爪蟾卵母细胞表达hERG1b钾通道的电生理学特性研究

目的建立人类ether-a-go-go相关基因1b（human ether-a-go-go-related gene 1b,hERG1b）钾通道在爪蟾卵母细胞上的异源性表达方法和hERG1b钾通道电流记录方法,研究hERG1b钾通道的电生理学特性。方法将编码hERG1b亚基的cRNA注入爪蟾卵母细胞中表达hERG1b钾通道,使用双电极电压钳技术记录全细胞电流,分析hERG1b钾通道电流门控动力学特征和电生理特性。结果与结论利用双电极电压钳技术,记录到hERG1b钾电流,该电流具有内向整流特性,胞外钾离子浓度升高可增大hERG1b钾通道电流幅度。与hERG1a钾通道电流相比,hERG1b钾通道电流的去激活速度明显加快,表现出快速去激活的电生理特征。     

爪蟾卵母细胞表达的大鼠ASIC3电生理学特性研究

目的:研究大鼠酸感受离子通道亚基3 (acid-sensing ion channel subunit 3,ASIC3)组成的同聚体离子通道的电生理学特性.方法:将编码ASIC3亚基的cRNA注射到爪蟾卵母细胞中以表达ASIC3离子通道,使用双电极电压钳技术记录H 诱发电流,分析电流的药理学和门控动力学特征,并观察了Ca2 、Na 及K 离子对H 诱发电流的影响.结果:在注射ASIC3亚基cRNA的爪蟾卵母细胞上,降低胞外液pH值可诱导出内向电流.pH值越小则H 诱发的电流幅度越大,pH50＝5.83.H 诱发电流可被阿米洛利(氨氯吡咪)可逆性阻断,电流呈现快速和稳态失活,其失活常数分别为τ1＝(1.5±0.7) ms,τ2＝(364.5±1.3) ms.提高胞外Ca2 浓度可降低H 诱发的电流幅度,IC50＝9.16 mmol/L.当细胞外液中无Na 时,H 基本上不能诱发出内向电流;当同时去除细胞外液中Na 和K 时,H 可诱发出外向电流.结论:成功建立了特异性表达大鼠ASIC3亚基同聚体离子通道细胞模型.ASIC3除了对Na 通透外,对K 具有一定程度的通透.胞外Ca2 对ASIC3的开放具有抑制性作用.     

不同偶联模式的兴奋性氨基酸受体所介导非洲爪蟾卵母细胞跨膜电流的相互影响

目的 研究不同偶联模式的兴奋性氨基酸受体所介导非洲爪蟾卵母细胞跨膜电流的相互影响.方法 用异硫氰酸胍-酚-氯仿法从成年大鼠脑中提取总RNA,以寡聚脱氧胸苷酸纤维素亲和层析法分离出mRNA并注射入非洲爪蟾卵母细胞使表达,通过全细胞双电极电压钳位法,分别以M-胆碱受体Carb,谷氨酸离子型受体激动剂kainate(KA),代谢Ⅰ型受体激动剂quisqualate(QA)及代谢Ⅲ型受体激动剂L-phosphoserine(L-SOP)两两同时灌流有受体表达的非洲爪蟾卵母细胞.结果 3种偶联模式的受体激动后产生的膜电流具有交叉脱敏现象.结论 在多种受体共存的细胞中,受体之间可能存在着广泛的相互作用.     

双孔钾离子通道TREK-1功能性串联二聚体载体的构建

目的：探讨在双孔钾离子通道（K2P）TREK-1（TWIK related K ＋ channel 1）分子间加入柔性 linker 构建串联二聚体载体的可行性。方法利用 PCR 技术在两个单体 TREK-1分子之间加入一个 linker,构建串联二聚体载体（pGH19-tdTREK-1）。将上述载体体外转录成 cRNA 并显微注射至爪蟾卵母细胞,培养24～48 h 后采用双电极电压钳技术记录电流。观察胞外钡离子和不同 pH 值外液对该串联二聚体通道表达的影响,并与天然二聚体通道的反应相比较。结果串联二聚体 TdTREK-1可在爪蟾卵母细胞中高效表达,该通道形成的电流可被胞外钡离子抑制,也可被胞外液酸化所抑制,而该串联二聚体通道对这些胞外刺激因素的反应程度与天然二聚体相似。结论人为加入柔性 linker 序列构建串联二聚体 TREK-1通道并不影响通道的表达及门控特性,证明该实验方案可行。这将为操作单个亚基,进而深入研究 TREK-1的结构与功能打下良好的基础。     

老年大鼠耳蜗核乙酰胆碱受体的表达

分别自青年和老年大鼠耳蜗核提取多聚腺嘌呤信使核糖核酸,注入非洲爪蟾卵母细胞表达有功能的乙酰胆碱受体,利用电压钳方法记录ＡＣｈ受体电流。推论移植的老年大鼠耳蜗核ＡＣｈ受体功能的下降可能与老年性聋的发病有关。     

人源GABAA受体的体外构建与功能评价

目的 建立不同亚型的人源γ-氨基丁酸A型受体(GABAAR)离体表达与功能评价技术,为化合物筛选和作用机制研究奠定基础.方法 将各种GABAA受体亚基cRNA混合注射入爪蟾卵母细胞内,分别实现α1β2、α1β2γ2、α632、α6p2δ受体亚型的表达,用双电极电压钳记录全细胞电流.结果 与结论 酶切鉴定提示,酶切产物与目的质粒基因片段大小相同;膜片钳记录显示,不同受体亚型均可被GABA激活并被相应工具药变构调节.因此,人源GABAA受体离体表达与功能评价技术建立成功.     

