前列腺精子结合蛋白的二维凝胶电泳及质谱分析

【目的】应用精子膜蛋白亲和层析柱分离金黄地鼠前列腺精子结合蛋白，并用二维电泳和质谱对金黄地鼠前列腺精子膜结合蛋白进行初步分析。【方法】应用附睾精子膜亲和层析柱纯化前列腺精子结合蛋白，采用二维凝胶电泳对纯化的前列腺精子膜结合蛋白进行蛋白分离和图像分析，并结合基质辅助激光解吸附电离飞行时间质谱分析及数据库搜索从而鉴定部分蛋白质斑点；RT—PCR检测已鉴定蛋白在前列腺及未受精卵子中的mRNA的表达。【结果】附睾精子膜蛋白亲和层析柱可纯化出前列腺结合蛋白，前列腺附睾精子膜蛋白结合蛋白的二维凝胶电泳蛋白图谱。分离的蛋白质斑点为30个，质谱鉴定出2个蛋白点，一为热休克蛋白105，另一羰基还原酶（NADPH）-3。前列腺和未受精卵中均表达热休克蛋白105和羰基还原酶-3的mRNA。【结论】自前列腺分泌物中分离出精子膜结合蛋白，金黄地鼠前列腺中精子结合蛋白经双向电泳及飞行质谱分析鉴定出蛋白热休克蛋白和羰基还原酶。     

前列腺精子结合蛋白的二维凝胶电泳及质谱分析

 【目的】应用精子膜蛋白亲和层析柱分离金黄地鼠前列腺精子结合蛋白，并用二维电泳和质谱对金黄地鼠前列腺精子膜结合蛋白进行初步分析。【方法】应用附睾精子膜亲和层析柱纯化前列腺精子结合蛋白，采用二维凝胶电泳对纯化的前列腺精子膜结合蛋白进行蛋白分离和图像分析，并结合基质辅助激光解吸附电离飞行时间质谱分析及数据库搜索从而鉴定部分蛋白质斑点；RT—PCR检测已鉴定蛋白在前列腺及未受精卵子中的mRNA的表达。【结果】附睾精子膜蛋白亲和层析柱可纯化出前列腺结合蛋白，前列腺附睾精子膜蛋白结合蛋白的二维凝胶电泳蛋白图谱。分离的蛋白质斑点为30个，质谱鉴定出2个蛋白点，一为热休克蛋白105，另一羰基还原酶（NADPH）-3。前列腺和未受精卵中均表达热休克蛋白105和羰基还原酶-3的mRNA。【结论】自前列腺分泌物中分离出精子膜结合蛋白，金黄地鼠前列腺中精子结合蛋白经双向电泳及飞行质谱分析鉴定出蛋白热休克蛋白和羰基还原酶。     

大鼠青光眼慢性高眼压视网膜组织的二维凝胶电泳分析

目的应用二维凝胶电泳对SD大鼠青光眼慢性高眼压视网膜组织的蛋白进行初步分析.方法分离大鼠青光眼慢性高眼压视网膜组织,以对侧眼视网膜组织为对照,经样本初处理后,进行二维凝胶电泳和凝胶银染色,然后分析电泳图谱,观察蛋白的分离效果,对比、分析其表达蛋白质点的差异,寻找SD大鼠视网膜中与慢性高眼压相关的蛋白质.结果用化学裂解法结合超声波粉碎,可以裂解视网膜神经组织,使其大部分蛋白质得到溶解,成功建立对大鼠青光眼慢性高眼压眼及对照眼视网膜组织进行样本处理、二维凝胶电泳的基本方法和条件,得到两组视网膜组织的二维凝胶电泳图谱.各组织蛋白在小胶上均能显示120～290个左右的斑点.两组视网膜组织的凝胶图谱呈现出9个斑点的差异.结论二维凝胶电泳可以有效分离、显示视网膜特异表达蛋白质.通过双向电泳,我们发现SD大鼠视网膜蛋白质表达与对照眼相比有质和量的差异.     

