【www.zhangdahai.com--入党自传】
[摘要] 目的 探讨卡托普利对糖尿病大鼠肾脏醛糖还原酶基因表达的影响及其意义。方法 建立糖尿病大鼠模型,分为卡托普利干预组和对照组,15周后处死大鼠,从肾脏皮质抽提mRNA,逆转录分别用Cy5和Cy3标记,成为两组cDNA探针。cDNA探针与基因芯片杂交,扫描仪扫描,用软件进行分析。结果 与对照组相比,干预组醛糖还原酶基因表达下调。结论 卡托普利可能通过下调醛糖还原酶的表达产生肾脏保护作用。
[关键词] 基因芯片;卡托普利;糖尿病;大鼠;Sprague Dawley;基因表达;醛糖还原酶
[中图分类号] R966;R587 [文献标识码] A [文章编号] 1673-9701(2009)36-18-02
Effect of Captopril on Kidney Aldose Reductase Gene Expression in Diabetic Rats
HUANG HuibinHOU JianmingCHEN GangWEN JunpingLIANG JixingLI Liantao
Department of Endocrinology,Fujian Provincial Hospital・Fujian Medical University,Fuzhou 350001,China
[Abstract] Objective To study the effect of captopril on the expression of aldose reductase gene in renal tissue from diabetic rats. Methods Diabetic rats were divided into captopril intervention group and control group. All diabetic rats were killed 15 weeks after intervention,and cDNA probes were prepared by labeling the captopril intervened tissue mRNA and the controled tissue mRNA with fluorescent Cy5 and Cy3 respectively through reverse transcription. The cDNA probes were hybridized against the gene chip and then the fluorescent signals were scanned. Results compared with the control group,the aldose reductase gene was down-regulated in the captopril intervention group. Conclusion Captopril may provide renal protection by down-regulating the expression of aldose reductase.
[Key words] DNA microarray;Captopril;Diabetes mellitus;Rats;Sprague Dawle;Gene expression;Aldose reductase
研究表明,醛糖还原酶(aldose reductase,AR)与糖尿病肾病(DN)的发生发展密切相关。血管紧张素转化酶抑制剂(ACEI)对糖尿病具有肾脏保护作用,但与AR之间的关系还不清楚。本课题用卡托普利对糖尿病大鼠进行干预,用基因芯片技术比较干预组与对照组间肾皮质AR基因表达的差异,探讨卡托普利是否可以通过改变AR的表达对糖尿病患者起肾脏保护作用。
1材料与方法
1.1糖尿病动物模型制作
雄性SD大鼠16只,体重(248±14.7)g,空腹12h后按60mg/kg体重腹腔注入链脲佐菌素STZ,日1次。注射48h后检测血糖浓度,5周后血糖浓度持续大于16.6mmol/L,表明糖尿病成模。16只大鼠随机分成干预组和对照组各8只,干预组按每天1.5mg/kg给予卡托普利口服,15周后将大鼠处死,取肾脏皮质冻存备用。
1.2探针制备
用Trizol(Invitrogen公司)抽提肾脏皮质总RNA,用mRNA药盒(Quagen 公司)纯化mRNA。参照Schena等[1]的方法反转录并标记cDNA探针。用Cy5-dUTP标记干预组,用Cy3-dUTP标记对照组。将两组cDNA探针混合,乙醇沉淀后溶解在20μL5×SSC+0.2%SDS杂交液中。
1.3杂交及洗涤
将基因芯片(上海博星基因芯片有限公司)和两组混合的杂交探针分别在95℃水浴中变性5min,将探针加在基因芯片上,用盖玻片封片,置于42℃杂交15～17h,然后揭开盖玻片,分别用2×SSC+0.2%SDS,0.1%SSC+0.2%SDS,0.1%SSC洗涤10min,室温晾干后扫描。
1.4检测
用ScanArray5000扫描芯片,用ImaGene3.0软件分析扫描结果中Cy5和Cy3两种荧光信号的强度和比值。用管家基因对信号进行均衡和修正[2]。
1.5判定标准
基因差异表达的判定标准:(1)Cy5和Cy3信号比值的自然对数的绝对值>0.69(即Cy5/Cy3 >2.0或2.0或 本研究通过基因芯片技术发现卡托普利可以使糖尿病大鼠AR表达下调,其意义在于:(1)预示ACEI的作用可能与AR存在关联,AR可能是ACEI的作用靶点;(2)DN之前应用ACEI可能可以通过下调AR的表达延缓DN的发生。
[参考文献]
[1] Schena M,Shalon D,Heller R,et al. Parallel human genome analysis:microarray-based expression monitoring of 1000 genes[J]. Pnas,1996, 93(20):10614-10619.
[2] Schena M, Shalon D, Dais RW, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray[J]. Science,1995,270(20):467-470.
[3] 朱禧星. 现代糖尿病学[M]. 上海:复旦大学出版社,2001:300.
[4] Tsukushi S,Katsuzaki T,Aoyama L,et al. Increased erythrocyte 3-DG and AGEs in diabetic hemodialysis patients:role of the polyol pathway[J]. Kidney Int,1999,55(5):1970-1976.
[5] Bravi MC,Pietrangeli P,Laurenti O,et al. Polyol pathway activation and glutathione redox status in non-insulin-dependent diabetic patients[J]. Metabolism,1997,46(10):1194-1198.
[6] Ishii H,Tada H,Isogai S. An aldose reductase inhibitor prevents glucose-induced increase in transforming growth factor-βand protein kinase activity in cultured human mesangial cells[J]. Diabetologia,1998,41(3):362-367.
[7] Cotter MA, Mirrlees DJ, Cameron NE. Neurovascular interactions between aldose reductase and angiotensin-converting enzyme inhibition in diabetic rats[J]. Eur J Pharm,2001,417(3):223-230.
(收稿日期:2009-09-09)
