真核表达载体pIRES2-EGFP-p93的构建及在卵巢癌细胞HO-8910中的表达

目的 :构建肿瘤抑制基因DOC - 2的真核表达载体pIRES2 -EGFP -p93,将其转入人源性卵巢癌细胞株HO - 8910中 ,并得到稳定表达。方法 :利用XhoI酶切含有p93cDNA的质粒得到p93cDNA基因片段 ,将其正向连接入真核表达载体pIRES2 -EGFP ,并通过酶切鉴定。用脂质体介导的方法 ,将真核表达载体pIRES2 -EGFP -p93转染人卵巢癌细胞系HO - 8910 ,通过G4 18筛选稳定表达克隆 ;分别在荧光显微镜下及用免疫组化法观察人DOC - 2蛋白在卵巢癌细胞系HO - 8910中的表达。结果 :构建了DOC - 2的真核表达载体pIRES2 -EGFP -p93,并通过酶切鉴定。在荧光显微镜下 ,转染了pIRES2 -EGFP -p93的人卵巢癌细胞HO - 8910发出绿色荧光 ,荧光全部集中于胞浆内。免疫细胞化学染色结果显示转染了pIRES2 -EGFP -p93的人卵巢癌细胞HO - 8910胞浆呈棕黄色 ,而转染空载体的细胞呈阴性染色。结论 :成功构建了真核表达载体转染了pIRES2 -EGFP -p93,并在人卵巢癌细胞系HO - 8910中稳定表达 ,为研究DOC - 2蛋白对卵巢癌细胞的作用奠定了基础     

人角化细胞生长因子-1的克隆及其在骨髓基质细胞中的表达

目的 从人胚肺成纤维细胞中克隆人角化细胞生长因子-1（KGF-1）编码区序列,构建真核荧光表达载体.方法 从人胚肺成纤维细胞中提取总RNA,通过反转录 PCR获取KGF-1 cDNA编码区序列,将其与pIRES2-EGFP质粒载体连接,构建重组表达载体pIRES2-EGFP/KGF-1,测序鉴定.将pIRES2-EGFP/KGF-1瞬时转染骨髓基质细胞（marrow stroma cells,MSCs）48 h后,免疫荧光法检测骨髓基质细胞是否存在KGF-1蛋白的表达.结果 构建的重组质粒pIRES2-EGFP/KGF-1经序列分析示与国外文献报道一致.KGF-1免疫荧光法激光共聚焦检测结果证实,pIRES2-EGFP/KGF-1转染骨髓基质细胞后,存在KGF-1蛋白的表达,定位于细胞浆内.结论 成功构建重组真核荧光表达载体pIRES2-EGFP/KGF-1,初步探讨了该基因在骨髓基质细胞中的表达,为其在皮肤组织工程的运用奠定了基础.     

骨唾液蛋白非融合荧光蛋白载体的构建及其在特异性骨转移乳腺癌细胞中的稳定表达

目的 构建骨唾液蛋白(BSP)非融合荧光表达载体,建立稳定高效表达BSP和荧光蛋白的乳腺癌细胞系,为进一步研究BSP在乳腺癌细胞骨转移中的作用奠定基础。方法 从构建好的pB-hBSP载体中用PCR方法扩增hBSP基因,通过BglⅡ和PstⅠ两个限制性酶切位点定向克隆至真核表达载体pIRES2一EGFP,构建重组载体pIRES2-hBSP-EGFP。利用脂质体转染的方法将构建好的重组质粒转入特异性骨转移的乳腺癌细胞株MDA-MB-23180中,并利用G418和流式细胞仪筛选阳性克隆。结果 成功构建hBSP和EGFP非融合表达的真核表达载体pIRES2-hBSP EGFP,并成功转染特异骨转移的乳腺癌细胞株,获得hBSP的稳定转染细胞株,荧光显微镜下可观察到荧光蛋白标记。结论 真核表达载体CRFS2-hBSP-EGFP的构建及hBSP稳定转染细胞株的获得为研究BSP在乳腺癌骨转移中的作用奠定了的基础。     

