卡托普利對(duì)糖尿病大鼠腎臟醛糖還原酶基因表達(dá)的影響及其意義

黃惠彬 侯建明 陳 剛 溫俊平 梁繼興 李連濤

[摘要] 目的 探討卡托普利對(duì)糖尿病大鼠腎臟醛糖還原酶基因表達(dá)的影響及其意義。方法 建立糖尿病大鼠模型,分為卡托普利干預(yù)組和對(duì)照組,15周后處死大鼠,從腎臟皮質(zhì)抽提mRNA,逆轉(zhuǎn)錄分別用Cy5和Cy3標(biāo)記,成為兩組cDNA探針。cDNA探針與基因芯片雜交,掃描儀掃描,用軟件進(jìn)行分析。結(jié)果 與對(duì)照組相比,干預(yù)組醛糖還原酶基因表達(dá)下調(diào)。結(jié)論 卡托普利可能通過(guò)下調(diào)醛糖還原酶的表達(dá)產(chǎn)生腎臟保護(hù)作用。

[關(guān)鍵詞] 基因芯片;卡托普利;糖尿病;大鼠;Sprague Dawley;基因表達(dá);醛糖還原酶

[中圖分類號(hào)] R966;R587 [文獻(xiàn)標(biāo)識(shí)碼] A [文章編號(hào)] 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,醛糖還原酶(aldose reductase,AR)與糖尿病腎病(DN)的發(fā)生發(fā)展密切相關(guān)。血管緊張素轉(zhuǎn)化酶抑制劑(ACEI)對(duì)糖尿病具有腎臟保護(hù)作用,但與AR之間的關(guān)系還不清楚。本課題用卡托普利對(duì)糖尿病大鼠進(jìn)行干預(yù),用基因芯片技術(shù)比較干預(yù)組與對(duì)照組間腎皮質(zhì)AR基因表達(dá)的差異,探討卡托普利是否可以通過(guò)改變AR的表達(dá)對(duì)糖尿病患者起腎臟保護(hù)作用。

1材料與方法

1.1糖尿病動(dòng)物模型制作

雄性SD大鼠16只,體重(248±14.7)g,空腹12h后按60mg/kg體重腹腔注入鏈脲佐菌素STZ,日1次。注射48h后檢測(cè)血糖濃度,5周后血糖濃度持續(xù)大于16.6mmol/L,表明糖尿病成模。16只大鼠隨機(jī)分成干預(yù)組和對(duì)照組各8只,干預(yù)組按每天1.5mg/kg給予卡托普利口服,15周后將大鼠處死,取腎臟皮質(zhì)凍存?zhèn)溆谩?/p>

1.2探針制備

用Trizol(Invitrogen公司)抽提腎臟皮質(zhì)總RNA,用mRNA藥盒(Quagen 公司)純化mRNA。參照Schena等[1]的方法反轉(zhuǎn)錄并標(biāo)記cDNA探針。用Cy5-dUTP標(biāo)記干預(yù)組,用Cy3-dUTP標(biāo)記對(duì)照組。將兩組cDNA探針混合,乙醇沉淀后溶解在20μL5×SSC+0.2%SDS雜交液中。

1.3雜交及洗滌

將基因芯片(上海博星基因芯片有限公司)和兩組混合的雜交探針?lè)謩e在95℃水浴中變性5min,將探針加在基因芯片上,用蓋玻片封片,置于42℃雜交15～17h,然后揭開(kāi)蓋玻片,分別用2×SSC+0.2%SDS,0.1%SSC+0.2%SDS,0.1%SSC洗滌10min,室溫晾干后掃描。

1.4檢測(cè)

用ScanArray5000掃描芯片,用ImaGene3.0軟件分析掃描結(jié)果中Cy5和Cy3兩種熒光信號(hào)的強(qiáng)度和比值。用管家基因?qū)π盘?hào)進(jìn)行均衡和修正[2]。

1.5判定標(biāo)準(zhǔn)

基因差異表達(dá)的判定標(biāo)準(zhǔn):(1)Cy5和Cy3信號(hào)比值的自然對(duì)數(shù)的絕對(duì)值>0.69(即Cy5/Cy3 >2.0或<0.5);(2)Cy5和Cy3信號(hào)值兩者皆大于200或其中之一大于800。Cy5/Cy3>2.0或<0.5分別表示干預(yù)組與對(duì)照組相比基因表達(dá)上調(diào)或是下調(diào)。

2結(jié)果

基因芯片結(jié)果顯示整張芯片雜質(zhì)或背景干擾均勻,管家基因均被檢測(cè)到,提示mRNA質(zhì)量良好,因此基因芯片表達(dá)結(jié)果可靠。在差異表達(dá)并且有功能的基因中,AR在對(duì)照組的信號(hào)值Cy3為3494.2,在干預(yù)組的信號(hào)值Cy5為803,

Cy5/Cy3=0.230,提示干預(yù)組與對(duì)照組相比,AR表達(dá)下調(diào)。

3討論

AR屬于還原型輔酶Ⅱ依賴性醛-酮還原酶族,廣泛分布于神經(jīng)、心臟、腎小球、視網(wǎng)膜、晶體和血管壁等非胰島素依賴性組織[3]。

已有大量研究表明,多元醇通路的激活與DN密切相關(guān)。AR是多元醇通路的限速酶。當(dāng)血糖濃度維持在正常生理水平時(shí),它對(duì)葡萄糖的親和力較低,此時(shí)葡萄糖很少轉(zhuǎn)化為山梨醇。在高血糖狀況下,磷酸己糖激酶被飽和,這時(shí)AR被激活,促使體內(nèi)的葡萄糖轉(zhuǎn)化為山梨醇。然而山梨醇脫氫酶的活力并未相應(yīng)地成比例增加,造成山梨醇的堆積。由于山梨醇不易透過(guò)細(xì)胞膜,細(xì)胞內(nèi)滲透壓升高,細(xì)胞腫脹,細(xì)胞外液的肌醇進(jìn)入受限,細(xì)胞內(nèi)肌醇下降,影響磷酸化過(guò)程,Na/K-ATP酶活性降低,細(xì)胞功能障礙。

新近的研究表明,多元醇激活還參與了蛋白非酶糖基化[4]、氧化應(yīng)激[5]、蛋白激酶C[6]激活等與DN有關(guān)的生化、病理生理過(guò)程,可能是DN發(fā)生發(fā)展的始動(dòng)因素。因此,AR的表達(dá)下調(diào)可以抑制多元醇通路的激活,延緩DN的發(fā)生發(fā)展。

AR被認(rèn)為是DN發(fā)生發(fā)展的因素之一,ACEI對(duì)DN的保護(hù)作用已被大量的循證醫(yī)學(xué)所證實(shí),但到目前為止ACEI與AR之間的關(guān)系很少報(bào)道。Cotter[7]等報(bào)道lisinopril(一種ACEI制劑)與AR抑制劑合用對(duì)改善糖尿病大鼠坐骨神經(jīng)血流和傳導(dǎo)速度有協(xié)同作用,但并未發(fā)現(xiàn)lisinopril改變AR抑制劑對(duì)多元醇通路的影響。

本研究通過(guò)基因芯片技術(shù)發(fā)現(xiàn)卡托普利可以使糖尿病大鼠AR表達(dá)下調(diào),其意義在于:(1)預(yù)示ACEI的作用可能與AR存在關(guān)聯(lián),AR可能是ACEI的作用靶點(diǎn);(2)DN之前應(yīng)用ACEI可能可以通過(guò)下調(diào)AR的表達(dá)延緩DN的發(fā)生。

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(收稿日期:2009-09-09)