感觉神经元特异性受体rMrgE基因的克隆与表达

目的 克隆感觉神经元特异性受体rMrgE基因并于HEK293细胞中表达.方法 利用聚合酶链式反应技术,从大鼠背根神经节总RNA中扩增出目的基因rMrgE,克隆入真核表达载体pcDNA3.1/myc-hisB中,构建真核表达rMrgE载体,瞬时转染人胚肾293细胞得到重组质粒表达细胞株.采用RT-PCR、免疫荧光和Western印迹检测对表达产物进行鉴定.结果 与结论含有rMrgE基因的重组质粒在人胚肾293细胞中获得表达,主要定位于细胞膜上.本研究为进一步探究rMrgE受体的功能奠定了基础.     

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.     

人CDK7基因真核表达载体的构建与鉴定

目的：构建带Myc标签的细胞周期蛋白依赖性激酶7（cyclin－dependent kinase 7, CDK7）的真核表达载体,获得其表达产物,并验证该激酶与已知相互作用蛋白P53之间的相互作用。方法应用PCR技术从人乳腺文库中扩增出CDK7全长编码区基因,将其克隆到pXJ－40载体中;继而重组质粒转染人胚肾293 T细胞,以SDS－PAGE和Western印迹鉴定表达情况;免疫共沉淀检测Myc－CDK7与FLAG－p53的相互作用。结果双酶切和基因测序鉴定显示,Myc－CDK7真核表达质粒克隆构建成功;SDS－PAGE和Western印迹结果表明,Myc－CDK7转染人胚肾293T细胞后成功表达;免疫共沉淀显示,重组Myc－CDK7与已知相互作用蛋白P53在蛋白质水平上具有相互作用,证实其具有生物学活性。结论成功构建Myc－CDK7的真核表达载体,为进一步探讨Myc－CDK7对细胞周期的调控奠定了实验基础。     

人β防御素2腺病毒表达载体的构建及其在大鼠真皮多能干细胞中的表达

目的 构建和鉴定人β防御素2（HBD2）的重组腺病毒表达载体，并观察其转染大鼠真皮多能干细胞（dMSCs）后的表达。方法反转录聚合酶链反应（RT-PCR）扩增HBD2全长cDNA，亚克隆至穿梭质粒（pAdTrack-CMV）中，然后转化含骨架质粒（pAdEasy-1）的大肠杆菌BJ5183，通过同源重组产生腺病毒载体质粒。PacⅠ酶切阳性的重组质粒后转染293细胞，包装出重组病毒载体。用所得重组腺病毒感染dMSCs细胞，并采用RT-PCR和荧光免疫组织化学检测其在dMSCs中的表达情况。结果 经测序、酶切及聚合酶链反应（PCR）鉴定，表明已成功地扩增到HBD2全长cDNA，并将其顺序克隆到穿梭质粒和骨架质粒中，进而组装出腺病毒表达载体。经RT-PCR和免疫组织化学证实构建的病毒载体可感染dMSCs，并有效表达HBD2。结论 本实验成功地构建了含HBD2基因的腺病毒表达载体，并证实其可在dMSCs中表达。     

线粒体抗病毒信号蛋白MAVS真核表达质粒的构建及表达

目的:构建线粒体抗病毒信号蛋白(mitoehondrial antiviral signaling protein,MAVS)真核表达质粒.方法:从人胚肾细胞系HEK293T细胞的总RNA中经过逆转录和PCR获得MAVS的全长cDNA双链片段.经过酶切后连接到真核表达载体pcDNA3/flag中,挑选出转化生成的阳性克隆进行测序,将序列正确的重组质粒命名为pcDNA3/flag-MAVS.借助脂质体转染peDNA3/flag-MAVS质粒到HEK293T细胞中,用Westem印迹检测目的蛋白的表达.同时用β干扰素的荧光素酶报告基因(IFN-β-luc)检测MAVS蛋白活性.结果:Western印迹实验证明pcDNA3/flag-MAVS可以在HEK293T细胞中表达MAVS,并且能使IFN-β报告基因活性明显增强.结论:构建了重组质粒peDNA3/flag-MAVS,在细胞中表达MAVS后具有刺激IFN-β转录的生物活性.     

