神经型一氧化氮合酶在早期糖尿病大鼠肾组织中的表达

Expression of neuronal nitric oxide synthase in renal tissues of diabetic rats in early stage

目的:观察早期糖尿病大鼠肾组织内神经型一氧化氮合酶的表达变化。方法:实验于2005-03/10在锦州医学院科学实验中心与组织胚胎学实验室完成。60只两三月龄大鼠随机数字表法分成糖尿病模型组和正常对照组,每组30只。禁食12h后糖尿病组一次性腹腔注射链脲佐菌素50mg/kg,建立链脲佐菌素诱导的糖尿病大鼠模型,对照组注射相同体积的柠檬酸缓冲液。分别在第1,2,4周3个时间点处死动物,取肾组织,采用亚硝酸还原酶法检测一氧化氮的含量;采用免组织化学染色法检查致密斑处神经型一氧化氮合酶的表达,采用Mivnt细胞图像分析系统测定一氧化氮合成酶在致密斑处阳性细胞的灰度值。结果:在实验过程中,无动物死亡,全部进入结果分析。①1周时糖尿病模型组大鼠肾组织神经型一氧化氮合成酶含量明显低于正常对照组,差异有显著性意义[(1068.01±61.61),(2329.99±200.62)mmol/L,P<0.05];2,4周时糖尿模型病组大鼠肾组织神经型一氧化氮合成酶含量逐渐升高,2周时与对照组差异无显著性意义[(2334.59±192.40),(2372.11±193.34)mmol/L,P>0.05];4周时差异有显著性意义[(4939.51±133.44),(2407.30±174.94)mmol/L,P<0.05]。②1周病程时糖尿病模型组大鼠肾皮质一氧化氮水平低于正常对照组,差异有显著性意义[(8.56±1.25),(11.23±2.08)μmol/L,P<0.05],2周病程时高于同期正常对照组,差异有显著性意义[(13.71±1.86),(9.93±2.01)μmol/L,P<0.05],4周病程时显著高于同期正常对照组,差异有极显著性意义[(16.29±2.46),(10.99±1.64)μmol/L,P<0.01]。③各病程糖尿病模型组大鼠血糖均高于正常对照组大鼠,差异有显著性意义(P<0.01)。结论:在糖尿病大鼠肾脏病变的早期,肾组织内一氧化氮合成减少,可能主要是由于神经型一氧化氮合酶合成减少造成的。

AIM： To observe the expression of neuronal nitric oxide synthase （nNOS） in the renal tissues of diabetic rats in the early period. METHODS： The experiment was conducted in the Center of Science and Experiment and the Department of Histology and Embryology, Jinzhou Medical College between March and October 2005. Totally 60 rats of 2-3 months old were randomly divided into diabetic model group and normal control group, with 30 rats in each group. After 12-hour fasting, the rats in diabetic model group were used to make diabetic models by intraperitoneal injection of 50 mg/kg streptozotocin （STZ） once, while rats in normal control group were given citric acid buffer of the same volume. Rats were executed respectively at the first, second and fourth weeks to fetch renal tissues. Nitrite reductase was applied to detect the content of nitrogen monoxide （NO）, while immunohistochemical staining was used to measure the expression of nNOS in macula densa （MD） cells. The gray values of nNOS positive neurons in MD were,assayed with Mivnt cell image analysis system. RESULTS： Totally 60 rats entered the result analysis without death.（1）The nNOS content of rats in diabetic model group was obviously lower than that of normal control group at the first week, with the significant difference [（1 068.01±61.61）, （2 329.99±200.62） mmol/L, P 〈 0.051, but gradually increased at the seco.nd and fourth weeks in diabetic model group. There was insignificant difference at the second week [（2 334.59±192.40）, （2 372.11±193.34） mmol/L, P 〉 0.05], whereas significant difference at the fourth week [（4 939.51±133.44）, （2 407.30±174.94） mmol/L, P 〈 0.05] between two groups. （2）Renal cortex NO level was lower in diabetic model group than in normal control group at the first week, with the significant difference [（8.56±1.25）, （11.23±2.08） μmol/L, P 〈 0.05], higher than that of normal control group at the second week, with the significant difference [（13.71±1.86）, （9.93±2.01） μmol/L, P 〈 0.05], and remarkably higher than that of normal control group at the fourth week, with the remarkably significant difference [（16.29±2.46）, （10.99±1.64） μmol/L, P 〈 0.01].（3） The blood sugar of rats were higher in diabetic model group than in normal control group at three time I~.ints, with the significant difference （P 〈 0.01）. CONCLUSION： The reduced expressions of nNOS in renal tissues maybe contribute to the decrease of NO in diabetic rats with renal disease of early coume.