肝癌特异性结合肽的筛选及鉴定

目的探讨利用噬菌体随机肽库筛选肝癌细胞特异性结合多肽或肝癌细胞优势结合多肽的可行性。方法①利用噬菌体随机十二肽库，对人肝癌细胞系HepG2和人正常肝细胞系L02进行4轮差异筛选、富集，获得与肝癌细胞亲和的噬菌体克隆。②用酶联免疫吸附测定(ELISA)法测定其与肝癌细胞、正常肝细胞的亲和力，进一步筛选出高度亲和肝癌细胞的特异性阳性噬菌体克隆。比较其与其他肿瘤细胞的亲和力。③提取阳性噬菌体DNA并进行DNA序列测定，获得与肝癌细胞亲和的多肽序列。④用免疫荧光共聚焦成像进一步确定展示阳性多肽序列的噬菌体与肝癌细胞的结合部位。⑤根据筛选的多肽序列合成十六肽WH16，利用竞争抑制实验鉴定其与肝癌细胞的结合。结果筛选及ELISA结果示得到与肝癌细胞特异性结合并内化入肝癌细胞的噬菌体克隆，测序结果示56．67％的被检噬菌体展示相同的多肽序列FLLEPHLMDTSM，推测FLEP是肝癌细胞特异性结合肽的基序。免疫荧光共聚焦成像进一步确定展示序列FLLEPHLMDTSM的噬菌体与肝癌细胞特异性结合并内化入细胞内。竞争抑制实验示WH16能有效抑制展示序列FLLEPHLMDTSM的噬菌体克隆与肝癌细胞的结合。结论利用噬菌体随机肽库获得能与肝癌细胞特异性结合的多肽序列FLLEPHLMDTSM，据此合成的十六肽WH16与肝癌细胞有亲和力。     

利用噬菌体肽库筛选脂多糖的模拟表位

目的：利用噬菌体肽库获得脂多糖的模拟表位。方法：用脂多糖免疫大耳白兔获得抗血清，从中纯化抗脂多糖的多克隆抗体。以纯化的抗体IgG为筛选靶分子，对噬菌体随机十二肽库进行亲和筛选。经3轮筛选后，随机挑取25个克隆测序，并进行噬菌体ELISA结合实验及脂多糖竞争抑制实验，以确定阳性克隆。结果：获得12种序列，ELISA实验结果显示其中6个为阳性克隆，且经脂多糖竞争抑制实验进一步证实。结论：获得了6个脂多糖的模拟表位，为脂多糖抗原表位的研究及疫苗研究提供了线索。     

VEGF受体3高亲和噬菌体融合肽的筛选及亲和功能研究

目的:利用噬菌体展示技术,筛选VEGF受体3(VEGFR3)胞外区蛋白高亲和噬菌体融合肽,并测量其亲和常数。方法:用噬菌体展示固相结合法,经过4个循环的筛选,ELISA法鉴定噬菌体单克隆的亲和性,测定亲和力高的阳性噬菌体融合蛋白的DNA序列,竞争法ELISA检测优势克隆与靶蛋白的特异性,并通过非竞争性ELISA固项法,得到融合肽与VEGFR3的抗原抗体结合反应曲线,计算融合肽的亲和常数。结果:4轮亲和筛选,噬菌体出现良好的富集,8个阳性克隆中,经测序有4个克隆序列相同(phage-WHGSLKQNLWWY);竞争性ELISA显示噬菌体融合肽与VEGFR3具有良好的亲合性,并能被VEGF-D分子抑制,融合肽的亲和常数为(1.505±0.184)×107M-1。结论:噬菌体12肽库筛选的VEGFR3高亲和融合肽(phage-WHGSLKQNLWWY)可为多肽分子的改造及靶向药物的开发提供依据。     

应用噬菌体表面展示技术筛选丙型肝炎病毒核心蛋白抗原模拟表位

为筛选丙型肝炎病毒（HCV）核心蛋白（core)特异性噬菌体12肽模拟表位，应用噬菌体表面展示技术，以抗－HCV核心蛋白抗原的单克隆抗体作为固相筛选分子，对人工化学合成的噬菌体随机12肽库进行5轮“吸附－洗脱－扩增”的筛选过程，随机挑取50个克隆，经噬菌体酶联免疫吸附法（ELISA）鉴定，并进行交叉反应实验以及竞争抑制性结合实验，最后对所选克隆进行DNA序列分析，以确定HCV核心抗原的模拟表位。经免疫学鉴定后，从随机筛选的50个克隆中确定10个阳性克隆，进行DNA序列测定，确定氨基酸序列XRQXXPXXXHXX为HCV核心的模拟表位。本实验为开展用HCV模拟表位探索HCV的防治研究创造了条件。     