用荧光强度反映Cbfal活性成骨细胞模型的构建

目的 构建稳定表达由60SE2启动子驱动的增强型绿色荧光蛋白(EGFP)的MG63细胞株,并筛选出能反映Cbfal活性的细胞株.方法 合成60SE2启动子的序列,并将其构建到T载体中:然后通过双酶切得到启动子片段;与去除CMV启动子的报告基因载体pEGFP-Nl的酶切片段连接,构建真核表达载体;并转染到MG63细胞系,经G418筛选得到了稳定转染60SE2-EGFP基因的MG63细胞株.用不同浓度的IGF-I和VD,处理,通过EGFP荧光强度分析,ALP活性测定,筛选出能够响应IGF-I和VD3的细胞株.结果 构建了pUC-60SE2扩增载体和p60SE2-EGFP真核表达载体,通过稳定转染以及IGF-I、VD_3筛选获得一株荧光强度随IGF-I和VD_3处理而增强的细胞株.OSE-MG63.结论 构建了能反应Cbfal活性的成骨细胞模型,为进一步研究微重力对Cbfal的转录活性及信号传导途径的影响奠定了基础.     

pIRES2-EGFP-hBMP2载体在人骨髓基质细胞中的表达及意义

目的：研究人骨形成蛋白hBMP2真核表达载体pIRKS2-EGFP-hBMP2在人骨髓基质细胞系HFCL中的表达及意义。方法：利用DNA重组技术，将hBMP2片段克隆到真核表达载体pIRES2-EGFP中；EeoRI/BamHI双酶切鉴定；脂质体介导的方法转染HFCL．细胞；荧光显微镜下观察转染细胞；免疫组化染色检测hBMP2基因的表达。结果：经酶切鉴定显示，成功地构建了重组载体pIRES2-EGFP-hBMP2。转染HFCL 48h后在荧光显微镜下观察到转染的HFCL细胞发绿色荧光。免疫组化染色显示转染hBMP2基因的HFCL细胞浆内有细的棕色颗粒，而转染空载体的HFCL细胞免疫组化呈阴性。结论：所构建的pIRES2-EGFP-hBMP2真核表达载体在HFCL细胞中有良好的表达，为进一步研究转基因骨髓基质细胞对放射和化疗所致的造血和骨微环境破坏的改善奠定了基础。     

阿片受体δ-FLAG-pIRES2-EGFP表达载体的构建及其在CHO细胞中的表达

目的 构建带FLAG标签的大鼠δ型阿片受体的pIRES2-EGFP表达质粒(δ-FLAG-pIRES2δEGFP),并实现其在CHO细胞中的表达.方法 以含大鼠全长δ阿片受体基因的δ-pMD20-T载体为模板,设计引物并行PCR扩增,在δ基因C端引入FLAG标签,AT克隆至pMD20-T载体中,测序鉴定,经酶切连接入pIRES2-EGFP中,将获得的δ-FLAG-pIRES2-EGFP载体转染CHO细胞,应用荧光显微镜观察EGFP表达情况,并采用细胞免疫荧光和蛋白印迹法检测δ-FLAG的表达.结果 测序及酶切结果表明获得了带FLAG标签的δ基因,成功构建δ-FLAG-pIRES2-EGFP表达质粒,在荧光显微镜下,转染细胞可以观察到绿色荧光.应用免疫荧光和蛋白印迹法可观察到目的 基因表达.结论 成功构建了δ-FLAG-pIRES2-EGFP表达质粒,并在CHO细胞中实现了高效表达.     

用荧光强度反映Cbfa1活性成骨细胞模型的构建

目的构建稳定表达由6OSE2启动子驱动的增强型绿色荧光蛋白(EGFP)的MG63细胞株,并筛选出能反映Cbfa1活性的细胞株。方法合成6OSE2启动子的序列,并将其构建到T载体中;然后通过双酶切得到启动子片段;与去除CMV启动子的报告基因载体pEGFP-N1的酶切片段连接,构建真核表达载体;并转染到MG63细胞系,经G418筛选得到了稳定转染6OSE2-EGFP基因的MG63细胞株。用不同浓度的IGF-I和VD3处理,通过EGFP荧光强度分析,ALP活性测定,筛选出能够响应IGF-I和VD3的细胞株。结果构建了pUC-6OSE2扩增载体和p6OSE2-EGFP真核表达载体,通过稳定转染以及IGF-I、VD3筛选获得一株荧光强度随IGF-I和VD3处理而增强的细胞株-OSE-MG63。结论构建了能反应Cbfa1活性的成骨细胞模型,为进一步研究微重力对Cbfa1的转录活性及信号传导途径的影响奠定了基础。     