不同亚基组成的N型钙离子通道电学特性研究

目的:建立不同辅助亚基与功能亚基重组体瞬时表达的N型钙离子通道细胞模型,观察不同亚基组成的N型钙离子通道的电学特征,揭示不同辅助亚基对功能亚基的调节作用.方法:将N型钙离子通道亚基α1B/α2δ/β1b的cDNA分别体外转录为cRNA后,注射至去除滤泡膜的非洲爪蟾卵母细胞中,表达48 h后使用双电极电压钳技术记录其电流.结果:向注射N型钙通道cRNA的细胞以一系列去极化刺激可诱发90%的细胞记录到内向电流.其电流可被N型钙离子通道特异性拮抗剂ω-CTX MVⅡA (1 μmol/L)完全阻断.α1B单独表达时可形成功能性N型钙离子通道,通过记录到电流后而得出电流-电压曲线(I-V曲线),可知产生电流峰值的膜电压约为20 mV(21.17±1.44)mV(n=7);α1B/β1b共表达时I-V曲线方向为超极化,电流达到峰值的电压为(12.35±0.88) mV(n=7,P＜0.01);功能亚基与α2δ亚基共表达时,I-V曲线稍向正电位方向,电流达到峰值的电压为(23.25±1.25) mV(n=7,P＜0.01);在α1B/α2δ/β1b共表达时,尽管有α2δ亚基存在时,β1b亚基仍显著地使I-V曲线向超级化方向(17.08±1.86) mV(n=7).本实验也观察了不同重组体电流的稳态失活电压,其中α1B表达电流的半数失活电压为-12.0 mV,α1B/α2δ表达电流的半数失活电压为-13.5 mV,α1B/β1b表达电流的半数失活电压为-51.4 mV,而α1B/α2δ/β1b共表达时电流半数失活电压为-54.9 mV.结论:成功地建立不同亚基组成的N型钙离子通道在爪蟾卵母细胞的功能性表达,不同的辅助亚基与功能亚基组合体现了通道不同的动力学特性,表明N型钙通道在体内可能具有功能的多样性.这些结果为今后深入研究N型钙通道奠定了基础.     

大鼠壁细胞K+离子通道特性的研究

目的研究大鼠壁细胞K^＋离子通道的特性，探讨其在生理病理过程中的作用。方法分离大鼠壁细胞，采用膜片钳方法，对大鼠壁细胞K^＋离子通道进行全细胞电生理记录，分析壁细胞K^＋离子通道的基本特性。结果10mM氯化TEA部分阻断壁细胞外向去极化K^＋离子通道电流。10mM乙酸TEA、10mM氯化钡、10mM乙酸钡能使壁细胞外向去极化K^＋离子通道电流增加。结论大鼠壁细胞的全细胞K^＋离子电流具有电压依赖性，兼有外向整流性和内向整流性。     

豚鼠初级精母细胞延迟外向钾离子电流的研究

应用机械分离技术获得豚鼠单离初级精母细胞后,进行全细胞式膜片钳记录。结果表明;在标准盐溶液中,当钳制电位－90mV,指令电压（－60～＋140）mV,步阶电压20mV时,可记录到外向离子电流。将标准盐溶液换成含4mmol／L的TEA盐溶液,外向电流显著抑制。冲洗换液后大部分电流恢复,表明此电流为外向K离子流。     

α-芋螺毒素Lt1.1的克隆、合成及靶点鉴定

目的 发现作用于神经元烟碱型乙酰胆碱受体(nAChR)的拮抗剂,为研制新型镇痛药和神经生物学工具奠定基础.方法 根据芋螺毒素A超家族DNA中保守的内含子及3′端非转录区(3′UTR)设计引物,从中国南海信号芋螺(Conus litteratus)中克隆获得新芋螺毒素Lt1.1序列.固相法合成Rink树脂肽,经裂解、空气氧化折叠、纯化后获得目的肽,利用两步氧化折叠法鉴定其二硫键连接方式.将神经元nAChR各亚基的cRNA在爪蟾卵母细胞中表达,利用双电极电压钳检测通道电流.结果 获得一种新α-芋螺毒素Lt1.1序列(GCCSHPACNVNNPDIC-NH2),其二硫键连接方式为"C1-C3、C2-C4",作用靶点为nAChR α3β2和α3β4亚型,IC50 分别为166.76和190.00 nmol/L.结论 Lt1.1是一种典型的4/7型α-芋螺毒素,其选择性抑制了nAChR α3β2和α3β4亚型.     

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.     

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）     

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.