MMP-2，9及TIMP-1在慢性肾衰竭大鼠动脉钙化形成中的表达
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摘要
实验一MMP-2,9及TIMP-1在慢性肾衰竭大鼠动脉钙化形成中的表达
目的:建立慢性肾衰竭血管钙化大鼠模型;探讨MMP-2、9及TIMP-1是否参与慢性肾衰竭动脉血管钙化的形成。
方法:(1)用2%腺嘌呤每日250-300mg/kg灌胃制备慢性肾衰竭血管钙化模型;(2)应用RT-PCR测定主动脉MMP-2、9及TIMP-1mRNA水平;(3)应用Von Kossa染色观察慢性肾衰竭大鼠主动脉、肾动脉、股动脉、冠状动脉的钙化情况;(4)应用免疫组织化学方法检测TIMP-1、OPN、Ⅰ型及Ⅳ型胶原蛋白在钙化主动脉的表达。以上测定及血清生化标本的采集时间点为:灌胃后第2、4、6、8周。
结果:(1)大鼠2周后出现明显肾衰竭,血尿素氮、肌酐、血磷和钙磷乘积持续增高; (2) Von Kossa染色显示,6周后主动脉、肾动脉、股动脉、冠状动脉血管中层出现不同程度的钙化; ( 3)钙化主动脉MMP9/TIMP-1比值较正常血管升高,而MMP2/TIMP-1比值较之下降;(4)在钙化的主动脉中层平滑肌细胞TIMP-1蛋白表达增多,并出现OPN的阳性表达。动脉血管发生钙化时Ⅰ型胶原蛋白表达增多,Ⅳ型胶原蛋白表达减少。
结论:(1)大鼠腺嘌呤模型接近尿毒症血管钙化的发病机理,是很好的研究慢性肾衰竭血管钙化的动物模型。(2)慢性肾衰竭发生血管钙化时,ECM发生重构,Ⅰ型胶原增多,Ⅳ型胶原减少,并且出现骨特异性蛋白OPN的阳性表达;MMP-2、MMP-9、TIMP-1比例失衡参与此过程的发生。
实验二CKD患者血清、尿TIMP-1水平和CKD分期的关系
目的:探讨慢性肾脏病患者血清、尿TIMP-1水平和CKD分期的关系和意义。
方法:用酶联免疫法(ELISA)双夹心法测定122例慢性肾脏病不同CKD分期患者和40名健康对照血清、尿TIMP-1水平,同时测定血胱蛋白酶抑制剂C(CystC)、甲状旁腺激素(PTH)、血脂等指标,并对TIMP-1水平和CKD分期及其它指标之间进行比较及相关分析。
结果:慢性肾脏病患者血清及尿TIMP-1水平增高(p<0.01),且随着CKD分期的增加而升高(p<0.05),特别是血清TIMP-1在CKD早期就明显增高。血清、尿TIMP-1水平与肾小球滤过率呈负相关(r分别为-0.647、-0.437,p<0.01)。
结论:血清TIMP-1水平是评价慢性肾脏病肾纤维化程度一个潜在很好的指标。
Part One The Expressions of MMP-2,9 and TIMP-1 in Rats Vascular Calcification with Chronic Renal Failure
Objective: To establish vascular calcification model of rat with Chronic Renal Failure; and to examine whether MM-P2,9 and TIMP-1 is involved in vascular calcification in chronic renal failure. Methods:
(1)Chronic renal failure was induced in Wistar rats by received stomach irritation daily with 2% adenine 250-300mg/kg for 6 weeks.
(2)Applying the methods of RT-PCR ,we observed the changes of MMP-2,9 and TIMP-1 mRNA .
(3)Von Kossa stained vessels for the presence of calcification.
( 4) TIMP-1、OPN、Type I andⅣCollagen were analyzed by immunhistochemistry. All above and serum biochemical parameters were examined at 2、4、6 and 8 weeks.
