醋氨酚大鼠肝细胞损伤及其机制的研究

醋氨酚(AAP)引起大鼠肝细胞膜损伤发生在肝细胞还原型谷胱甘肽(GSH)明显下降之后,提示GSH下降是AAP损伤肝细胞的原因之一。同步测定培养大鼠肝细胞GSH和胞浆自由钙离子浓度([Ca~(2+)f]c)的结果证明:加10mmol/L AAP培养2.5小时可引起GSH显著降低,[Ca~(2+)f]c明显升高。二巯基苏糖醇(DTT)5mmol/L可使加AAP后,细胞内GSH仍维持较高水平,[Ca~(2+)f]c升高几乎被完全拮抗。首次直接证明AAP所致的[Ca~(2+)f]c升高与GSH等巯基物质被消耗有关。     

S-nitrosoglutathione诱导胸腺细胞凋亡的机制

目的 研究NO供体亚硝酰谷胱甘肽(GSNO)诱导胸腺细胞凋亡与p53表达的相互关系。方法 体外培养：BALB／C小鼠胸腺细胞，用地塞米松(DXM)作为阳性对照，再分别加不同剂量GSNO及NO抑制剂，孵育3h。用透射电镜观察胸腺细胞形态；用琼脂糖凝胶电泳检测DNA梯型带纹；采用末端脱氧核酸转移酶(TdT)介导的末端标记在流式细胞仪上检测胸腺细胞凋亡；用RT-PCR的方法分析抑癌基因p53的表达在此过程中的变化及作用。结果胸腺细胞在无巯基S-77培养液中孵育可产生自然凋亡，用不同处理后在透射电镜下可见各组早、中、晚期不同阶段的凋亡细胞的形态，琼脂糖电泳显示DXM组出现较多中、晚期凋亡的胸腺细胞，有明显的DNA梯形条纹，加不同剂量的NO和NO抑制剂组则较弱，NO浓度愈高则梯形条纹愈不明显，0．3mmol／L和0．6mmol／L GSNO呈早中期凋亡形态学变化，1．2mmol／LGSNO主要呈早期改变；PCR结果同DNA电泳结果基本一致；流式细胞仪检测各组的凋亡率也有相同的趋势，各组之间差异有显性。结论 胸腺细胞在无巯基培养液内孵育3h会产生自然凋亡，GSNO诱导胸腺细胞凋亡随浓度增加反而抑制胸腺细胞凋亡，NOS抑制剂可抑制凋亡，NO供体GSNO通过p53的表达来诱导细胞凋亡是NO引起细胞凋亡的可能机制之一。     

谷氨酰胺下调大鼠肝细胞诱导型一氧化氮合酶的表达

目的 利用原代培养的大鼠肝细胞,观察谷氨酰胺(Gln)在体外对白细胞介素-1β(IL-1β)刺激下一氧化氮合酶(iNOS)过度表达的影响.方法 原位胶原酶灌注法获取原代大鼠肝细胞,分别用生理盐水、IL-1β(1 nmol/L)以及IL-1β(1 nmol/L)联合不同浓度的Gln(2、5、10 mmol/L)刺激24 h,留取细胞和堵养液,采用生化法测定培养液一氧化氮(NO)的水平,采用实时定量逆转录多聚酶链反应(RT-PCR)法及蛋白质印迹法检测肝细胞内iNOS信使RNA和蛋白质水平,采用电泳迁移率转变试验检测肝细胞核内核因子κB(NF-κB)的活性.结果 IL-1β刺激后培养液NO的平均浓度为72.7 μmol/L明显高于生理盐水组41.7 μmol/L,肝细胞iNOS蛋白和mRNA水平分别增加了35%和90%,同时给予Gln能够抑制iNOS的过度活化减少NO的产生,并且这种抑制作用随着Gln浓度的增加而增强;IL-1β刺激后肝细胞核内NF-κB的结合活性增强约50%,Gln对NF-κB的活化没有抑制作用,在5、10 mmol/L浓度时反而对NF-κB有进一步的激活.结论 谷氨酰胺能够下调肝细胞在炎症应激诱导下iNOS基因的过度表达,减少NO的产生,这一作用不是通过NF-κB信号通路实现的.     

