蛋白酶体功能障碍对多巴胺能PC12细胞的特异性损伤

目的探讨蛋白酶体抑制剂lactacystin对多巴胺能PC12细胞的特异性损伤.方法不同浓度的lactacystin(1、5、10、15和20μmoL/L)分别处理多巴胺能PC12细胞和胶质瘤U251细胞24 h,MTT法检测细胞活力;50 μmol/L的lactacystin处理U251细胞24 h,MTT法检测细胞活力;10、20 μmol/L lactacystin处理PC12细胞,Western Blot检测细胞内多泛素化蛋白含量;单胺氧化酶B抑制剂selegiline(500μmol/L)和特异性酪氨酸羟化酶抑制剂α-MT(1 mmol/L)提前4 h预处理PC12细胞,再与10 μmol/Llactacystin共同作用24 h,MTT法检测细胞活力,Western Blot检测多泛素化蛋白含量.结果Lactacystin呈剂量依赖性损伤多巴胺能PC12细胞,其对胶质瘤U251细胞无毒性作用,而且其毒性与细胞内多泛素化蛋白生成增加相关.用以增加细胞内多巴胺含量的selegiline和用以减少细胞内多巴胺含量的α-MT都导致lactacystin毒性增强.结论蛋白酶体功能障碍特异性损伤多巴胺能细胞,而其特征性的神经递质多巴胺在其易感性中的作用复杂.     

蛋白酶体抑制剂lactacystin诱导PC12细胞凋亡以及半胱氨酸蛋白水解酶3活化的实验研究

目的探讨蛋白酶体功能缺陷是否能诱导多巴胺能细胞发生凋亡及其可能的作用机理。方法应用高选择性的蛋白酶体抑制剂lactacystin(0、1μmol/L、5μmol/l或10μmol/L)处理大鼠嗜铬细胞瘤PC12细胞株24h,四甲基偶氮唑蓝(MTT)比色法检测细胞活力的改变。10μmol/Llactacystin作用24h后,Hoechst荧光染色观察凋亡细胞的核形态学改变,流式细胞仪测定细胞凋亡百分率。用Western印迹法检测10μmol/Llactacystin作用0、24或48h时PC12细胞内半胱氨酸蛋白水解酶3活化片段的动态变化。结果5μmol/L或10μmol/Llactacystin作用24h后,PC12细胞的活力(87%±6%和26%±6%)显著低于对照组(P<0.05)。Hoechst荧光染色显示10μmol/Llactacystin作用24h后部分细胞呈现凋亡细胞核形态学改变;流式细胞仪证实31.4%±4.0%的PC12细胞发生了凋亡,与对照组(7.5%±0.8%)比较差异有统计学意义(P<0.01)。Western印迹法显示对照组细胞内仅有极少量的半胱氨酸蛋白水解酶3活化片段表达;10μmol/Llactacystin作用48h的PC12细胞内半胱氨酸蛋白水解酶3活化片段的表达明显较多。结论蛋白酶体抑制剂lactacystin能诱导多巴胺能细胞PC12发生凋亡,半胱氨酸蛋白水解酶3蛋白酶的激活可能参与lactacystin诱导的PC12细胞凋亡过程;蛋白酶体的功能缺陷在帕金森病发病机制中可能发挥重要作用。     

蛋白酶体抑制剂对中脑脑片的毒性作用及其机制研究

目的:观察蛋白酶体抑制剂对体外培养的大鼠中脑脑片黑质多巴胺(DA)能神经元的早期毒性作用,利用这种新型的组织模型探讨蛋白酶体功能异常在帕金森(PD)发病中的作用。方法:制备Wistar大鼠体外中脑黑质脑片的长期培养体系,加入蛋白酶体抑制剂lactacystin(0.1,0.5,1.0,5.0μmol/L)作用24 h后,测定培养基中乳酸脱氢酶(LDH)活力水平观察脑片活力。TH免疫组化观察黑质细胞变性缺失,α-synuclein免疫组化观察α-synuclein的表达,TUNEL法检测多巴胺能神经元凋亡。结果:经0.1,0.5,1.0和5.0μmol/L浓度的lactacystin作用24 h后,脑片黑质部位TH阳性神经元数量减少,α-synu-clein表达率增加,凋亡检测显示,5.0μmol/L的lactacystin组部分黑质细胞出现TUNEL染色阳性。结论:蛋白酶体抑制剂lactacystin对脑片中DA能神经元具有毒性作用,能诱导黑质细胞凋亡,其作用具有浓度依赖性。蛋白酶体功能缺陷在帕金森病发病机制中可能发挥重要作用。     