Results:
(1)Serum urea nitrogen、creatinine concentrations and inorganic phosphate progressive increased significantly (P<0.05) in all model rats compared with those of control rats after 2 weeks,and calcium-phosphorus product was increased also.
(2) Medial calcification was found in the aorta、femoral、renal artery and coronary arteries after 6 weeks.
(3)MMP-2/TIMP-1 ratio was lower than control group,and MMP9/ TIMP1 ratio showed opposite changes.
(4)The expression of TIMP-1 in calcified aortic smooth muscle cells in the medial layer was increased obviously by immunhistochemistry, and there were positive immunostaining for OPN and type I collagen increased,but absent staining for type IV collagen in these same areas .
Conclusion : The morphology of arteriosteogenesis in uraemic rats made by adenine approximate to uraemic patient, so it may serve as a useful model for arteriosteogenesis of chronic renal failure . The imbalance of MMP-2,9 /TIMP-1 involved in vascular calcification of chronic renal failure.
PartTwo Relationship between serum、urine tissue inhibitor of metalloproteinases-1(TIMP-1) levels and the stages of CKD
Objective: To investigate the relationship and significances of serum and urine TIMP-1 level and different stages of CKD.
Methods: Serum and urine level of TIMP-1 were measured by enzyme-linked immunoadsorbent assay ( ELISA ) double-antibody- sandwich method in 122 CKD patients and 40 healthy controls,CystC、PTH and LDL were determined simultaneously . Correlationanalysis between TIMP-1 level and degree of renal function were performed.
Results: Serum and urine TIMP-1 in CKD group was significantly higher than that healthy control group(p<0.01). There was correlation between serum、urine TIMP-1 and different stages of CKD. Especially,TIMP-1 in serum obviously increased in early stage of CKD.The corelation of Serum and urine TIMP-1 with glomerular filtration rate was positive( r=-0.647、-0.437,respectively, p<0.01).
Conclusion : Serum concentrations of TIMP-1 may be used as a fine potential maker for diagnosis of renal fibrosis.
目的:建立慢性肾衰竭血管钙化大鼠模型;探讨MMP-2、9及TIMP-1是否参与慢性肾衰竭动脉血管钙化的形成。
方法:(1)用2%腺嘌呤每日250-300mg/kg灌胃制备慢性肾衰竭血管钙化模型;(2)应用RT-PCR测定主动脉MMP-2、9及TIMP-1mRNA水平;(3)应用Von Kossa染色观察慢性肾衰竭大鼠主动脉、肾动脉、股动脉、冠状动脉的钙化情况;(4)应用免疫组织化学方法检测TIMP-1、OPN、Ⅰ型及Ⅳ型胶原蛋白在钙化主动脉的表达。以上测定及血清生化标本的采集时间点为:灌胃后第2、4、6、8周。
结果:(1)大鼠2周后出现明显肾衰竭,血尿素氮、肌酐、血磷和钙磷乘积持续增高; (2) Von Kossa染色显示,6周后主动脉、肾动脉、股动脉、冠状动脉血管中层出现不同程度的钙化; ( 3)钙化主动脉MMP9/TIMP-1比值较正常血管升高,而MMP2/TIMP-1比值较之下降;(4)在钙化的主动脉中层平滑肌细胞TIMP-1蛋白表达增多,并出现OPN的阳性表达。动脉血管发生钙化时Ⅰ型胶原蛋白表达增多,Ⅳ型胶原蛋白表达减少。
结论:(1)大鼠腺嘌呤模型接近尿毒症血管钙化的发病机理,是很好的研究慢性肾衰竭血管钙化的动物模型。(2)慢性肾衰竭发生血管钙化时,ECM发生重构,Ⅰ型胶原增多,Ⅳ型胶原减少,并且出现骨特异性蛋白OPN的阳性表达;MMP-2、MMP-9、TIMP-1比例失衡参与此过程的发生。
实验二CKD患者血清、尿TIMP-1水平和CKD分期的关系
目的:探讨慢性肾脏病患者血清、尿TIMP-1水平和CKD分期的关系和意义。
方法:用酶联免疫法(ELISA)双夹心法测定122例慢性肾脏病不同CKD分期患者和40名健康对照血清、尿TIMP-1水平,同时测定血胱蛋白酶抑制剂C(CystC)、甲状旁腺激素(PTH)、血脂等指标,并对TIMP-1水平和CKD分期及其它指标之间进行比较及相关分析。
结果:慢性肾脏病患者血清及尿TIMP-1水平增高(p<0.01),且随着CKD分期的增加而升高(p<0.05),特别是血清TIMP-1在CKD早期就明显增高。血清、尿TIMP-1水平与肾小球滤过率呈负相关(r分别为-0.647、-0.437,p<0.01)。
