肾阳虚大鼠模型的水通道蛋白1改变
【摘要】 目的:探讨中医学肾阳虚证的物质基础。 方法:运用腺嘌呤灌服和氢化可的松肌肉注射2种方法制备大鼠肾阳虚模型(分别称为模型Ⅰ和模型Ⅱ),100只Wistar大鼠随机分为正常对照组,模型Ⅰ组,模型Ⅱ大、中和小剂量组。造模结束后,常规生化法检测血肌酐(serum creatinine,SCr)、血尿素氮(blood urea nitrogen,BUN)、尿肌酐(urine creatinine,UCr)含量,冰点比重法检测尿渗透压(urine osmotic pressure,Uosm)、高岭土法检测尿17羟皮质类固醇含量。实时逆转录聚合酶链式反应检测肾脏组织中水通道蛋白1表达。 结果:两种模型大鼠均出现类似肾阳虚证表现。与正常对照组和模型Ⅱ各剂量组比较,模型Ⅰ组大鼠SCr和BUN含量升高(P0.05),UCr含量和Uosm降低(P0.05)。模型Ⅰ组大鼠尿17羟皮质类固醇含量和肾组织AQP1表达比正常对照组下降(P0.05)。与正常对照组比较,模型Ⅱ中剂量组SCr含量增加(P0.05),尿17羟皮质类固醇含量降低(P0.05),而模型Ⅱ各剂量组大鼠肾组织AQP1表达升高(P0.05),并显示出一定的剂量依赖关系,以高剂量组升高最为明显。 结论:水通道蛋白可能是肾阳虚证的物质基础之一。
Methods: Two kinds of rat models of deficiency of kidneyyang were induced by adenine intragastric administration (model Ⅰ) and hydrocortisone intramuscular injection (model Ⅱ). One hundred rats were randomly divided into normal control group, model Ⅰ group, lowdose model Ⅱ group, mediumdose model Ⅱ group and highdose model Ⅱ group. After model establishment, contents of serum creatinine (SCr), blood urea nitrogen (BUN), and urine creatinine (UCr) were detected by automatic biochemistry analyzer; freezing point method was used for 24hour urinary osmotic pressure (Uosm) testing and kaolinite method was used to detect the content of urinary 17hydroxycorticosteroid (17OHCS). Expression of aquaporin1 in renal tissue was observed by using real time reverse transcription polymerase chain reaction.
Results: The rats of model groups had the characteristics of kidneyyang deficiency syndrome. The contents of SCr and BUN were significantly higher in model Ⅰgroup than in normal control group and three model Ⅱ groups (P0.05), and UCr and Uosm were significantly lower (P0.05). The level of urinary 17OHCS and expression of aquaporin1 in renal tissue were decreased in the model Ⅰ group (P0.05) as compared with the normal control group. Compared with the normal control group, the content of SCr was increased and urinary 17OHCS was decreased in the mediumdose model Ⅱ group, but the expression of aquaporin1 was increased in three model Ⅱ groups with a dosedependent manner.
Conclusion: Aquaporin1 may be one of the material foundations of kidneyyangdeficiency syndrome.