丙炔苯丙胺对多巴胺能神经细胞的保护机制

目的探讨丙炔苯丙胺对多巴胺能神经细胞的保护机制。方法采用神经生长因子(NGF)将大鼠嗜铬细胞瘤细胞(PC12细胞)诱导分化成多巴胺能神经元的细胞模型,经鱼藤酮处理后给予不同浓度的丙炔苯丙胺,观察细胞形态的改变,采用四甲基偶氮唑盐(MTT)法检测细胞活性及代谢状态,2',7'-二氯荧光黄双乙酸盐(DCFH-DA)检测细胞内活性氧(ROS)、还原型谷胱甘肽((GSH)含量及蛋白酶体功能。结果与鱼藤酮组比较, 50μmol／L浓度的丙炔苯丙胺作用24 h后,细胞恢复活力,570 nm处光密度值为0．39±0．01。与未处理组比较,鱼藤酮组ROS水平显著升高(P<0．01),丙炔苯丙胺组较鱼藤酮组显著降低(P<0．01)。鱼藤酮组的GSH含量为(104.76±0．88)μmol／L,显著低于丙炔苯丙胺组的(130．21±0．84)μmol／L(P<0．01)。与未处理组比较,鱼藤酮组的细胞蛋白酶体活性明显降低(P<0．01),丙炔苯丙胺组蛋白酶体活性恢复。结论丙炔苯丙胺能够明显减少鱼藤酮诱导的PC12细胞死亡,其机制可能是抑制氧化应激及保护蛋白酶体。     

去甲斑蝥素调节Hippo/YAP信号通路对U251胶质瘤细胞恶性生物学行为的影响

目的 探究去甲斑蝥素（NCTD）对U251胶质瘤细胞恶性生物学行为的影响及分子机制。方法 用不同浓度的NCTD处理U251细胞后,MTT法检测NCTD对U251细胞的细胞毒性。体外培养U251细胞,分为正常对照（NC）组、MST1/2抑制剂XMU-MP-1组（5μmol/L）、25μmol/L NCTD组、50μmol/L NCTD组、100μmol/L NCTD组、NCTD+XMU-MP-1组（100μmol/L NCTD+5μmol/L XMU-MP-1）。给予相应的干预后,细胞克隆形成实验检测细胞增殖活力,流式细胞术检测细胞凋亡,Transwell小室实验检测细胞迁移、侵袭能力;Western blot检测Hippo/Yes相关蛋白（YAP）和Wnt/β-连环蛋白（β-catenin）信号通路相关蛋白[哺乳动物Ste-20样激酶（MST）1/2、p-MST1/2、大型肿瘤抑制因子（LATS）1/2、p-LATS1/2、YAP、p-YAP、糖原合酶激酶-3β(GSK-3β)、p-GSK-3β、β-catenin]的表达。结果 与NC组相比,25、50、100、200μmol/L N...     

蛋白酶体抑制剂MG132对PC12细胞的增殖抑制作用

目的:了解蛋白酶体抑制剂MG132对多巴胺能细胞PC12增殖和凋亡的影响. 方法:用不同浓度的MG132处理PC12细胞3 d,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)微量比色法检测MG132对PC12细胞增殖的影响,通过姬姆萨染色法和流式细胞术检测MG132对PC12细胞凋亡的影响. 结果:在作用时间相同(3 d)的条件下,随着MG132浓度的增高(0, 1, 2.5, 5, 10, 20 μmol/L),对PC12细胞增殖的抑制作用逐渐增强. 当MG132浓度达到2.5 μmol/L时,对PC12细胞的抑制率超过40%, IC50＝3.78 μmol/L. 同时,MG132能够明显诱导PC12细胞凋亡:姬姆萨染色显示,浓度为2.5 μmol/L的MG132作用3 d,细胞凋亡率为24.7%,而对照为1.3%;流式细胞术分析结果表明,浓度为2.5 μmol/L的MG132作用3 d,细胞凋亡率为25.6%,而对照为0%. 结论:蛋白酶体抑制剂MG132能够抑制多巴胺能细胞PC12的增殖并诱导细胞凋亡,蛋白酶体活性的丧失可能影响到多巴胺能细胞的生存.     