小鼠肝细胞核磷酸化蛋白质组的提取和双向电泳分离

目的提取小鼠肝细胞核磷酸化蛋白并利用双向凝胶电泳分离小鼠肝细胞核磷酸化蛋白。方法提取小鼠肝细胞核蛋白并利用金属磷酸盐亲和层析树脂纯化出磷酸化蛋白后,进行一维等电聚焦分离和二维聚丙烯酰胺凝胶电泳分离,并挑选其中一个蛋白斑点进行质谱分析。结果成功提取了肝细胞核磷酸化蛋白,通过双向凝胶电泳分离技术成功建立了小鼠肝细胞核磷酸化蛋白质组图谱。质谱鉴定结果证实被挑选蛋白斑点是小鼠核磷酸化蛋白。结论磷酸化蛋白纯化技术结合二维凝胶电泳分离技术是研究肝细胞核磷酸化蛋白质组的有效方法,为进一步全面研究和鉴定小鼠肝细胞核磷酸化蛋白的功能打下了基础。     

运用蛋白质组学技术研究结肠黏膜、腺瘤及腺癌组织蛋白的差异表达

【目的】通过比较结肠正常黏膜、腺瘤、腺癌组织之间的蛋白质组表达差异，寻找结肠腺癌相关的蛋白质，筛选结肠癌早期诊断的分子标志物。【方法】运用比较蛋白质组学技术，对8例结肠癌患者（伴有腺瘤）的正常黏膜、腺瘤、腺癌组织提取的总蛋白进行双向电泳（2-D），分析凝胶图片选择两两间差异表达超过2倍的蛋白质进行MALDI-TOF／TOF质谱分析和生物学信息分析。【结果】成功建立结肠正常粘膜、腺瘤、腺癌的双向凝胶电泳图谱，在凝胶电泳图谱中平均蛋白质斑点数分别为3066、2986和3289,其中两两间表达差异超过2倍的斑点共有31个，质谱分析和数据库检索共鉴定出18种蛋白质，包括keratin 8、S100A6、protein disulfide isomerase等。从功能分析，这些差异蛋白与癌细胞的发生、增殖、分化、转移等相关。【结论】比较蛋白质组学能较好地提示结肠腺癌与腺瘤及正常组织间的蛋白质表达差异,而对相关差异表达蛋白的研究有可能为结肠癌的早期诊断和治疗提供新的分子靶向。     

正常人尿蛋白质组学研究

目的 初步建立正常人尿液蛋白质双向电泳图谱,分析正常尿蛋白基本组成,为疾病下尿液蛋白差异研究作参考。方法 用丙酮沉淀法对4例正常人尿液中蛋白质进行浓缩,用固相pH梯度双向凝胶电泳技术分离蛋白样品,凝胶考马斯亮蓝染色,GS-800 Calibrated Densitometer凝胶成像系统获取图像,PDQuest2D分析软件对图像进行背景消减、斑点检测、匹配、斑点位置重复性的系统分析,寻找表达蛋白。应用基质辅助激光解析电离飞行时间质谱(MALIDI-TOF)得到相应的肽指纹图谱,搜索相关数据库对蛋白质点进行鉴定。结果 对染色明显且独立的30个蛋白斑点进行鉴定,并对结果进行数据库检索,共有28个点获得有效匹配,包括各种血清白蛋白、免疫分子以及补体分子、载脂与转运蛋白、肽酶S1家族、细胞结构蛋白、蛋白酶抑制因子等,鉴定出的蛋白质大部分PI在4-7之间。结论 应用蛋白质组学方法分析正常人尿液蛋白质组成成分可行,初步建立了正常人尿液28个蛋白质图谱,为分析疾病状态下尿液蛋白质成分的改变提供了基础资料。     

