以大肠埃希菌tolC蛋白为靶标的适配体的筛选及结构分析

目的:筛选大肠埃希菌外排泵tolC蛋白的核酸适配体.方法:重组表达大肠埃希菌外排泵外膜蛋白tolC,利用指数富集的配体系统进化技术(stematic evolution of ligand by exponential enrichment,SELEX)从单链DNA文库中筛选出一组能够特异性与其结合的核酸适配体.利用FITC荧光素标记技术检测每轮单链DNA文库与靶蛋白的亲和力,直至亲和力的上升趋于饱和,将最后一轮筛选产物克隆测序,并用DNAman软件分析其二级结构.结果:经过12轮筛选,单链DNA文库与靶标蛋白的亲和力趋向稳定,将第12轮筛选产物克隆测序,对获得的23个适配体进行分析.一级结构分析显示23个适配体并无共同的保守序列,但分别有3对和2对适配体序列完全一致.二级结构预测分析表明,茎环结构为适配体主要的结构形式,提示其可能是适配体与tolC蛋白蛋白特异性结合的基础.根据二级结构特点可将23个适配体分为4个家族,其中20号适配体与靶标蛋白亲和力最高.结论:成功利用SELEX技术筛选获得了特异结合tolC的高亲和力的核酸适配体,为大肠埃希菌耐药干预及机制研究奠定了基础.     

胃癌血清适配体的筛选及其鉴定

目的:以羧基化琼脂磁珠为筛选介质,利用RT-PCR和靶标替换消减SELEX技术从胃癌血清中筛选得到高特异性?强亲和力的适配体?方法:以离心超滤(50 000超滤管)法处理后的胃癌血清作为靶分子,羧基化琼脂磁珠为载体,先将寡核苷酸文库与反筛磁珠(结合正常血清)结合,取上清再与筛选磁珠(结合胃癌血清)结合,洗去未结合的寡核苷酸分子,对结合在磁珠上的寡核苷酸分子进行分离和扩增,λ酶切法制备ssDNA次级库,进行10轮筛选,将第10轮的文库扩增得到dsDNA,利用胶回收试剂盒回收纯化目的片段的dsDNA,并与pMDTM18-T载体连接,转化大肠杆菌DH5α感受态细胞,挑取阳性克隆,测序获得适配体序列,同时用流式细胞术测定胃癌血清适配体的Kd值?结果:经过10轮筛选后,得到20条与胃癌血清结合的适配体?结论:特异性检测表明,筛选得到的胃癌血清适配体与胃癌血清的结合解离常数均在纳摩尔级水平,其中5?7?16?17?18号适配体能高特异性?强亲和力结合胃癌血清,与正常人血清不结合?该研究为胃癌的早期诊断提供了实验基础?     

肝癌血清肿瘤标志物适配体的筛选及其亲和力和特异性的鉴定

目的：采用指数富集的配基系统进化技术（SELEX）筛选获得一组已知和未知肝癌血清肿瘤标志物的适配体,为肝癌的早期诊断提供新的便捷方法。方法：收集50例首次经诊断证实为原发性肝癌的患者血清,以及50例体检无异常的正常人血清,并分别等比例混合制备成肝癌混合血清和正常人混合血清,作为筛选的靶标分子,磁珠作为分离载体。先将磁珠?正常人血清复合物与ssDNA文库结合,取上清再与磁珠?肝癌血清结合,经过洗脱、分离与肝癌血清结合的特异性ssDNA,并进行扩增,链霉亲和素磁珠法制备次级ssDNA,进行9轮筛选,将9轮筛选获得的饱和文库与PMD18?T载体连接进行转化、挑选单克隆送上海生工进行测序,同时利用流式细胞术测定肝癌血清肿瘤标志物适配体的亲和力（Kd值）。结果：经9轮筛选,成功分离出200个核酸序列,其中序列不同的有10个。特异性检测表明,筛选得到的肝癌血清肿瘤标志物适配体与肝癌血清的结合解离常数均在纳摩尔级水平,其中Seq?1、Seq?16、Seq?17、Seq?56、Seq?72号适配体能高特异性结合肝癌血清,与正常人血清不结合,再通过200例肝癌血清和200例正常人血清进一步鉴定5条肝癌血清肿瘤标志物适配体检出肝癌的阳性率,阳性检出率达91%以上。结论：利用随机单链寡核苷酸文库成功获得与肝癌患者血清特异性结合的适配体,所获得的适配体具有拮抗肝癌血清的能力,有可能为肝癌的早期诊断提供新的便捷方法。     

