kg/d共20周。观察小鼠的存活率、
蛋白尿、肾功能与肾脏病理改变。利用免疫组化、明胶酶谱以及
原位酶谱法检测肾脏明胶酶A、B的表达及活性变化。同时利用
免疫组化与RT-PCR方法检测狼疮小鼠肾脏凝血酶受体PAR-1的
表达，以改良的MSB染色法检测纤维蛋白的沉积。为了明确局部
沉积的纤维蛋白是否可以调控明胶酶的表达，通过纤维蛋白与系
膜细胞体外共培养，利用明胶酶谱法、纤维蛋白酶谱法、RT-PCR
以及纤维蛋白平板法现察纤维蛋白诱导活化系膜细胞表达明胶
酶A、B以及tPA与uPA的作用，明确明胶酶活化与PA/纤溶酶
系统之问的关系。结果：水蛭能够延长狼疮小鼠的存活率(70%.
50%)、减少蛋白尿(40%:80%)并减轻肾小球与小血管的病变。
与8周龄狼疮小鼠比较，28周龄狼疮小鼠肾小球内明胶酶A、B
的表达、活化以及明胶酶的净活性均明显增加，与狼疮性肾炎的
病理损害密切相关，水蛭能够抑制明胶酶A、B的表达及活化。8
2
车由进修丰阮博士学仁企父 中又扔要
周龄狼疮小鼠肾脏没有纤维蛋白的沉积，在肾小球内皮细胞、系
膜细胞、上皮细胞与小血管内皮细胞有广泛的凝血酶受体N卜1
的表达。而28周龄狼疮小鼠肾小球系膜区、内皮下与血管内有
广泛的纤维蛋白的沉积，肾小球内皮细胞、系膜细胞与小血管内
皮细胞凝血酶受体 PAR八的表达却明显减少，提示 PARl受到凝
血酶的持续活化。水蛙可以减少肾脏局部纤维蛋白的沉积，抑制
凝血酶受体的活化。体外实验发现，纤维蛋白能够诱导系膜细胞
明胶酶 A、B的表达及活化，纤维蛋白还可以诱导 t PA与 ［JPA的
表达，总 PA活性明显增加。利用抑肽酶阻断 PA／纤溶酶的活性
可以完全抑制酶原形式明胶酶A、B的活化。结论：MRL／lpr 自
发性狠疮小鼠肾脏明胶酶A、B的表达明显增加，在狼疮性肾炎
的发展中扮演重要角色。凝血酶、纤维蛋白与明胶酶A、B的表
达与活化关系密切。水蛀阻断凝血酶的活性，通过抑制PAR习的
活化以及减少纤维蛋白的沉积这两种机制抑制狠疮性小鼠肾脏
明胶酶A、B的表达与活化，这可能是水蛀减轻MRL／l pr自发性
狼疮小鼠’肾脏病理损害的主要作用机制之一。
Coagulation and fibrinolytic processes are involved in the
pathogenesis of lupus nephritis The presence of thrombin
activation and fibrin deposition in the glomeruli may be
associated with more severe diseases We have found that hirudo reduce
fibrin deposition and ameliorate renal lesions in primary
glomerulonephntis In the present study we investigated its effect on
inhibiting disease progression in MRL lpr/lpr mice MRL lpr/lpr mice
were admimstered daily with hirudo (3g/kgld orally) at the age of 8
weeks After treatment for 20 weeks, a progressive reduction in positive
proteinuria number/total mice (40% vs 80%, proteinuria over 300mgIdl
as positive) and a prolonged survival rate (70% vs 50%) were found
when compared with the control group Histological analysis of kidney
tissues indicated that hirudo could inhibit the mesangial proliferation and
renal sclerosis that was evident in murine lupus nephntis No significant
differences were observed in the tubular and interstitial lesions Using
SDS-PAGE gelatin zymography, we have identified increased
expressions of both latent and activated form enzymes of MMP-2 and
MMP-9 in unne and kidney extraction Immunohistochemical staining
showed both MMP-2 and MMP-9 were obviously up-regulated within
glomerulus in control group To determine the net gelatinase activities in
murine lupus kidney, film in situ zymography was used, we found a
markedly increased gelatinase activities in munne lupus nephritis,
consistent with the immunohistochemical staining Hirudo suppressed
4
the expression of both latent and activated form of MMP-2 and MMP-9
We compared thrombin receptor PAR-i expression in glomerulus using
immunohistochemistry and PAR-i mRNA expression by RT-PCR
analysis among the groups With the development of murine lupus
nephntis, we observed an increase in thrombin receptor mRNA, while
thrombin receptor protein expression was strikingly down regulated,
suggesting its continuous activation and degradation Hirudo inhibited
PAR-i activation significantly correlated with the reduced fibrin
deposition To investigate the effects of covering fibnn on the expression
of MMP-2 and MMP-9 in cultured glomerular mesangial cells (GMC)
RT-PCR and gelatin zymography were used to detect the changes of
MMP-2 and MMP-9 in GMC at levels of mRNA expressions and protein
activities The relationship between plasminogen activator/plasmin and