PCGF1过表达慢病毒载体的构建及稳定过表达A549细胞系的建立

目的 构建人多梳家族环指蛋白1(PCGF1)基因的慢病毒载体,建立稳定表达PCGF1基因的细胞系.方法 根据人PCGF1序列设计并合成引物,以A549细胞cDNA为模板,PCR扩增目的基因.双酶切目的基因并插入pLVX-IRES-puro质粒,对重组质粒进行慢病毒包装,用包装好的病毒感染A549细胞系,通过嘌呤霉素筛选阳性表达细胞,Western blotting验证PCGF1表达情况.结果 重组质粒经测序分析正确,瞬时转染293T细胞后,Western blotting验证PCGF1表达明显升高.包装慢病毒颗粒后感染A549细胞,经嘌呤霉素筛选后,Western blotting验证得到PCGF1稳定过表达的A549细胞系.结论 成功构建了pLVX-IRES-PCGF1-puro慢病毒过表达载体和PCGF1稳定过表达的A549细胞系,为进一步研究PCGF1的功能奠定了基础.     

重组腺病毒载体Ad5-hVEGF165-EGFP的构建与鉴定

目的人血管内皮生长因子（的hVEGF165）为靶基因,增强型绿色荧光蛋白（EGFP）基因构建体的复制缺陷型腺病毒载体,为基因治疗牙周进一步再生奠定了基础。方法质粒的pDC316-的VEGF165为模板,PCR扩增基因片段进行消化,获得的hVEGF165用于连接到含有绿色荧光蛋白标记基因重组质粒的消化方法的pDC316-MCMV-EGFP质粒载体,PCR鉴定和双酶切证实成功构建重组质粒;使用ADMAX包装系统,改造的质粒共转染用质粒骨架293包装细胞系和重组病毒的扩增;扩增病毒的离子交换纯化;TCID50测定病毒粒子和效价的数目;的荧光的荧光显微镜重组腺病毒表达。结果 PCR鉴定,酶切分析和测序证实成功构建人VEGF基因的携带绿色荧光蛋白标记（的hVEGF165）重组质粒的pDC316-的hVEGF165-MCMV-EGFP和同源重组腺病毒Ad5的-的hVEGF165-EGFP的成功。扩增和纯化后,腺病毒颗粒的数量测量5.4×1011VP/mL,约2.0,为1.8×1010CCID50/mL的滴度OD260/OD280值。结论成功构建携带hVEGF165基因重组腺病毒载体Ad5-hVEGF165-EGFP并获得高滴度病毒颗粒,为hVEGF165基因功能研究以及细胞移植、基因治疗提供有效的工具。     

DC-SIGN真核表达载体的构建及其稳定转染BHK21细胞系的建立

目的 构建DC-SIGN分子真核表达载体,获得可稳定表达DC-SIGN分子的BHK21细胞系.方法 以pUNO-hDC-SIGN1Aa质粒为模板,通过PCR方法扩增人DC-SIGN基因,经过Not Ⅰ和BamH Ⅰ双酶切之后,将其克隆入真核表达载体pIRES-neo,构建真核表达载体pIRES-neo-DC-SIGN,以Not Ⅰ和BamH Ⅰ双酶切以及测序验证重组质粒的正确性.将重组质粒以Lipo-fectamine~(KM) 2000转染到BHK21细胞,通过G418及有限稀释法进行阳性克隆的筛选,建立稳定表达DC-SIGN分子的BHK21细胞系,利用流式细胞仪、Western blotting及免疫荧光法检测DC-SIGN分子的表达.结果 双酶切鉴定证明DC-SIGN基因已经成功克隆入真核表达载体pIRES-neo中,测序结果表明EC-SIGN基因与原序列一致.pIRES-neo-DC-SIGN转染BHK21细胞后,获得了急定表达DC-SIGN分子的细胞系.流式细胞仪检测结果显示,阳性克隆中DC-SIGN分子的表达率为85.42%.免疫荧光结果显示,DC-SIGN分子主要表达于细胞膜表面.Western blotting能检测到DC-SIGN蛋白表达的特异条带.结论 成功构建了稳定、高效表达DC-SIGN分子的BHK21细胞系,为后续DC靶向性疫苗研究奠定了基础.     

