一氧化氮合酶在糖尿病大鼠视网膜中表达模式的研究

目的 研究一氧化氮合酶(NOS)在8周糖尿病大鼠视网膜的表达,探讨其与一氧化氮在糖尿病视网膜病变(DR)中可能的分子作用机制.方法 采用限制片段差异显示PCR(RFDD-PCR)技术建立正常和8周糖尿病大鼠视网膜基因表达谱,经生物信息学分析两者差异,初步确定NOS三种亚型-eNOS、nNOS和iNOS为DR相关基因,并以半定量RT-PCR和免疫组化方法进行验证.结果 RFDD-PCR结果显示,糖尿病组eNOS和nNOS表达下调,iNOS表达上调.RT-PCR结果显示,糖尿病组eNOS和nNOS表达比正常组明显降低(eNOS:0.23±0.03,0.32±0.03,P＜0.05;0.25±0.02,0.36±0.02,P＜0.05),iNOS比正常组明显增高(0.27±0.02,0.20±0.03,P＜0.05).免疫组化结果显示,正常组eNOS、nNOS和iNOS阳性细胞均见于内核层(INL)和节细胞层(GCL),eNOS阳性细胞也分布于血管内皮层;糖尿病组eNOS和nNOS阳性细胞较正常组明显减少(eNOS:14.33±3.19,22.13±3.60,P＜0.05;nNOS:21.87±3.62,34.40±7.09,P＜0.05),iNOS较正常组明显增多(17.60±2.58,11.73±2.70,P＜0.05).结论 eNOS、nNOS和iNOS表达变化与DR发生发展有关.     

L-精氨酸对大鼠尾核一氧化氮合酶基因表达的影响

目的 研究尾核中一氧化氮在痛觉调制中的作用机制.方法 大鼠尾核内微量注射L-精氨酸、Nω-硝基-L-精氨酸甲酯、生理盐水,应用逆转录-聚合酶链反应测定4,8,12,24和48 h大鼠尾核nNOS mRNA表达的变化;新生Wistar大鼠尾核神经元原代培养液中加入L-精氨酸、Nω-硝基-L-精氨酸甲酯、生理盐水,观察给药12 h尾核nNOS mRNA表达的变化.结果 大鼠尾核给药24 h内L-精氨酸组nNOS mRNA表达较正常增强(P＜0.05),而Nω-硝基-L-精氨酸甲酯组nNOS mRNA表达较正常减弱(P＜0.05),48 h恢复正常.体外尾核原代神经元培养,nNOS mRNA表达的变化趋势和在体实验相一致.结论 中枢神经系统尤其是尾核中NOS活性是一氧化氮作为神经递质或调质参与中枢痛觉调制的关键因素之一.     

当归芍药散对阿霉素肾病综合征大鼠一氧化氮及其合酶表达影响

目的 :尾静脉注射阿霉素法建立肾病综合征(Nephrotic Syndrome,NS)大鼠,探讨当归芍药散对肾病综合征大鼠一氧化氮合酶表达的影响及其机制研究。方法 :将雄性SD大鼠复制为NS模型,随机分为阳性对照组、模型组和当归芍药散组,另设正常对照组。灌胃治疗后,HE染色观察肾脏组织病理变化;BCA法检测24 h尿蛋白量;分光光度法测定尿液NO含量;WB法检测肾组织内皮型一氧化氮合酶(endothelial NOS,eNOS)、诱导型一氧化氮合酶(inducibleNOS,iNOS)和神经型一氧化氮合酶(neural NOS,nNOS)的表达。结果 :模型组与正常组比较,尿蛋白量显著增加(P0.01),肾脏病理损伤严重;各治疗组与模型组比较尿蛋白含量下降明显,差异具有显著性(P0.01);模型组与正常组比较大鼠尿液NO含量降低显著(P0.01),当归芍药散组与模型组比较NO含量显著增加(P0.01);与正常组比模型组iNOS、nNOS和eNOS蛋白表达均降低,且差异具有显著意义(P0.01);与模型组比较各给药组能显著增加iNOS、nNOS和eNOS蛋白表达,差异具有极显著意义(P0.01)。结论 :当归芍药散可以提高肾病综合征大鼠NOS的表达,增加大鼠体内NO含量,减少尿蛋白含量,延缓肾病综合征病理进程。     