the activation of MMP-2 and -9 were also investigated Fibnn treatment
induced a dramatic expression of activated MMP-2 and MMP-9 in GMC
in a dose and time dependent manner Fibrin zymography showed fibnn
also induced the expression of tissue type plasminogen activator (tPA) as
well as urokinase type plasminogen activator (uPA) Inhibition of the
activities of plasmin by aprotimn can totally block the conversion from
pro-MMP to active MMP These in vitro and in vivo results suggested
that PAR-i activation and fibnn deposition might play an important role
in regulating the MMP-2/-9 expressions in munne lupus nephritis,
contributing to the remodeling of extracellular matrix Hirudo delays the
development of glomerulonephntis and improves survival in MRL
lpr/lpr mice at least by suppressing the expressions of MMP-2/-9 via
inhibiting thrombin receptor PAR-i activation and fibrin deposition
B(MMP-2与MMP-9)的表达变化，明确水蛭的调节作用及机理。
方法：8周龄狠疮小鼠60只，随机分为治疗组与对照组。治疗
组经饲料服用生水蛭粉3g/kg/d共20周。观察小鼠的存活率、
蛋白尿、肾功能与肾脏病理改变。利用免疫组化、明胶酶谱以及
原位酶谱法检测肾脏明胶酶A、B的表达及活性变化。同时利用
免疫组化与RT-PCR方法检测狼疮小鼠肾脏凝血酶受体PAR-1的
表达，以改良的MSB染色法检测纤维蛋白的沉积。为了明确局部
沉积的纤维蛋白是否可以调控明胶酶的表达，通过纤维蛋白与系
膜细胞体外共培养，利用明胶酶谱法、纤维蛋白酶谱法、RT-PCR
以及纤维蛋白平板法现察纤维蛋白诱导活化系膜细胞表达明胶
酶A、B以及tPA与uPA的作用，明确明胶酶活化与PA/纤溶酶
系统之问的关系。结果：水蛭能够延长狼疮小鼠的存活率(70%.
50%)、减少蛋白尿(40%:80%)并减轻肾小球与小血管的病变。
与8周龄狼疮小鼠比较，28周龄狼疮小鼠肾小球内明胶酶A、B
的表达、活化以及明胶酶的净活性均明显增加，与狼疮性肾炎的
病理损害密切相关，水蛭能够抑制明胶酶A、B的表达及活化。8
2
车由进修丰阮博士学仁企父 中又扔要
周龄狼疮小鼠肾脏没有纤维蛋白的沉积，在肾小球内皮细胞、系
膜细胞、上皮细胞与小血管内皮细胞有广泛的凝血酶受体N卜1
的表达。而28周龄狼疮小鼠肾小球系膜区、内皮下与血管内有
广泛的纤维蛋白的沉积，肾小球内皮细胞、系膜细胞与小血管内
皮细胞凝血酶受体 PAR八的表达却明显减少，提示 PARl受到凝
血酶的持续活化。水蛙可以减少肾脏局部纤维蛋白的沉积，抑制
凝血酶受体的活化。体外实验发现，纤维蛋白能够诱导系膜细胞
明胶酶 A、B的表达及活化，纤维蛋白还可以诱导 t PA与 ［JPA的
表达，总 PA活性明显增加。利用抑肽酶阻断 PA／纤溶酶的活性
可以完全抑制酶原形式明胶酶A、B的活化。结论：MRL／lpr 自
发性狠疮小鼠肾脏明胶酶A、B的表达明显增加，在狼疮性肾炎
的发展中扮演重要角色。凝血酶、纤维蛋白与明胶酶A、B的表
达与活化关系密切。水蛀阻断凝血酶的活性，通过抑制PAR习的
活化以及减少纤维蛋白的沉积这两种机制抑制狠疮性小鼠肾脏
明胶酶A、B的表达与活化，这可能是水蛀减轻MRL／l pr自发性
狼疮小鼠’肾脏病理损害的主要作用机制之一。
Coagulation and fibrinolytic processes are involved in the
pathogenesis of lupus nephritis The presence of thrombin
activation and fibrin deposition in the glomeruli may be
associated with more severe diseases We have found that hirudo reduce
fibrin deposition and ameliorate renal lesions in primary
glomerulonephntis In the present study we investigated its effect on
inhibiting disease progression in MRL lpr/lpr mice MRL lpr/lpr mice
were admimstered daily with hirudo (3g/kgld orally) at the age of 8
weeks After treatment for 20 weeks, a progressive reduction in positive
proteinuria number/total mice (40% vs 80%, proteinuria over 300mgIdl
as positive) and a prolonged survival rate (70% vs 50%) were found
when compared with the control group Histological analysis of kidney
tissues indicated that hirudo could inhibit the mesangial proliferation and
renal sclerosis that was evident in murine lupus nephntis No significant
differences were observed in the tubular and interstitial lesions Using
SDS-PAGE gelatin zymography, we have identified increased
expressions of both latent and activated form enzymes of MMP-2 and
MMP-9 in unne and kidney extraction Immunohistochemical staining
showed both MMP-2 and MMP-9 were obviously up-regulated within
glomerulus in control group To determine the net gelatinase activities in
murine lupus kidney, film in situ zymography was used, we found a