外源性NO在细胞水平对GluR6 S-亚硝基化的作用

目的观察外源性一氧化氮（NO）供体硝普钠（SNP）和亚硝基谷胱甘肽（GSNO）在细胞水平对GluR6 S-亚硝基化的影响。方法将本事构建的pEGFP—GluR6质粒转染HEK293细胞24h后,加不恫浓度外源性NO供体SNP和GSNO处理30min后收集蛋白,用生物素转换法检测GluR6S—亚硝基化水平。结果给了不同浓度SNP时GluR6的s一亚硝基化均增高,并存给予1mmol／LSNP时其亚硝基化水平达最高。另外,给予200μmoL／L GSNO时GluR6的S-亚硝基化也增高。结论在过表达GIuR6的HEK293细胞中,外源性NO供体SNP和GSNO均能使GluR6 S-亚硝基化水平增高。结果提示,在体外,外源性NO能直接诱导GluR6 S-亚硝基化。     

一氧化氮诱导对肝细胞内环境稳定的影响

目的研究肝细胞诱导性一氧化氮(NO)及外源性NO对肝细胞内环境稳定的影响。方法用内毒素及细胞因子诱导培养大鼠肝细胞内源性NO及应用外源NO供体硝普钠(SNP)与原代肝细胞作用,观察肝细胞丙二醛(MDA)、还原型谷胱苷肽(GSH)及细胞内游离钙的变化规律。结果内毒素+细胞因子作用肝细胞24h后,肝细胞MDA增高7.97倍及GSH降低57.9%,一氧化氮合酶(NOS)竞争性抑制剂NG-甲基-L-精氨酸使肝细胞MDA及GSH分别逆转43.5%和98.4%。异搏定显著抑制内源性NO合成及相关氧应激,A23187的作用不显著,而氯丙嗪和α-生育酚并不导致内毒素+细胞因子诱导肝细胞释放的NO下降。SNP作用肝细胞表现为可逆性增强氧化态,但外源NO显著抑制K+去极化及肝细胞生长素诱导动员的肝细胞游离钙瞬增。结论在内毒素及细胞因子导致肝细胞代谢和功能应激反应中,诱导NO、独立介导脂质过氧化和抗氧化能力下降,可能具有起始关键作用;NO抑制肝细胞内游离钙动员,可能通过负反馈调节与蛋白激酶C相关的NOS诱导而作为一种重要的肝细胞内伺服调控机制。     

艾纳香二氢黄酮对脂质过氧化损伤大鼠原代培养肝细胞的保护作用

研究 5种艾纳香二氢黄酮类化合物对过氧化损伤的原代培养肝细胞的保护作用。胶原酶灌流法分离大鼠肝细胞。以 CCl4或 Fe SO4 Cys损伤原代培养肝细胞 ,1× 10 - 4 和 1× 10 - 5 mol/ L 的 5种艾纳香二氢黄酮均能明显抑制受损伤细胞的转氨酶逸出、MDA产生及 GSH耗竭。其中以 2 ,5 -二羟基艾纳香二氢黄酮的作用最强 ,在 CCl4所致肝细胞损伤实验中 ,其抑制 MDA产生的 ED5 0 约为 2 .6× 10 - 6 mol/ L ,保护 GSH的 ED5 0 约为 1.0 8× 10 - 5 mol/ L ,抑制 AL T逸出细胞的 ED5 0 约为 9.39× 10 - 6 mol/ L     

