临床膀胱癌组织中miRNA的差异表达研究

目的寻找并鉴定在膀胱癌及癌旁组织中差异表达的微RNA（miRNA）。方法结合miRNA芯片技术寻找膀胱癌及癌旁组织中差异表达的miRNA,并通过荧光定量PCR进行验证。结果在癌与癌旁组织中共检测到115条存在显著性差异的miRNA,其中膀胱癌组织中下调42条,上调73条。结论膀胱癌组织与癌旁组织中miRNA表达谱存在较大差异,miRNA与膀胱癌的发生发展存在一定的相关性。     

Poly（A）加尾-RNase H消化-RT-PCR法分析应激诱导生成5′端tRNA半分子

目的：建立一种简便、快速检测应激诱导细胞生成的5′端转移RNA（ tRNA）半分子的方法。方法提取应激诱导细胞RNA,用poly（ A）加尾酶在RNA分子3′端加尾后,加入与待测tRNA的3′端部分序列互补的简并DNA探针杂交,经RNase H消化与DNA探针互补的tRNA序列,再由oligo（ dT） n锚定引物进行逆转录获得互补DNA（ cDNA）,以5′端tRNA半分子和锚定引物的序列为PCR反应的上下游引物,PCR扩增检测5′端tRNA半分子。结果经poly（ A）加尾-RNaseH消化-RT-PCR-电泳等步骤,可以实现对5′端tRNA半分子的检测分析。结论 poly （ A）加尾-RNaseH消化-RT-PCR法的建立实现了5′端tRNA半分子的简便、快速检测分析,为tRNA半分子生物学功能研究和检测分析提供了工具。     

ErbB4基因miRNA干扰质粒的构建和有效性鉴定

目的构建ErbB4基因特异性miRNA表达载体并检测其有效性。方法设计合成大鼠ErbB4基因特异性miRNA前体寡核苷酸,构建ErbB4基因特异性的重组miRNA干扰质粒。从大鼠脑分离提取总RNA,经RT-PCR构建靶序列质粒,并将其与miRNA干扰质粒共转染HEK293T细胞,经实时定量RT-PCR技术鉴定其干扰有效性。结果与结论针对ErbB4基因的miRNA干扰质粒构建成功,并能够有效抑制其靶基因的表达。该质粒为后续ErbB4基因特异性miRNA慢病毒表达载体的构建及ErbB4的相关功能研究奠定了基础。     

ErbB4基因miRNA干扰质粒的构建和有效性鉴定

目的构建ErbB4基因特异性miRNA表达载体并检测其有效性。方法设计合成大鼠ErbB4基因特异性miRNA前体寡核苷酸,构建ErbB4基因特异性的重组miRNA干扰质粒。从大鼠脑分离提取总RNA,经RT-PCR构建靶序列质粒,并将其与miRNA干扰质粒共转染HEK293T细胞,经实时定量RT-PCR技术鉴定其干扰有效性。结果与结论针对ErbB4基因的miRNA干扰质粒构建成功,并能够有效抑制其靶基因的表达。该质粒为后续ErbB4基因特异性miRNA慢病毒表达载体的构建及ErbB4的相关功能研究奠定了基础。     

慢病毒载体介导E6AP基因敲低乳腺癌细胞株的构建及其功能检测

目的：构建E6AP基因的小干扰RNA（siRNA）真核表达载体,并检测其对乳腺癌细胞ZR75-1生长的影响。方法利用RNA干扰（RNAi）技术设计并合成两条E6AP基因的siRNA,并将其与siRNA表达载体pSIH-H1连接。经过酶切和测序鉴定成功后,人胚肾细胞293T包装E6AP siRNA的慢病毒,然后感染乳腺癌细胞ZR75-1,建立敲低E6AP表达的稳定细胞株。稳定细胞株建立后通过实时定量PCR（ qRT-PCR）和Western 印迹等方法检测RNAi的干扰效果,并利用细胞生长实验检测E6AP siRNA对乳腺癌细胞生长的影响。结果 DNA测序证明,成功构建了E6AP siRNA的表达载体。实时定量PCR和Western 印迹实验证明,构建的siRNA确能有效抑制E6AP基因的表达,并建立了敲低E6AP表达的稳定细胞株。生长实验表明,E6AP siRNA可有效抑制细胞的生长。结论成功构建E6AP基因的siRNA真核表达载体,转染细胞后能有效地抑制E6AP基因的表达,且抑制细胞ZR75-1的生长。     