从噬菌体随机十二肽库中筛选NF-κB(p65)亲和肽的研究

目的：采用噬菌体展示技术筛选核转录因子-κB（nuclear factor-κB，NF-κB）亚基p65亲和肽。方法：以p65亚基为靶分子，利用噬菌体随机十二肽库进行亲和筛选，用ELISA法进行阳性克隆结合特异性验证。结果：经过3轮亲和筛选后，随机挑选15个噬菌体克隆，对其亲和力做ELISA试验，其中6个克隆显示较强的阳性结果。将上述6个阳性克隆DNA测序，序列均为CGTCAGCCTCGGCGAATAAGTATCCGAAGGAGTAAA，相应的氨基酸序列为FTPSDTYSPRLT。竞争性ELISA的结果显示，抑制率随p65亚基浓度的增大而升高。结论：从噬菌体随机肽库中筛选到能与NF-κBp65亚基特异性结合的克隆，并确定了其核心序列。     

从噬菌体随机七肽库中筛选hERG钾通道亲和肽的研究

目的获得与hERG钾通道特异性结合的功能性亲和肽。方法以hERG1a亚基第3个细胞外环（L3）为靶蛋白分子,对噬菌体随机七肽库进行亲和筛选,ELISA法检测噬菌体克隆与靶蛋白的亲和性,使用全自动膜片钳技术观察亲和肽的活性。结果与结论经过3轮亲和筛选后,随机挑选15个噬菌体克隆对其亲和性做ELISA鉴定,均显示较强的阳性结果。将上述阳性克隆进行DNA测序得到3种不同小肽序列。电生理学研究表明,合成的3个小肽均能显著抑制hERG钾电流,为靶向hERG钾通道抗肿瘤研究提供了新的研究思路。     

噬菌体展示技术筛选甲型H1N1流感病毒抗原模拟表位的研究

目的筛选特异性甲型H1N1流感病毒抗原模拟表位。方法应用噬菌体表面展示技术,以2009年甲型H1N1流感康复患者的血清作为固相筛选分子,对人工合成的噬菌体随机环七肽库进行3轮吸附-洗脱-扩增筛选。从顶部琼脂噬菌斑中随机挑取32个克隆至处于对数生长期的大肠埃希菌中培养,采用ELISA,交叉反应以及竞争抑制性实验筛选并鉴定阳性克隆,对阳性克隆进行DNA序列分析,对其编码的展示于噬菌体表面的氨基酸序列与流感病毒血凝素(HA)基因进行同源性比较,确定甲型H1N1流感病毒抗原的模拟表位。结果从随机挑选的32个克隆中得到12个阳性克隆,确定氨基酸序列PLHARLP为甲型H1N1流感病毒抗原的模拟表位,其表位由HA的第52,53,59,60,61,83,271位氨基酸共同构成。结论用噬菌体环七肽库筛选得到甲型H1N1流感病毒的模拟表位,为开展用流感病毒模拟表位探索新的流感防治方法奠定了基础。     

用抗体捕获法从噬菌体肽库中筛选志贺毒素B亚基结合分子

目的：避免生物素化对志贺毒素Ｂ亚基结构功能和生物学活性的影响，从噬菌体肽库中筛选与志贺毒素Ｂ亚基结合的短肽分子。方法：采用抗体捕获法。结果：经过四轮筛选，ＥＬＩＳＡ结果表明，与ＳｔｘＢ结合的噬菌体得到了有效富集，在８６个随机挑选的克隆中，４９个与ＳｔｘＢ结合，阳性克隆的序列也表现出一定的偏向性，在结构明确的２９个克隆中，有１２个克隆（４１％）所编码的短肽序列完全一致。结论：抗体捕获法是有效的。运用噬菌体表面显示技术筛选目的分子时，对于生物素化或其他固定化策略影响到结构功能和生物学活性的某些配体分子而言，抗体捕获法是一种较好的选择，可能具有广泛的适用性。     