适于人表皮组织蛋白质组分析的双向电泳技术

 【目的】 建立适于人表皮组织的双向电泳技术,并应用质谱技术对人表皮组织蛋白质组进行初步分析?【方法】 利用组织匀浆法制备人表皮组织总蛋白?固相pH 梯度胶条进行双向电泳?PDQuest 7.4软件比较和分析电泳图谱,利用质谱技术鉴定部分蛋白斑点?【结果】 成功建立了适用于人表皮组织蛋白质组分析的双向电泳技术,获得和比较了人表皮组织双向电泳四种染色图谱?人表皮组织的蛋白质在pH5～8范围内分布均匀,分子量集中在15～100 ku之间?银染图谱获得的蛋白斑点数为586±14;对质谱兼容的几种染色方法进行比较分析,结果表明Neuhoff 胶体考染更适合于人表皮组织蛋白质的双向电泳分析,其在上样量为350 μg时蛋白斑点数达394±9,匹配率为85.53%?利用基质辅助激光解析电离飞行时间质谱技术成功获得了Neuhoff 胶体考染的两个蛋白斑点的肽质量指纹图谱,并鉴定为Caspase 14 前体和27 ku热激蛋白1?【结论】建立的表皮组织双向电泳技术及其图谱可为皮肤蛋白质组学研究提供参考,为人皮肤蛋白质组学平台的构建奠定良好基础?     

血清蛋白双向凝胶电泳-质谱分析诊断早期肺腺癌的临床研究

目的分析肺腺癌患者与正常人血清双向凝胶电泳的差异蛋白质,从而寻找肺腺癌早期特异性的诊断指标。方法用固相IPG梯度双向凝胶电泳分离肺腺癌患者及正常人血清总蛋白,用PDQuest凝胶图像软件分析,以识别差异蛋白质点。用基质辅助激光解吸电离飞行时间质谱仪（MALDI—TOF—MS）分析获得肽质量指纹图,然后利用Mascot查询软件搜寻数据库鉴定蛋白质。结果①获得了重复性较好的双向电泳银染图谱。②经PDQuest7．1软件匹配分析,共筛选出22个差异的蛋白点,对7个差异蛋白点进行胶内原位酶解-质谱指纹图分析,获得了7张肽质量指纹图谱,查询数据库鉴定出1个蛋白质-血清淀粉样蛋白A（SAA）。结论①固相pH梯度双向凝胶电泳分离血清总蛋白获得了重复性较好的双向电泳凝胶图谱。②肺腺癌患者血清和正常人血清的蛋白质组表达存在明显差异,这些差异蛋白点为进一步建立人肺癌的2-DE数据库奠定了基础。③SAA蛋白很可能是肺癌的一个潜在标志物。     

双向电泳-质谱法寻找类风湿关节炎疾病相关蛋白

目的建立正常人及类风湿关节炎(rheumatoid arthritis,RA)患者血浆蛋白质的双向凝胶电泳图谱,并分析其差异表达的蛋白质,寻找RA疾病相关蛋白,以阐明RA的发病机制.方法采用固相pH梯度(immobilized pH gradient,IPG)双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)分离正常人及类风湿关节炎患者血浆总蛋白质,凝胶经银染显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,对差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行鉴定.结果获得正常人及类风湿关节炎患者血浆蛋白双向凝胶电泳图谱,平均蛋白质点数分别为592和563,匹配率分别为89%和87%,通过比较分析,差异表达蛋白质点数为24,选取15个点进行质谱鉴定,成功鉴定7个蛋白质,其中6个蛋白质表达上调.结论两者间存在一些差异表达的蛋白质,为阐明RA的发病机制打下了坚实的基础.     

脾肾虚型重症肌无力患者血清双向电泳图谱的建立及分析

目的:建立脾肾虚型重症肌无力患者血清蛋白质双向电泳图谱,观察脾肾虚型重症肌无力患者与正常人血清蛋白质差异表达情况。方法:采用反复冻融法抽提血清蛋白质,以优化的双向电泳技术分离血清蛋白质,建立脾肾虚型重症肌无力患者与正常人血清蛋白质双向电泳图谱,并采用二维电泳图谱专用软件对所得结果进行图谱分析。采用基质辅助激光解吸电离-飞行时间质谱法对两组间比较所得目标蛋白质点进行质谱鉴定。结果:通过双向电泳,建立血清蛋白质双向电泳图谱,选取具有明显差异的蛋白点21个进行质谱鉴定,共鉴定出8种蛋白质。结论:通过蛋白质组技术快速建立脾肾虚型重症肌无力患者血清蛋白表达图谱及质谱分析,可为进一步研究其发病机制及治疗手段提供实验基础。     

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.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

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.     

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.