结论:血清TIMP-1水平是评价慢性肾脏病肾纤维化程度一个潜在很好的指标。
Part One The Expressions of MMP-2,9 and TIMP-1 in Rats Vascular Calcification with Chronic Renal Failure
Objective: To establish vascular calcification model of rat with Chronic Renal Failure; and to examine whether MM-P2,9 and TIMP-1 is involved in vascular calcification in chronic renal failure. Methods:
(1)Chronic renal failure was induced in Wistar rats by received stomach irritation daily with 2% adenine 250-300mg/kg for 6 weeks.
(2)Applying the methods of RT-PCR ,we observed the changes of MMP-2,9 and TIMP-1 mRNA .
(3)Von Kossa stained vessels for the presence of calcification.
( 4) TIMP-1、OPN、Type I andⅣCollagen were analyzed by immunhistochemistry. All above and serum biochemical parameters were examined at 2、4、6 and 8 weeks.
Results:
(1)Serum urea nitrogen、creatinine concentrations and inorganic phosphate progressive increased significantly (P<0.05) in all model rats compared with those of control rats after 2 weeks,and calcium-phosphorus product was increased also.
(2) Medial calcification was found in the aorta、femoral、renal artery and coronary arteries after 6 weeks.
(3)MMP-2/TIMP-1 ratio was lower than control group,and MMP9/ TIMP1 ratio showed opposite changes.
(4)The expression of TIMP-1 in calcified aortic smooth muscle cells in the medial layer was increased obviously by immunhistochemistry, and there were positive immunostaining for OPN and type I collagen increased,but absent staining for type IV collagen in these same areas .
Conclusion : The morphology of arteriosteogenesis in uraemic rats made by adenine approximate to uraemic patient, so it may serve as a useful model for arteriosteogenesis of chronic renal failure . The imbalance of MMP-2,9 /TIMP-1 involved in vascular calcification of chronic renal failure.
PartTwo Relationship between serum、urine tissue inhibitor of metalloproteinases-1(TIMP-1) levels and the stages of CKD
Objective: To investigate the relationship and significances of serum and urine TIMP-1 level and different stages of CKD.
Methods: Serum and urine level of TIMP-1 were measured by enzyme-linked immunoadsorbent assay ( ELISA ) double-antibody- sandwich method in 122 CKD patients and 40 healthy controls,CystC、PTH and LDL were determined simultaneously . Correlationanalysis between TIMP-1 level and degree of renal function were performed.
Results: Serum and urine TIMP-1 in CKD group was significantly higher than that healthy control group(p<0.01). There was correlation between serum、urine TIMP-1 and different stages of CKD. Especially,TIMP-1 in serum obviously increased in early stage of CKD.The corelation of Serum and urine TIMP-1 with glomerular filtration rate was positive( r=-0.647、-0.437,respectively, p<0.01).
Conclusion : Serum concentrations of TIMP-1 may be used as a fine potential maker for diagnosis of renal fibrosis.
引文
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