人转化铁受体基因的克隆和表达

目的探讨人转化铁受体基因（hTfR）克隆和高效表达的方法及临床意义。方法用RT-PCR法从人胚肝、肺组织中克隆hTfR基因，构建重组表达质粒pcDNA3-hTfR和pEGFP-C1-hTfR，转染人胚肾上皮HEK293细胞。用Western印迹法检测pcDNA3-hTfR转染HEK293细胞后hTfR蛋白的表达情况；用荧光显微镜和共聚焦荧光显微镜观察重组pEGFP-C1-hTfR质粒转染HEK293细胞后hTfR蛋白的表达水平及亚细胞定位。结果经过DNA测序证实，克隆的hTfR基因为全长cDNA序列（2332bp）。Western印迹法检测到转染细胞能有效表达190×10^3的hTfR蛋白，荧光显微镜和共聚焦荧光显微镜观察到绿色荧光蛋白（GFP）-hTfR融合蛋白主要定位于细胞膜。结论从人胚肝、肺组织中可以成功克隆hTfR基因，该基因转染HEK293细胞后可获得hTfR蛋白的高效表达。hTfR主要定位于细胞膜。     

SP110基因过表达慢病毒载体的构建与包装

目的 构建核体蛋白SP110基因过表达慢病毒载体,并进行慢病毒包装,为研究SP110在结核病中的作用机制奠定基础。方法 利用PCR技术获得SP110基因序列并将其插入到慢病毒穿梭质粒LV5中,获得重组慢病毒质粒V-SP110,经酶切鉴定及DNA测序鉴定后,用RNAi-Mate将重组质粒和慢病毒包装质粒系统(pGag/Pol、pRev、pVSV-G)包装成重组慢病毒颗粒,共转染293T细胞,包装产生慢病毒。通过荧光显微镜或FACS计数荧光细胞,结合稀释倍数计算病毒滴度。结果 酶切鉴定结果显示产生约1 650 bp的片段,片段大小与SP110基因cDNA大小一致。DNA测序比对测序结果与预期SP110基因序列完全一致,说明SP110基因正确插入载体中,成功构建了SP110基因过表达载体。经293T细胞包装后,成功获得病毒滴度为1.0×108 TU/mL的重组慢病毒LV5-SP110。结论 SP110基因过表达慢病毒载体构建与包装成功完成,为进一步探讨SP110基因在结核病发生发展中的作用提供工具。     

凋亡相关斑点样蛋白ASC真核表达质粒的构建及表达

目的构建凋亡相关的斑点样蛋白（apoptosis-associated speck-like protein containing CARD,ASC）真核表达质粒。方法从人胚肾细胞系HEK293T细胞的总RNA中经过逆转录和PCR获得ASC的全长cDNA双链片段,经过酶切后连接到真核表达载体pcDNA3/flag中,挑选出转化生成的阳性克隆测序,将序列正确的重组质粒命名为pcDNA3/flag-ASC。脂质体转染pcDNA3/flag-ASC质粒到HEK293T细胞中,用Western印迹检测目的蛋白的表达。同时用免疫沉淀和免疫印迹方法检测ASC蛋白与前半胱天冬酶（pro-caspase）-1的相互作用,并用ELISA方法检测ASC蛋白对IL-1β分泌的影响。结果 Western印迹实验证明pcDNA3/flag-ASC可以在HEK293T细胞中表达ASC,并且可以与pro-caspase-1结合,使IL-1β分泌明显降低。结论构建了重组质粒pcDNA3/flag-ASC,在细胞中表达ASC后具有与pro-caspase-1结合的能力,并具有降低IL-1β分泌的生物活性。     

人野生型和突变型CITED2真核表达质粒的构建与表达

目的 构建人转录辅助因子CITED2突变型(c.573-578del6)及野生型真核表达质粒并检测两种重组质粒CITED2蛋白的表达情况.方法 分别以健康儿童和CITED2基因突变的先天性心脏病患儿血细胞DNA为模板,PCR定向克隆扩增野生型和突变型CITED2编码链,分别T/A克隆至pMD19-T simple质粒上,筛选出野生型和突变型CITED2的T载体重组质粒.应用DNA重组技术将T载体重组质粒上的CITED2基因片段亚克隆入真核表达载体pEGFP-C1,构建pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2真核表达质粒,并分别转染至HEK293细胞,24h后在荧光显微镜下观察质粒转染情况,48h后应用流式细胞仪检测转染效率,Western blotting检测CITED2蛋白的表达.结果 成功构建了人野生型pEGFP-C1-wtCITED2和突变型pEGFP-C1-mtCITED2真核表达质粒.转染至HEK293细胞后24h,在荧光显微镜下可观察到各转染组EGFP的表达,转染后48h流式细胞仪检测转染效率为50％ ～ 60％,Western blotting检测可见CITED2蛋白与EGFP的融合表达.结论 成功构建了人转录辅助因子CITED2突变型及野生型真核表达质粒,并检测到CITED2蛋白的表达.     