环境毒素导致多巴胺能神经元蛋白水解应激的实验研究

目的探讨环境毒素诱发蛋白水解应激在多巴胺能神经元变性死亡中的作用。方法6-羟多巴（6-OHDA）、1-甲基-4-苯基吡啶离子（MPP^＋）、鱼藤酮作用于神经生长因子诱导的PC12细胞,MTT法检测各种药物的神经毒性作用,应用共聚焦免疫荧光双标染色观察药物处理后细胞内仪．突触核蛋白及泛素化蛋白的表达及聚集。荧光分光光度计测量各组细胞蛋白酶体中各蛋白水解酶活性的变化。结果3种神经诱变剂处理PC12细胞后,细胞活力呈浓度依赖性下降,其中6-OHDA 100μmol／L、MPP^＋ 75μmol／L、鱼藤酮20nmol／L作用24h使细胞活力分别下降52％、44％、40％（P〈0．01）。共聚焦显微镜观察显示药物处理后细胞胞质内α-突触核蛋白及泛素表达上调,并形成蛋白颗粒物。酶活性测定提示MPP^＋75μmol／L、鱼藤酮20nmol／L处理使胰蛋白酶,糜蛋白酶及PgH水解酶活性明显下降（P〈0．05）,而6-OHDA 100μmol／L处理使细胞蛋白酶水解能力轻度降低,差异无统计学意义（P〉0．05）,6-OHDA 200μmol／L则诱导3种蛋白酶活性进一步下降,荧光指数分别为7．23±0．58,79．60±2．73和4．23±0．49（正常组为13．90±1．75,99．30±5．23和6．90±0．60,P〈0．01）。结论神经诱变剂可导致多巴胺能神经元泛素蛋白酶体功能失调及仪．突触核蛋白和泛素化蛋白的积聚,诱发蛋白水解应激异常状态。     

Aβ1-42诱导U251细胞趋化因子RANTES的研究

目的 探讨β淀粉样蛋白1-42(Aβ1-42)体外诱导人神经胶质瘤细胞(U251)细胞炎性反应的分子学机制. 方法 U251细胞经常规去血清培养,采用终浓度为0.2～4.0 μmol/L Aβ1-42处理U251细胞24 h,以四唑盐比色(MTT)法测定细胞存活率;2 μmol/L Aβ1-42处理U251细胞12、24、36、48 h后,采用硝酸还原酶法检测一氧化氮(NO)含量;采用双抗体夹心ELISA法测定细胞RANTES蛋白表达水平;免疫细胞化学染色法检测NF-κB和STAT1的表达.结果 随着Aβ1-42剂量的增加,细胞的存活率降低(P<0.05);用2 μmol/L Aβ1-42处理U251细胞12、24、36、48 h,结果显示NO的生成于24 h达高峰,此后逐渐下降;同浓度Aβ1-42处理U251细胞24 h后,RANTES的表达增加4倍(P<0.01);2 μmol/L Aβ1-42刺激U251细胞24 h后细胞内NF-κB P65和STAT1从胞浆移至胞核且表达明显. 结论 Aβ1-42降低体外培养的U251细胞存活率,诱导NO和RANTES的释放,提示Aβ1-42诱导的U251细胞趋化因子RANTES产生可能与NF-κB和STAT1的活化有关.     

姜黄素对胶质瘤细胞ATP酶的影响

目的:探讨姜黄素(curcumin)对人脑胶质瘤U-251细胞ATP酶的影响及诱导胶质瘤细胞凋亡的机制.方法:分别用20、40、80和100μmol/l,姜黄素处理U-251细胞24 h.用MTT法测定姜黄素对U-251细胞增殖的抑制作用;流式细胞仪检测细胞凋亡,比色法测定ATP酶活力.结果:姜黄素抑制U251细胞的增殖;诱导U25I细胞凋亡;姜黄素明显使细胞质膜ATP酶活力明显降低.结论:姜黄素通过抑制细胞质膜ATP酶活性可诱导胶质瘤细胞凋亡和抑制增殖.     

蝙蝠葛苏林碱调控Akt/mTOR信号通路对胶质瘤细胞增殖及自噬的影响

目的 探讨蝙蝠葛苏林碱（DAS）对胶质瘤U251和U87细胞生长与增殖的影响及其分子机制。方法用2.5、5.0和10.0μmol/L DAS(DAS组)、10.0μmol/L替莫唑胺（TMZ组）和空白对照（对照组）处理U251和U87细胞。采用CCK-8法检测DAS对细胞活力的影响,细胞集落形成和Edu实验检测DAS对细胞增殖的影响,流式细胞术检测DAS对细胞凋亡的影响,透射电镜观察自噬小体的形成,Western blotting检测DAS对胶质瘤细胞凋亡、自噬和Akt/mTOR信号通路相关蛋白表达的影响。结果 CCK-8结果显示,与对照组比较,不同浓度DAS组胶质瘤细胞活力逐渐降低（P <0.05）,DAS组细胞活力低于TMZ组（P <0.05）。胶质瘤U251和U87细胞IC50分别为5.11μmol/L和6.35μmol/L。Edu和克隆结果显示,DAS能抑制胶质瘤细胞增殖,且随着药物浓度升高细胞增殖逐渐减少（P <0.05）。流式细胞术结果显示,DAS能诱导胶质瘤细胞凋亡,且随着药物浓度升高细胞凋亡增加（P <0.05）。DAS能增加胶质瘤细胞Bax、L...     

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.