L-硝基精氨酸对大鼠局灶性脑缺血损伤的保护作用及其机制

目的:研究非选择性一氧化氮合酶(NOS)抑制剂L-硝基精氨酸(L-NA)对局灶性脑缺血大鼠脑组织的保护作用及其机制。方法:线栓法复制大鼠大脑中动脉脑缺血模型;采用RT-PCR法检测大鼠缺血0,2,6,12,24 h脑组织中[内皮型NOS(eNOS)、神经型NOS(nNOS)及诱导型NOS(iNOS)]基因表达的变化;TTC染色法测定大鼠脑梗死体积,HPLC法测定纹状体、海马、皮层中氨基酸含量。结果:正常对照组可见eNOS和nNOS表达,eNOS于脑缺血后2 h达到高峰,nNOS于6 h达到高峰;正常对照组未见iNOS表达,但在脑缺血后开始表达,缺血后12 h达到高峰;缺血后12 h给予L-NA治疗3 dL-NA组中梗死体积/全脑体积(Ⅳ%)显著低于相应的缺血组;缺血组天门冬氨酸、谷氨酸、甘氨酸、GABA的含量明显高于假手术组,缺血12 h治疗3 d时,L-NA组中纹状体、海马、皮层中天门冬氨酸、谷氨酸的含量显著低于缺血组,GABA的含量显著高于缺血组,海马中甘氨酸含量显著高于缺血组。结论:缺血后期应用L-NA对脑缺血有治疗作用,可能与兴奋性氨基酸合成、释放相对减少,抑制性氨基酸合成、释放相对增加有关。     

内皮型一氧化氮合酶基因多态性与脑梗死的研究

一氧化氮合酶(Nitric Oxide Synthase，NOS)的一个重要指征是不仅合成一氧化氮(Nitric Oxide，NO)，也能产生超氧离子。目前已确定的NOS的亚型有三种，根据不同的基因编码分别称为神经元型NOS(neuronal NOS，nNOS)、诱导型NOS(inducibl eNOS，iNOS)、内皮型NOS(endothel ialNOS，eNOS)。三种NOS亚型在血管内皮细胞中均可表达，但在生理情况下，血管内皮细胞中的eNOS合成和释放NO无疑是血管张力的主要调节因子。而nNOS与iNOS产生的NO则主要产生氧化作用，具神经毒性作用。     

安氟醚对大鼠耳蜗一氧化氮合酶表达的影响

目的:观察安氟醚对大鼠耳蜗一氧化氮合酶(NOS)表达的影响,探讨其对耳蜗影响的可能机制。方法:30只Wistar大鼠随机分为3组(n=10),对照组(C组)持续吸入纯氧;低浓度安氟醚组(E1组)持续吸入1.5%安氟醚+纯氧;高浓度安氟醚(E2组)持续吸入3.0%安氟醚+纯氧,30min后处死大鼠,免疫组织化学方法检测耳蜗螺旋器、血管纹和螺旋神经节诱生型NOS(iNOS)、内皮型NOS(eNOS)和神经元型NOS(nNOS)的表达水平。结果:与C组比较,E1组耳蜗螺旋器、螺旋神经节iNOS、eNOS和nNOS以及血管纹eNOS表达均下调,E2组耳蜗螺旋器、血管纹和螺旋神经节iNOS、eNOS和nNOS表达下调(P<0.05或0.01);E2组耳蜗螺旋神经节eNOS和nNOS表达组较E下调明显(P<0.05)。结论:安氟醚可浓度依赖性地下调大鼠耳蜗NOS表达,从而影响耳蜗功能。     

甲状腺激素对新生鼠脑NOS基因表达的调节

目的"观察不同日龄新生鼠脑一氧化氮(NO)含量,一氧化氮合酶(NOS)活性的变化及甲状腺激素对nNOS基因表达的调节. 方法"采用丙基硫氧嘧啶(PTU)灌注孕母鼠造成甲减模型.采用NO、NOS的生化测定法以及半定量逆转录聚合酶链反应(RT-PCR). 结果"生化测定法显示,不同年龄段大鼠随着年龄的增加NO含量上升(P＜0.01),NOS的活性上升(P＜0.01);同一年龄段仔鼠,甲减组较正常组NO含量下降(P＜0.01),NOS活性下降(P＜0.01);RT-PCR测定显示,幼鼠nNOS mRNA水平随着年龄的增加而上升;同一年龄组,甲减组较正常组nNOS mRNA表达下降. 结论"提示甲状腺激素对nNOS mRNA表达有上调作用;NO信号系统可能参与鼠脑发育阶段甲状腺激素缺乏所致的脑损害过程.     