感觉神经元特异性受体rMrgD基因的克隆与表达

目的克隆rMrgD基因并于HEK293细胞中表达。方法利用聚合酶链反应技术,从总RNA中扩增出目的基因,克隆入真核表达载体pcDNA3.1中,构建真核表达rMrgD载体,瞬时转染人胚肾293细胞得到重组质粒表达细胞株。通过免疫荧光和Western印迹检测对表达产物进行鉴定。结果直接和间接免疫荧光实验皆表明rMrgD主要定位于细胞膜上,Western印迹检测证实rMrgD在HEK293细胞主要以单体和二聚体形式存在。结论含有rMrgD基因的重组质粒在人胚肾293细胞中获得表达,主要定位于细胞膜上。本研究为进一步探究rMrgD受体的功能奠定了基础。     

Optimization of the system of medical service of students of Cadet Schools of the Ministry of Defense of the Russian Federation

One of the factors of the successful military career guidance Cadet schools students is preserving and promoting their health. Medical support of children and adolescents aged 10-17 years should include the full range of medical and preventive measures defined for this group. The state of providing outpatient care for pupils at the Cadet School in St. Petersburg was studied. These results show that full medical care in accordance with the standards can be based only on children's health clinics. It is important that the organization of medical support pupils cadet schools should be cooperate with civilian health care.     

带状疱疹83例误诊原因分析

带状疱疹是由水痘—带状疱疾病毒引起的皮肤科常见疾病。其主要的病理损害,一是受累神经的严重炎症性浸润,继而导致受侵犯神经节内神经细胞变性、坏死;二是皮肤的水泡。迅速抑制神经节和相应的感觉神经纤维的充血、水肿和坏死,防止粘连形成,达到迅速镇痛、改善皮损,缩短病程及防止后遗症的发生是治疗的关键。因而,尽早明确诊断,     

Estimation of accumulated dose of radiation by method of ESR-spectrometry of dental enamel of mammals

ESR-spectrometry was used to investigate radiation-induced paramagnetic centers in enamel of mammals: carnivores (polar bear and fox), ungulates (reindeer, European bison, moose), and man. Values at half the microwave power saturation of the radiation signal, P_1/2, evaluated at room temperature, was found to range from 16 to 26 mW for animals and man. A new approach to discrimination of the radiation induced signal from the total ESR spectrum of reindeer enamel is proposed. ‘Dose-response’ dependencies of enamel of different species mammals were measured within the dose range from 0.48 up to 10.08 Gy. Estimations of ‘radiosensitivity’ enamel of carnivores and ungulates showed good agreement with radiosensitivity enamel of man by ESR method.     

Analysis of the results of the international comparison of activity measurements of a solution of Fe

The results of an international comparison of activity measurements of a solution of ⁵⁵Fe organized by the BIPM in 2005 are reported and analysed. This exercise, which follows the procedures of the CIPM mutual recognition arrangement to update older comparisons, is a renewal of the comparison organized by the BIPM that took place in 1978. A EUROMET comparison was organized in 1996 specifically to compare activity measurements of a ⁵⁵Fe solution by means of liquid-scintillation techniques. Results of these three comparisons are presented and discussed in this paper.

The radionuclide solution was provided by the NPL, which also distributed the samples to the participants. The activity of the ampoules was measured by 16 laboratories using 12 methods producing 25 results. Some general considerations on uncertainty assessments pertaining to the different techniques used are drawn. The outcome of four different estimators is compared from which the presence of at least one outlier can be confirmed. Further measurements should be made to try to reduce the discrepancy between the results. To date the outcome of the present comparison does not show an improvement to that of the 1996 comparison.     

Evaluation of the results of various technics of non-surgical therapy of varicocele

A new method of non-surgical treatment of varicocele syndrome is described: it consists in sclerotherapy of spermatic vein by trans-femoral percutaneous catheterization with balloon-catheters. In 8 cases venous thrombosis has been induced by direct electric clotting. The techniques and a 6 months follow-up are discussed. It is pointed out that this procedure should be considered as the method of choice for tubular lesions and sub-fertility prophylaxis in young people and in childhood.     

延迟性脾破裂误漏诊原因分析

目的探讨延迟性脾破裂误漏诊原因和预防措施.方法回顾性分析总结12例延迟性脾破裂中的诊断和误漏诊的经验与教训.结果本组延迟性脾破裂的误漏诊5例(41.66%).对多发伤与脾破裂并存可能认识不足,外伤史轻微或伤员隐瞒外伤史,缺乏腹痛-缓解-突然再腹痛的典型病史,缺乏“对冲性脾破裂”力学分析和整体化诊断思路等为其误漏诊的主要原因.结论详细的外伤史和全面系统检查,重视腹以外多发伤掩盖腹内脏器伤及延迟性脾破裂可能.确立外伤-腹内脏器伤-脾破裂整体化诊断思路.不间断地辅以B超检查脾形态学变化和腹内有无积液,腹腔穿刺确定有无血腹、X线胸腹部检查观察左侧胸肋角和膈肌运动情况、必要时CT检查以尽早发现脾包膜下血肿,降低延迟性脾破裂误漏诊率.