markedly increased gelatinase activities in munne lupus nephritis,
consistent with the immunohistochemical staining Hirudo suppressed
4
the expression of both latent and activated form of MMP-2 and MMP-9
We compared thrombin receptor PAR-i expression in glomerulus using
immunohistochemistry and PAR-i mRNA expression by RT-PCR
analysis among the groups With the development of murine lupus
nephntis, we observed an increase in thrombin receptor mRNA, while
thrombin receptor protein expression was strikingly down regulated,
suggesting its continuous activation and degradation Hirudo inhibited
PAR-i activation significantly correlated with the reduced fibrin
deposition To investigate the effects of covering fibnn on the expression
of MMP-2 and MMP-9 in cultured glomerular mesangial cells (GMC)
RT-PCR and gelatin zymography were used to detect the changes of
MMP-2 and MMP-9 in GMC at levels of mRNA expressions and protein
activities The relationship between plasminogen activator/plasmin and
the activation of MMP-2 and -9 were also investigated Fibnn treatment
induced a dramatic expression of activated MMP-2 and MMP-9 in GMC
in a dose and time dependent manner Fibrin zymography showed fibnn
also induced the expression of tissue type plasminogen activator (tPA) as
well as urokinase type plasminogen activator (uPA) Inhibition of the
activities of plasmin by aprotimn can totally block the conversion from
pro-MMP to active MMP These in vitro and in vivo results suggested
that PAR-i activation and fibnn deposition might play an important role
in regulating the MMP-2/-9 expressions in munne lupus nephritis,
contributing to the remodeling of extracellular matrix Hirudo delays the
development of glomerulonephntis and improves survival in MRL
lpr/lpr mice at least by suppressing the expressions of MMP-2/-9 via
inhibiting thrombin receptor PAR-i activation and fibrin deposition
引文
1 Cameron JS Lupus nephritis an histoncai perspective 1968-1998 J Nephrol 12 (suppl 2) S29-S41, 1999
2 McLaughlin JR, Bombardier C, Farewell VT, Gladman DD, Urowitz MB Kidney
biopsy in systemic lupus erythematosus Ⅲ Survival analysis controlling for clinical and laboratory variables Arthritis Rheum 37 559-67, 1994
3 Bono L, Cameron JS, Hicks JA The very long-term prognosis and complications of lupus nephritis and its treatment QJM92 211-8,1999
4 Perampalam S, Wang L, Myers-Mason N, Yeow JN, Stanietsky N, Phillips J, Weitz JI, Ackerley C, Levy GA, Cole EH Identification of a unique glomerular factor X activator in murine lupus nephritis J Am Soc Nephrol 10 2332-41, 1999
5 Wharram BL, Fitting K, Kunkel SL, Remick DG, Merritt DE, Wiggins RC Tissue factor expression in endothelial cells/mono-cyte cultures stimulated by lipopolysaccharide and/or aggregated IgG J Immunol 146 1437-1445,1991
6 Yamamoto K, Loskutoff DJ, Saitor H Renal Expression of fibrinolytic genes and tissue factor in a murine model of renal disease as a function of age Semin Thromb Hemost 24 261-268, 1998
7 Zacharias U, He CJ, Hagege J, Xu Y, Sraer JD Thrombin and phorbol ester induce internahzation of thrombin receptor of human mesangial cells through different pathways Exp Cell Res 216 371-379, 1995
8 Grand RJA, Turnell AS, Grabham PW Cellular consequences of thrombin receptor activation Biochem J 31 353-368, 1996