基孔肯雅病毒中和表位的筛选及表达

目的:寻找基孔肯雅病毒中和表位,并将其表达为重组融合蛋白.方法:对噬菌体展示12肽库进行了生物淘洗,随机挑取噬菌体克隆,使用 ELISA方法筛选并鉴定阳性克隆,分析阳性克隆的DNA序列,将其翻译成氨基酸序列进行同源性比较.将其中一个表位连同M13噬菌体PⅢ蛋白结构域Ⅰ～Ⅱ的编码序列用PCR技术扩增后插入原核表达载体pQE30,用IPTG诱导融合蛋白表达.结果与结论:从经过3轮淘洗的噬菌体中随机挑选123个克隆,从中筛选到20个阳性克隆,其中包含7种不同的氨基酸序列,这些序列缺乏同源性,表明该单抗表位应为构象型表位.在室温培养条件下成功地在大肠杆菌周质腔表达了一种可溶性的表位融合蛋白,经亲和层析得到了纯化的表位融合蛋白.     

丙型肝炎病毒NS4A抗独特型人源单链可变区抗体的筛选与鉴定

为探索新型治疗方法，制备抗丙型肝炎病毒（HCV）非结构蛋白NS4A的抗独特型人源单链可变区抗体（抗－Id scFv),采用噬菌体表面展示技术，将抗HCV NS4A单克隆抗体固相包被于Nunc板，应用半合成的人源单链可变区抗体文库技术，从噬菌体单链可变区抗体库中经过5轮“吸附－洗脱－扩增”筛选过程，随机挑选82个克隆，利用酶联免疫吸附法（ELISA）、交叉反应和竞争抑制实验，对其进行免疫学检测和鉴定，获得与HCV NS4A单克隆抗体结合活性较强的抗－Id scFv阳性克隆，并对HCV NS4A特异性抗－Id scFv的编码基因进行序列测定分析。结果：经过筛选82个克隆中有40株克隆ELISA的吸光度（A450nm)值较高，将这些噬菌体上清与牛血清白蛋白（BSA）进行交叉反应，确定其中有7株交叉反应较弱，结合2次ELISA重复实验的A值及竞争抑制实验结果，最后确定1株阳性克隆。提取质粒，进行DNA序列测定，DNA为789bp。本实验结果提示，用噬菌体抗体库技术能够成功地获得抗HCV NS4A的抗－Id scFv，为开展用抗－Id scFv防治丙型肝炎的研究创造了条件。     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

Prevalence and severity of hip and groin pain in sub‐elite male football: a cross‐sectional cohort study of 695 players

The purpose of this study was twofold: (a) to investigate the prevalence of hip and groin pain in sub‐elite male adult football in Denmark and (b) to explore the association between prevalence and duration of hip and groin pain in the previous season with the Copenhagen Hip and Groin Outcome Score (HAGOS) in the beginning of the new season. In total 695 respondents from 40 teams (Division 1–4) were included. Players completed in the beginning of the new season (July–Sept 2011) a self‐reported paper questionnaire on hip and/or groin pain during the previous season and HAGOS. In total 49% (95% CI: 45–52%) reported hip and/or groin pain during the previous season. Of these, 31% (95% CI: 26–36%) reported pain for >6 weeks. Players with the longest duration of pain during the previous season had the lowest HAGOS scores, when assessed at the beginning of the new season, P < 0.001. This study documents that half of sub‐elite male adult football players report pain in the hip and/or groin during a football season. The football players with the longest duration of pain in previous season displayed the lowest HAGOS scores in the beginning of the new season.     

Bronchial stump fistula :treatment with covered retrievable hinged metallic stents-preliminary clinical experience

Objective To evaluate the preliminaily clinical efficacy and retrievability of a retrievable hinged covered metallic stent in the treatment of the bronchial stump fistula （BSF）. Methods Between April 2003 and March 2005, 8 patients with bronchial stump fistula after pneumonectomy or lobectomy were treated with two types （A and B） of retrievable hinged covered metallic stents. Type A stent was placed in 6 patients and type B in 2 under fluoroscopic guidance. The stent was removed with a retrieval set when BSF was healed or complications occurred. Results Stent placement in the bronchial tree was technically successful in all patients, without procedure-related complications. Immediate closure of the BSF was achieved in all patients after the procedure. Stents were removed from all patients but one. Removal of the stents was difficult in two patients due to tissue hyperplasia. Patients were followed up for 6 - 21 months. Placement of the stents remained stable in all patients except one due to severe cough. Permanent closure of BSF was achieved in 7 （87.5%） of 8 patients. Conclusion Use of a retrievable hinged covered expandable metallic stent is a simple, safe, and effective procedure for closure of the BSF. Retrieval of the stent seems to be feasible. （J Intervent Radiol, 2007, 16： 253-257）