The role of interventional radiology in trauma

Introduction Interventional Radiology has evolved into a specialty having enormous input into the care of the traumatized patient.In all hospitals,regardless of size,the Interventional Radiologist must consider their relationships with the trauma service in order to     

Sexual homicide: Brief review of the literature and case report involving rape,genital mutilation and human arson

The objective of the current paper is to report a new case of sexual murder involving human arson and summarize the literature on the phenomenon of sexual homicide. The present case study is unprecedented in Greece and a rarity in international literature due to the fact that the victim suffered genital mutilation and incineration while still alive. The evaluation consisted of 176 articles; 53 were reviewed by the authors. The results revealed sparse, but significant, research findings. The authors discuss the limitations regarding research, incidence of the phenomenon, crime-scene patterns, offender characteristics (killing methods, motive inferences, sociodemographic data, classifications, psychopathology, modus operandi), and victim selection. The incidence of the phenomenon is unclear (1–4%) due to non-standardized criteria. It is an expression of displaced anger or sexual sadism and/or a way to elude detection (ancillary benefit). Most offenders (in their first kill) and victims were in their late 20s to early 30s and belong to Caucasian populations. Personal weapons were commonly used against women, strangulation is the prevalent killing method against children, and firearms against men. Most of the sexual homicide perpetrators are non-psychotic at the time of the attack, but experience personality pathology, primitive defenses, pathological object relations, and withdrawal into fantasy in order to deal with social isolation.     

Extension du modèle puissance–temps limite pour estimer la production d’énergie aérobie et anaérobie lors de l’exercice intense

Aim

Examine how the modelling of the relation between power and time to exhaustion can provide an estimation of the production of aerobic and anaerobic energy during intense exercise.

Current knowledge

The hyperbolic model made it possible to define the critical power corresponding to the maximal rate of energy renewed by aerobic metabolism. A new model distinguishing the critical power from the maximal aerobic power has been built to estimate more precisely the anaerobic contribution. Data from middle distance runners and subjects tested on cycle ergometer showed a relative contribution of anaerobic metabolism arising from critical power and increasing until around 10 % of total power when aerobic energy production reaches its maximum.

Prospects

Considering the slow component of oxygen uptake would provide a more precise analysis of energy production and transformation during exercise at high intensity.     

Beziehungen zwischen Aufprallgeschwindigkeit,Fahrzeugbeschädigungen,Frakturen und „Wurfweite” bei 50 tödlichen Fußgänger-PKW-Unfällen

Zusammenfassung Aus 160 Obduktionen von Fußgängern, die durch PKW getötet worden waren, sind 50 nach folgenden Gesichtspunkten ausgewählt worden:Auffahrunfälle Erwachsener mit gesicherten Angaben in den Gerichtsakten über Fahrzeugbeschädigungen, Zusammenstoßstellen, Endlage der Fußgänger und Bremsspuren. Aus diesen Daten wurden Ausgangs- und Aufprallgeschwindigkeiten berechnet sowie die Wurfweite der Fußgänger gemessen.Die Ausgangsgeschwindigkeiten, nach der Berechnung zwischen 32 und 95 km/h, lagen meistens etwas höher als die angegebenen Geschwindigkeiten. Zwischen Ausgangs- und Aufprallgeschwindigkeiten waren die Differenzen größer. Ab 12 km/h Aufprallgeschwindigkeit kam es bereits zu tödlichen Verletzungen. Auffallend ist die zweigipfelige Verteilung der Häufigkeit sowohl bei der Einteilung nach den Aufprallgeschwindigkeiten als auch nach den Deformationsarbeiten (Aufprallgeschwindigkeit: 26% bei 51–60 km/h und 22% bei 21 bis 30 km/h; Deformationsarbeit: 34% bei 39–200 kpm und 30% bei 701–1000 kpm).Bei Aufprallgeschwindigkeiten über 50 km/h wurden doppelt so viel Knochenbrüche an der Wirbelsäule und am Becken als bei Aufprallgeschwindigkeiten unter 50 km/h festgestellt.Bei 44 Fußgängerunfällen lag ein Drittel innerhalb der Erwartungsgrenze der Wurfweite nach Fiala, je ein Drittel aber darüber bzw, darunter.Das Beschädigungsbild und die Anstoßverletzungen geben Hinweise zur Ermittlung der Aufprallgeschwindigkeit, vor allem wenn weitere Berechnungsgrundlagen fehlen.