雄激素致无排卵大鼠卵巢nNOS、eNOS、ET-1的表达

目的 观察雄激素致无排卵大鼠(ASR)血浆及卵巢血管舒张因子一氧化氮(NO)、一氧化氮合酶(nNOS、eNOS)、环磷酸鸟苷(cGMP)、血管收缩因子内皮素1(ET-1)、血管紧张素Ⅱ(ATⅡ)的变化,探讨卵巢血管舒-缩因子在无排卵发病机制中的作用.方法 建立雄激素致无排卵大鼠模型,分为模型组和正常组.采用放射免疫法测定模型组、正常组大鼠血浆NO、cGMP、ET-1、ATⅡ以及血清雌二醇(E2)、孕酮(P)、睾酮(T)含量;采用免疫组化法检测大鼠卵巢nNOS、eNOS、ET-1表达;采用HE染色观察大鼠卵巢组织形态学变化.结果 与正常组比较,模型组大鼠血浆NO、cGMP含量降低(P<0.01),血浆ET-1、ATⅡ含量升高(P<0.01).血清E2、T含量及E2/P显著升高(P<0.01),血清P含量无统计学差异(P>0.05);与正常组比较,模型组大鼠卵巢颗粒细胞以及髓质nNOS、eNOS表达减弱(P<0.01),ET-1表达增强(P<0.01);正常组大鼠卵巢皮质可见发育期各级卵泡及黄体;模型组大鼠卵巢皮质卵泡囊状扩张,颗粒细胞层极薄.结论 雄激素致无排卵大鼠血浆及卵巢血管舒张因子降低、收缩因子增高,可能使卵巢血液供应障碍、颗粒细胞凋亡,导致卵泡在排卵前闭锁,无排卵的发生.     

内皮型一氧化氮合酶基因在犬内皮细胞的转染和表达

目的探讨转染内皮型一氧化氮合酶(eNOS)基因后内皮细胞(EC)的功能变化.方法采用脂质体法转染eNOS基因于实验犬EC;RT-PCR和免疫组化法检测转染效果;分别采用比色法和酶联免疫法检测细胞培养液一氧化氮合酶(NOS)、一氧化氮(NO)和vWF的浓度;并观察转染细胞生长增殖情况.结果 RT-PCR产物电泳和测序及免疫组化法检测证实转染效果满意;转染后EC培养液NOS和NO浓度在不同时间明显升高(120 h分别为33.53和32.99),与正常组对比差异显著(P<0.05).转染后细胞生长增殖和vWF含量无显著差异.结论通过脂质体法成功地将eNOS基因转染于实验犬EC;转染后eNOS基因在mRNA和蛋白质水平均高效表达;内皮细胞eNOS活性显著增强;转染后细胞生物学功能稳定.     

地塞米松对内毒素休克新生大鼠脑一氧化氮合成酶基因表达的调节

目的：探讨新生大鼠内毒素休克脑损伤时脑一氧化氮合成酶(NO6)三种亚型基因表达的变化及地塞米松(DEX)对其的调控作用。方法：在新生大鼠内毒素休克动物模型基础上，采用逆转录PCR及PCR技术，对脑组织中三型NOS mRNA及caspase—3 mRNA的表达进行半定量分析。结果：正常新生大鼠脑iNOS及eNOS mRNA无明显表达，nNOS mRNA、caspase—3 mRNA有一定程度表达。内毒素脂多糖(LPS)腹腔注射后2h，三种亚型NOS mRNA开始表达，于LPS6h达高峰，并持续至24h。caspase—3 mRNA于LPS腹腔注射后2h后表达逐渐增加，24h达高峰。DEX可抑制nNOS、iNOS及caspase—3 mRNA的表达，且以用药后2h最为明显，并持续至用药后24h。结论：内毒素休克脑损伤时，各型NOS均有表达，NO的产生是内毒素休克脑损伤时重要的病理生理机制之一。DEX通过抑制NOS、caspase—3 mRNA的表达部分实现其神经保护作用．     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.