人重组S100A8蛋白适配体的筛选和结构分析

目的:筛选人重组S100A8蛋白特异性的适配体。方法:采用配体指数级富集系统进化(systematic evolu-tion of ligand by exponential enrichment,SELEX)技术,以人重组S100A8蛋白为靶蛋白,以微孔板为筛选介质,从体外合成的随机单链DNA文库中筛选其适配体。利用生物素-链霉亲和素-辣根过氧化物酶系统检测每轮单链DNA文库与靶蛋白的亲和力直至亲和力的上升趋于饱和,将最后一轮筛选产物克隆测序,并进行生物信息学分析。结果:经过11轮筛选,单链DNA文库与人重组S100A8蛋白的亲和力趋向稳定,将第11轮筛选产物克隆测序,对获得的30个适配体进行分析。一级结构分析显示30个适配体并无共同的保守序列,但有3对适配体序列完全一致。二级结构预测分析表明,茎环和口袋结构为主要的结构形式,提示其可能是适配体与人重组S100A8蛋白特异性结合的基础。根据茎环和口袋的相对比例可将30个适配体分为4组,其中第1组中35号适配体与人重组S100A8蛋白亲和力最高。结论:获得了与人重组S100A8蛋白特异性结合的适配体群,为后续适配体的应用研究以及S100A8蛋白功能的研究奠定了基础。     

重组 PBP2a 转肽酶区蛋白核酸适配体的筛选及鉴定

目的：筛选和鉴定重组青霉素结合蛋白2a（PBP2a）转肽酶区蛋白的核酸适配体。方法利用指数富集的配体系统进化技术（SELEX）,以重组 PBP2a 转肽酶区蛋白为靶标,从单链 DNA 文库中筛选能与之特异性结合的核酸适配体。筛选产物克隆测序后,对其进行结构分析和特性鉴定。结果经过11轮筛选,单链 DNA 文库与靶标蛋白的亲和力趋向稳定,将第8、10轮筛选产物克隆测序,对获得的40个适配体进行分析,一级结构分析显示40个适配体并无共同的保守序列,二级结构预测分析表明,其可分为3个家族。其中13号适配体与重组 PBP2a 蛋白亲和力最高,并能与之特异性结合。结论利用 SELEX 技术成功筛选出特异性结合重组 PBP2a 转肽酶区蛋白的核酸适配体,为探索耐甲氧西林金黄色葡萄球菌感染的诊疗新途径奠定基础。     

人重组S100A8蛋白适配体的筛选和结构分析

目的：筛选人重组S100A8蛋白特异性的适配体。方法：采用配体指数级富集系统进化（systematic evolution of ligand by exponential enrichment,SELEX）技术,以人重组S100A8蛋白为靶蛋白,以微孔板为筛选介质,从体外合成的随机单链DNA文库中筛选其适配体。利用生物素-链霉亲和素-辣根过氧化物酶系统检测每轮单链DNA文库与靶蛋白的亲和力直至亲和力的上升趋于饱和,将最后一轮筛选产物克隆测序,并进行生物信息学分析。结果：经过11轮筛选,单链DNA文库与人重组S100A8蛋白的亲和力趋向稳定,将第11轮筛选产物克隆测序,对获得的30个适配体进行分析。一级结构分析显示30个适配体并无共同的保守序列,但有3对适配体序列完全一致。二级结构预测分析表明,茎环和口袋结构为主要的结构形式,提示其可能是适配体与人重组S100A8蛋白特异性结合的基础。根据茎环和口袋的相对比例可将30个适配体分为4组,其中第1组中35号适配体与人重组S100A8蛋白亲和力最高。结论：获得了与人重组S100A8蛋白特异性结合的适配体群,为后续适配体的应用研究以及S100A8蛋白功能的研究奠定了基础。     

琼脂磁珠消减SELEX技术筛选HIV gp41抗原适配体

目的 通过消减SELEX技术筛选得到高特异性、强亲和力的HIV gp41抗原适配体,为HIV的早期诊断提供了新的检测技术.方法 以琼脂磁珠为载体,HIV gp41抗原为靶标分子,利用消减SELEX技术和实时定量-PCR技术,筛选得到HIV gp41抗原适配体.结果 通过6轮筛选,筛选得到的次级ssDNA库通过PCR扩增得到dsDNA,dsDNA与pMDTM 18载体链接,进行克隆、测序,获得4条HIV gp41抗原适配体.获得的4条适配体亲和力(Kd)均在纳摩尔水平,其中15号适配体的亲和力最强,特异性检测表明筛选得到的适配体几乎只与HIV gp41抗原结合,不与其他非特异性蛋白结合.结论 利用随机单链寡核苷酸文库成功获得与HIV gp41抗原特异性结合的适配体,所获得的适配体具有拮抗HIV gp41抗原的能力.     