串联MS2衣壳蛋白和MS2特异结合RNA表达载体构建及应用

目的构建体内研究RNA-蛋白相互作用的表达载体。方法利用MS衣壳蛋白可与MS2结合位点（MS2bs）RNA特异性结合的特点,通过设计特异引物,构建pcDNA3．0-Flag．2XMS2和pcDNA3．0—12XMS2％s表达载体,以及表达MEG3非编码RNA的表达载体pcDNA3．0-EG3V1—12XMS2bs。共转染细胞,进行RNA免疫沉淀,所得RNA进行实时定量PCR分析验证。结果成功构建了pcDNA3．0-Flag-2XMS2和pcDNA3．0．12XMS2bs表达载体,实时定量PCR结果表明,与对照相比能明显富集MEG3V1非编码RNA分子。结论成功构建了体内研究RNA-蛋白质相互作用的表达载体,为RNA一蛋白相互作用研究提供了新的技术方法。     

志贺菌、沙门菌和霍乱弧菌多重PCR快速检测体系的建立

目的 建立快速检测志贺菌、沙门菌和霍乱弧菌的多重PCR方法.方法 根据志贺菌ipaH基因、沙门菌ipaB基因及霍乱弧菌EPSM基因设计特异性PCR引物,加热煮沸法制备DNA模板,进行PCR扩增及琼脂糖电泳检测.结果 应用所建立的多重PCR方法能分别或同时快速、特异地检测出志贺菌606bp、沙门菌314bp和霍乱弧菌482bp的目的基因.结论 初步建立了灵敏、特异的一步法检测志贺菌、沙门菌和霍乱弧菌的多重PCR体系,可用于高危腹泻致病菌的早期快速诊断.     

HBV-DNA提取液I的配制和应用评价

目的：探讨荧光定量PCR法检测HBV-DNA提取液I的配制并评价其应用效果。方法按照DNA提取原理及文献,筛选提取液I中有效成分PEG6000和NaCl的最佳浓度,与原厂家核酸提取液I比对并评估其应用效果。结果在不同分子量PEG配制的提取液中,以PEG6000效果最好;PEG6000和NaCl浓度分别为200g/L、250g/L组成的提取液I提取效果最佳;对30份不同浓度的HBV标本进行的检测结果显示,自配HBV-DNA提取液I与原厂家提取液无显著差异（P＞0.05）,两者相关性良好（r=0.96）,变异系数分别为3.8%和3.4%。结论自配200g/L PEG6000和250g/L NaCl浓度的HBV-DNA提取液I可用于HBV-DNA定量检测。     

实验性胸腹撞击伤后血浆TNF-α、IL-1基因水平的变化特点及意义

目的探讨胸腹撞击伤前后血浆肿瘤坏死因子-α（TNF-α）、白细胞介素-1（IL-1）的DNA和RNA水平动态变化特点及临床意义。方法随机选取新西兰兔12只,采用BIM-Ⅳ型生物撞击机撞击右侧胸壁及剑突部致伤。运用聚合酶链反应（PCR）和反转录多聚酶链反应（RT-PCR）技术监测胸腹撞击伤前后不同时相点血浆中TNF-α和IL-1的DNA和RNA的变化水平。结果胸腹撞击伤后动物血浆中TNF-α、IL-1的DNA和RNA水平的表达在30分钟内急剧升高,与伤前比较差异非常显著（P〈0．01）,直至8小时后或动物死亡时仍高于伤前水平（P〈0．05）。结论胸腹撞击伤后血浆TNF-α、IL-1的DNA和RNA的变化特点是变化发生早,灵敏度高、特异性强,检测方法简单、稳定性好。监测其动态变化对于了解和掌握胸腹伤的伤情预后具有重要的临床参考意义。     

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.     

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.     

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).     

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.