9 Papadimitriou E, Manolopoulos VG, Hayman GT, Margoudakis ME, Unsworth BR Thrombin modulates vectonal secretion of extracellular matrix proteins in cultured endothelial cells Am J Physiol 272 C1112-C1122, 1997
10 Yamabe H, Osawa H, Inuma H, Kaizuka M, Tamura N Thrombin stimulates production of transforming growth factor-beta by cultured human mesangial cells Nephrol Dial Transplant 12 438-442, 1997
11 Galis ZS, Kranzhofer R, Fenton JW, Libby P Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells Thromb Vasc Biol 17 483-489,1997
12 Kaplanski G, Fabrigoule M, Boulay Y, Dianrello CA, Bongrand P Thrombin induces endothelial type Ⅱ activation in vitro IL-1 and TNF-alpha-independent IL-8 secretion and E-selection expression J Immunol 158 5435-5441, 1997
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50 Pollak VE, Kant KS, Hanharan S Diffuse and focal proliferative lupus nephritis Treatment approaches and results Nephron 59 177-193,1991
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52 Troyer DA, Chandrasekar B, Barnes JL, Fernandes G Calorie restriction decreases platelet-derived growth factor (PDGF)-A and thrombm receptor mRNA expression in autoimmune murine lupus nephritis dm Exp Immunol 108 58-62, 1997
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57 Khamashta MA Primary prevention of thrombosis in subjects with positive antiphosphohpid antibodies J Autoimmun 15 249-253,2000
2 McLaughlin JR, Bombardier C, Farewell VT, Gladman DD, Urowitz MB Kidney
biopsy in systemic lupus erythematosus Ⅲ Survival analysis controlling for clinical and laboratory variables Arthritis Rheum 37 559-67, 1994
3 Bono L, Cameron JS, Hicks JA The very long-term prognosis and complications of lupus nephritis and its treatment QJM92 211-8,1999
4 Perampalam S, Wang L, Myers-Mason N, Yeow JN, Stanietsky N, Phillips J, Weitz JI, Ackerley C, Levy GA, Cole EH Identification of a unique glomerular factor X activator in murine lupus nephritis J Am Soc Nephrol 10 2332-41, 1999
5 Wharram BL, Fitting K, Kunkel SL, Remick DG, Merritt DE, Wiggins RC Tissue factor expression in endothelial cells/mono-cyte cultures stimulated by lipopolysaccharide and/or aggregated IgG J Immunol 146 1437-1445,1991
6 Yamamoto K, Loskutoff DJ, Saitor H Renal Expression of fibrinolytic genes and tissue factor in a murine model of renal disease as a function of age Semin Thromb Hemost 24 261-268, 1998
7 Zacharias U, He CJ, Hagege J, Xu Y, Sraer JD Thrombin and phorbol ester induce internahzation of thrombin receptor of human mesangial cells through different pathways Exp Cell Res 216 371-379, 1995
8 Grand RJA, Turnell AS, Grabham PW Cellular consequences of thrombin receptor activation Biochem J 31 353-368, 1996
9 Papadimitriou E, Manolopoulos VG, Hayman GT, Margoudakis ME, Unsworth BR Thrombin modulates vectonal secretion of extracellular matrix proteins in cultured endothelial cells Am J Physiol 272 C1112-C1122, 1997
10 Yamabe H, Osawa H, Inuma H, Kaizuka M, Tamura N Thrombin stimulates production of transforming growth factor-beta by cultured human mesangial cells Nephrol Dial Transplant 12 438-442, 1997
11 Galis ZS, Kranzhofer R, Fenton JW, Libby P Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells Thromb Vasc Biol 17 483-489,1997
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