Keywords: deficiency of kidneyyang; aquaporin1; rats。
肾在中医脏腑学说中占有重要地位,“肾为先天之本”和“生命之根”。体内水液的存留、分布与排泄作用,主要是靠肾的气化功能完成的,而气化作用的动力就是肾阳。本实验运用氢化可的松肌肉注射和腺嘌呤灌服两种方法制备大鼠肾阳虚模型[1],观察2种模型大鼠的肾功能、肾小管功能及水通道蛋白1的变化,探讨肾阳虚证与水通道蛋白的关系,为中医证型规范化及建立中医疗效评价体系提供实验依据。
1 材料与方法。
1.1 实验材料。
1.1.1 实验药物和主要试剂 腺嘌呤购自上海新兴化工试剂研究所,分子式C5H5N5,分子量为135.13,批号为061108,含量≥98%;层析试剂,1 g/瓶。注射用氢化可的松琥珀酸钠购自天津市生物化学制药厂,批号为20060202,50 mg/支。焦碳酸二乙脂(diethyl pyrocarbonate, DEPC)由Sigma公司提供,TRIzol、莫洛尼氏鼠白血病病毒(Moloney murine leukemia virus, MMLV)逆转录酶和Taq酶由美国Invitrogen公司提供,实时聚合酶链式反应(polymerase chain reaction,PCR)检测仪和ABI7300 Sequence Detection System由美国ABI公司提供。
1.1.2 实验动物 雄性Wistar大鼠100只,清洁级,体质量200~250 g,由上海中医药大学曙光医院实验动物中心提供,实验动物生产许可证号为SCXK(沪)20030003。
1.2 实验方法。
1.2.1 造模方法 参照文献[1]方法建立腺嘌呤致肾阳虚(模型Ⅰ)和氢化可的松致肾阳虚(模型Ⅱ)2种模型,将大鼠编号后根据随机数分为正常对照组,模型Ⅰ组,模型Ⅱ大、中和小剂量组。模型Ⅰ组20只,予2%腺嘌呤(20 mg/ml,生理盐水配制)2 ml灌胃,1次/d,连续4周。模型Ⅱ小剂量组20只,氢化可的松5 mg/(kg·d),中剂量组20只,氢化可的松20 mg/(kg·d),大剂量组20只,氢化可的松50 mg/(kg·d),1次/d,后肢肌肉注射,连续2周。正常组20只中10只予生理盐水2 ml灌胃,1次/d,连续4周;另外10只予生理盐水0.3 ml后肢肌肉注射,1次/d,连续2周。实验过程中所有大鼠自由饮水。
1.2.2 观察指标及检测方法 造模结束后,代谢笼留取24 h尿样,应用冰点渗透压仪检测尿渗透压(urine osmotic pressure, Uosm),应用高岭土法检测尿17羟皮质类固醇(17hydroxycorticosteroid,17OHCS)含量。用2%戊巴比妥钠(2 ml/kg)腹腔注射麻醉大鼠后腹主动脉取全血,在4 ℃条件下,3 000 r/min离心10 min,制备血清,检测各指标。血肌酐(serum creatinine,SCr)、血尿素氮(blood urea nitrogen,BUN)、尿肌酐(urine creatinine,UCr)应用全自动生化分析仪检测。每组抽取5只大鼠,迅速摘取肾脏,分离包膜后液氮中保存,应用实时PCR检测仪检测肾脏组织中水通道蛋白1(aquaporin1,AQP1)表达。经过组织总RNA的抽提,逆转录cDNA,进行PCR扩增。扩增体系如下:SYBRGreen Mix 32.5 μl,上游引物F 0.5 μl,下游引物R 0.5 μl,ddH2O 14.5 μl,cDNA模板2 μl,总体积50 μl。扩增条件:95 ℃ 10 min、95 ℃ 20 s、55 ℃ 30 s和72 ℃ 30 s,40个循环。数据采用ABI Prism 7300 SDS Software熔解曲线分析。引物序列 GAPDH–FP:5’CGT TGA CAT CCG TAA AGA CC3’;GAPDH–RP:5’AAC AGT CCG CCT AGA AGC AC3’。扩增片段长度328 bp。AQP1–FP:5’CGA GGT GAC CTG GGC ACC ATC CAT GAC3’;AQP1–RP:5’CTG CTC CAC CTT GGG CTT GCG ACC CAC3’。扩增片段长度286 bp。引物由广州达辉公司合成。
1.3 统计学方法 应用SPSS 11.0统计软件进行多样本两两比较的q检验或独立样本非参数检验进行两两比较。
2 结果。
2.1 一般情况观察 模型Ⅰ组大鼠造膜14 d后、模型Ⅱ组大鼠造膜10 d后开始出现不同程度的体质量下降、活动减少、反应迟钝、喜扎堆、畏寒喜暖、体毛枯萎和肛周污染的症状,并出现采食量减少和饮水量略增加。类似人的肾阳虚症状[1]。正常对照组大鼠未出现上述症状。