肝癌血清肿瘤标志物适配体的筛选及活性鉴定

目的:肝癌血清中含有已知和未知的标志物,可以作为SELEX筛选的靶标,获得一组已知和未知肝癌血清标志物的适配体,为肝癌早期诊断提供新的分子生物学检测方法。方法：收集50例经诊断证实为原发性肝癌患者的血清,以及50例体检无异常的正常人血清,并分别等比例混合制备成肝癌混合血清和正常人混合血清,作为筛选的靶标分子,磁珠作为分离载体。先将磁珠-正常人血清复合物与ssDNA文库结合,取上清再与磁珠-肝癌血清结合,经过洗脱、分离与肝癌血清结合的特异性ssDNA,并进行扩增,链霉亲和素磁珠法制备次级ssDNA,进行9轮筛选,将9轮筛选获得的饱和文库与PMD18-T载体连接进行转化、挑选单克隆团送上海生工进行测序,同时利用流式细胞术测定肝癌血清肿瘤标志物适配体的亲和力(Kd值)。结果：经9轮筛选,成功分离出200个核酸序列,其中序列不同的有10个。结论：特异性检测表明,筛选得到的肝癌血清肿瘤标志物适配体与肝癌血清的结合解离常数均在纳摩尔级水平,其中Seq-1、Seq-16、Seq-17、Seq-56、Seq-72号适配体能高特异性结合肝癌血清,与正常人血清不结合,再通过200例肝癌血清和200例正常人血清进一步鉴定5条肝癌血清肿瘤标志物适配体检出肝癌的阳性率,阳性检出率达91%以上,为肝癌的早期诊断提供了新方法。     

H22肿瘤细胞aptamer的筛选与结构分析

目的：获得与H22肿瘤细胞特异性结合的aptamer。方法：利用SELEX技术,以小鼠DC细胞为反筛选细胞,以H22肿瘤细胞为靶细胞,从体外合成的80bp随机单链DNA文库中筛选出能与H22肿瘤细胞特异结合的aptamer。采用荧光标记引物法检测aptamer与H22肿瘤细胞的亲和力：所获得的aptamer采用MACAWv2．05软件进行aptamer序列的一级结构同源序列比较,用DNASISv2．5软件计算分析序列最低二级结构能量值,获得其二级结构模拟图。结果：本实验设计的文库序列：5’-CGTCGCTGCACATTCCG—N46-CGCACAGCTGGGAGTAC-3’具有较高的扩增效率,适合于aptamer与以细胞为靶物质的筛选。经过11轮循环筛选,随机单链DNA文库与靶细胞结合的荧光强度从1％上升到59％,结合曲线进入平台期,表明结合已基本处于稳定状态,因此可以判断aptamer与靶细胞的结合基本已经处于饱和状态：对所获得的32个aptamer进行测序,然后进行一级结构和二级结构分析。一级结构分析获得5个保守序列：AGGGA、AGAAGG、GTGAXAA、ATAGT、CAAGG,其余10个aptamer无同源序列。二级结构分析表明,aptamer形成的茎环、凸环结构可能是与H22肿瘤细胞特异性结合的结构基础。亲和力检测结果表明第24号aptamer具有最强的亲和力。结论：利用随机单链寡核苷酸文库成功获得与H22肿瘤细胞特异结合的aptamer,其一级和二级结构与亲和力密切相关。     

SELEX技术筛选HCV非结构蛋白NS3的DNA适配子

目的 用配基指数富集的系统进化技术(SELEX)技术筛选丙型肝炎病毒(HCV)非结构蛋白NS3的DNA适配子.方法 构建随机ssDNA文库,两端为固定序列,中间为39 nt的随机序列,总长度为85 nt,将NS3蛋白包被在96孔板上,ssDNA与NS3蛋白进行亲和反应,洗去未结合的ssDNA,洗脱回收与NS3蛋白结合的DNA,PCR扩增后,磁珠法分离成单链.重复以上筛选步骤,直至筛选出高特异性和亲和力的核酸适配子.将核酸适配子连接质粒载体克隆转人大肠杆菌,挑选阳性克隆提取质粒外送到公司进行测序.结果 通过8轮筛选,成功筛选出DNA适配子,并获得其中6条克隆的DNA一级序列.6条适配子序列长度相同,均为85 nt,两端固定序列与设计相符,中间为随机序列,随机序列无共同保守系列.所有寡核苷酸适配子的二级结构以茎环和凸环为主.结论 以NS3蛋白作为药物研究的靶点,结合SELEX筛选技术研究其核酸适配子,具有巨大的潜力和研究价值.     

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.