一氧化氮诱导对肝细胞内环境稳定的影响

目的 研究肝细胞诱导性一氧化氮（NO）及外源性NO对肝细胞内环境稳定的影响。方法 用内毒素及细胞因子诱导培养大鼠肝细胞内源性NO及应用外源NO供体硝普钠（SNP）与原代肝细胞作用，观察肝细胞丙二醛（MDA），还原型从胱苷肽（GSH）及细胞内游离钙的变化规律。结果 内毒素 细胞因子作用肝细胞24h后，肝细胞MDA增高7.97倍及GSH降低57.9%，一氧化氮合酶（NOS）竞争性抑制剂，N^G-甲基-L-精氨酸使肝细胞MDA及GSH分别逆转43.5%和98.4%。异搏定显著抑制内源性NO合成及相关氧应激，A23187的作用不显著，而氯丙嗪和α-生育酚并不导致内毒素 细胞因子诱导肝细胞释放的NO下降，SNP作用肝细胞表现为可逆性增强氧化态，但外源NO显著抑制K^ 去极化及肝细胞生长素诱导动员的肝细胞游离钙瞬增，结论 在内毒素及细胞因子导致肝细胞代谢和功能应激反应中，诱导NO，独立介导脂质过氧化和抗氧化能力下降，可能具有起始关键作用；NO抑制肝细胞内游离钙动员，可能通过负反馈调节与蛋白激酶C相关的NOS诱导而作为一种重要的肝细胞内伺服调控机制。     

一氧化氮对白血病HL-60细胞作用的研究

目的 :探讨NO对髓系白血病细胞HL - 6 0的作用。方法 :以SNP为NO供体 ,采用MTT法检测细胞生长抑制率 ,光镜、电镜观察细胞形态学变化 ,DNA电泳、FCM检测凋亡率等方法。结果 :SNP在 0 .5～ 4 .0mmol/L浓度范围内能明显抑制HL - 6 0细胞的生长 ,此作用有浓度和时间依赖性 ;且在作用 12h后细胞出现凋亡 ,SNP2 .0mmol/L浓度作用 36h ,凋亡率达 6 8.71% ,DNA电泳出现梯形带。NO清除剂N -乙酰半胱氨酸能显著降低SNP的这种作用 (P <0 .0 1)。结论 :NO对HL - 6 0细胞有明显的生长抑制和诱导凋亡作用。     

外源性一氧化氮对精子运动能力的影响

目的 :探讨一氧化氮 (NO)对精子运动能力影响的机制 .方法 :采用NO供体硝普钠 (sodiumnitroprusside,SNP)与上游法获得优质精子共同体外孵育 ,在超高倍显微镜下观察不同时间和不同浓度对精子运动能力的影响 .用MTT法测定NO对精子细胞呼吸链的作用 .结果 :NO在低浓度下(SNP 5 0～ 1 0 0nmol·L -1 )明显促进精子运动 ,在中浓度下(SNP 1 5 0～ 2 0 0nmol·L -1 )对精子运动能力影响极微 ,高浓度下 (SNP >2 0 0nmol·L -1 )则对精子运动有显著的抑制作用 ,随孵育时间的延长 ,精子活率呈进行性下降趋势 .随SNP浓度的升高 ,精子细胞呼吸链活性进行性下降 .结论 :NO对精子运动能力的影响具有双重性和浓度依赖效应 ,对避孕及男性不育症的治疗有重要的参考价值     

血浆亚硝酸根及环磷酸鸟苷浓度预测早产的价值

目的　探讨血浆亚硝酸根 (NO2 - )及环磷酸鸟苷 (c GMP)浓度预测早产的价值。方法　采用示波极谱法和放射免疫法测定了 2 5 0例妊娠妇女 (2 10例正常妊娠妇女和 4 0例早产妇女 )血浆 NO2 - 和 c GMP浓度。结果　正常妊娠妇女从 2 8周至 36 + 6周 ,血浆 NO2 -和 c GMP浓度均逐渐增加。NO- 2 从 1.95 75± 0 .35 0 9增加至 2 .2 36 0±0 .3486μmol/ L ,c GMP从 31.16 2 5± 3.90 82增加至 4 2 .185 0± 11.3816μm ol/ L。血浆 NO2 - 浓度在 34～ 34+ 6 周达最高值 (2 .2 4 2 5± 0 .340 7μm ol/ L) ,血浆 c GMP浓度在 33～ 33+ 6周达最高值 (49.0 0 10± 8.6 719μm ol/ L)。早产妇女血浆 NO2 -和 c GMP浓度均低于相应孕周的正常妊娠妇女 (P<0 .0 1和 P<0 .0 5 )。结论　血浆 NO2 -和 c GMP浓度预测早产具有良好的可行性和应用前景     

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.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

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.