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Summary Fifty cases were selected from 160 autopsies performed on pedestrians who were accidentally killed by private motorcars; the cases were selected according to the following criteria:The cases were confined to impact accidents involving adults in which reliable evidence could be gathered from the Court records in relation to damage to the car, the place of collision, the final position of the injured pedestrian and the extent of skidmarks. The initial and collision speeds of the cars were calculated from these facts and the distance of projection of the pedestrians were measured.The initial speeds, calculated between 32 and 95 km/h, were, in most cases, higher than the declared speeds. The differences between initial and collision speeds were larger. Fatal injuries could arise from a collision velocity of only 12 km/h. A striking feature of the analysis was the distribution of two peaks of frequency whether these were classified according to the collision speeds or according to the deformation of the vehicle (collision speed: 26% at the speed of 51 to 60 km/h and 22% at the speed of 21 to 30 km/h; deformation work: 34% at the deformation work of 39 to 200 kpm and 30% at the deformation work of 701 to 1,000 kpm).Twice as many fractures of the vertebral column and pelvis were sustained at collision speeds over 50 km/h as were sustained under 50 km/h.In one third of 44 pedestrian accidents, the distance of projection was within the expected range described by Fiala; in one third the distance was above and in one third below the expected range.If additional data for calculation were lacking, the patterns in injury arising from the impacts gave indications from which it was possible to estimate the collision speed.

Stipendiat der Alexander v. Humboldt-Stiftung, Dozent Dr. med. S. Kamiyama, Dept. of Legal Medicine, School of Medicine, Chiba University, 313 Inohanacho, Chiba, Japan.     

Diagnóstico ecográfico del neumotórax

The ultrasonographic diagnosis of pneumothorax is based on the analysis of artifacts. It is possible to confirm or rule out pneumothorax by combining the following signs: lung sliding, the A and B lines, and the lung point. One fundamental advantage of lung ultrasonography is its easy access in any critical situation, especially in patients in the intensive care unit. For this reason, chest ultrasonography can be used as an alternative to plain-film X-rays and computed tomography in critical patients and in patients with normal plain films in whom pneumothorax is strongly suspected, as well as to evaluate the extent of the pneumothorax and monitor its evolution.     

Carbohydrate Supplementation during Exercise: Does It Help? How Much is Too Much?

KEY POINTS· Carbohydrate intake during exercise can delay the onset of fatigue and improve performance of prolonged exercise as well as exercise of shorter duration and greater intensity (e.g., continuous exercise lasting about 1h and intermittent high-intensity exercise), but the mechanisms by which performance is improved are different.     

磁共振DWI和动态增强鉴别颅底脊索瘤和侵袭性垂体瘤的应用

目的 探讨磁共振扩散加权成像(DWI)和动态增强在颅底脊索瘤和侵袭性垂体瘤(IPA)鉴别诊断中的应用价值.方法 搜集经手术病理证实且影像学有鞍区破坏的颅底脊索瘤患者15例、向鼻咽部侵犯的IPA患者20例.测量二者的表观扩散系数(ADC)值,绘制受试者工作特征曲线(ROC),分析动态增强曲线的类型,统计达峰时间(TTP)、增强峰值(EP)和最大对比增强率(MCER),分析各个参数在鉴别诊断中的价值.结果 颅底脊索瘤的ADC值为(1.274±0.07)×10-3mm2/s,高于IPA ADC值(0.672±0.03) ×10-3 mm2/s(P ＜0.001),ADC阈值为0.964×10-3mm2/s时,ROC曲线下面积为0.997,敏感度为93.3％,特异度为100％.颅底脊索瘤时间-信号强度曲线(TIC)Ⅰ型14例,TICⅢ型1例,此例TICⅢ型者TTP约40 s;IPA TIC Ⅰ型7例,TICⅢ型13例.颅底脊索瘤和IPA的EP、MCER差异均有统计学意义(P ＜0.001).结论 ADC值和TIC的类型及其相关参数(EP,MCER)有助于颅底脊索瘤和IPA之间的鉴别.     

Hip and pelvic injuries in the elderly

Fractures of the hip and pelvis are frequent and serious injuries in elderly patients. Due to the aging population, their incidence should double by 2050. Therefore, the social and economical implications of these fractures are significant. Delay in diagnosis increases the associated morbidity and mortality. The purpose is to review the imaging features of these fractures, the imaging techniques (projections, CT) to depict them and their classification based on severity.