人胎脑神经生长因子4基因与质粒载体重组体的体外构建

BACKGROUND： Neurotrophin-4 （NT-4） can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its activity in the central nervous system. Delivering NT-4 into the central nervous system v/a cells or vectors may have therapeutic benefit. OBJECTIVE： To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-NI. DESIGN, TIME AND SETTING： Neural genetic engineering experiment. The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008. MATERIALS： The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences; embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College. TRIzol RNA extraction Kit was purchased from Sigma （USA）, One Step RNA PCR Kit （AMV） etc. were from Takara （Dalian, China）. METHODS： Total RNA was extracted from human embryonic brain tissues using Trizol. The agarose gel electrophoresis showed two bands： 18 S and 28 S, which were essential subunits of total RNA. The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction. MAIN OUTCOME MEASURES： The sequencing results of the DNA in the recombinant of NT-4- pEGFP-NI. RESULTS： The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight. CONCLUSION： The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.     

动脉内皮细胞中TIE2启动CREG真核表达质粒pTIE2-CREG-EGFP-N1的表达

背景：血管内皮发生的分子机制是细胞及基因替代治疗的首要前提。 目的：构建一种通过血管内皮细胞特异蛋白TIE2启动子来启动表达的E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes, CREG)和增强绿色荧光蛋白(enhanced green fluorescence protein, EGFP)融合的真核表达质粒。 方法：根据GenBank中公布的TIE2基因的启动子序列,人工合成TIE2启动子DNA序列,经AseⅠ和NheⅠ双酶切后,亚克隆入表达质粒pEGFP-N1中构建pTIE2-EGFP-N1。同时,用BamHⅠ和EcoRⅠ 双酶切pcDNA3.1 myc-His/hCREG质粒得到CREG基因,亚克隆入质粒pTIE2-EGFP-N1中构建pTIE2-CREG-EGFP-N1,酶切鉴定。应用脂质体法将该质粒转染至体外培养的小鼠动脉内皮细胞,荧光显微镜下观察EGFP的表达;Western blot检测CREG蛋白的表达。 结果与结论：经酶切鉴定证实实验构建的pTIE2-CREG-EGFP-N1质粒正确;体外转染48 h,荧光显微镜下可见EGFP的表达,Western blot检测到CREG蛋白的表达。说明实验成功构建了pTIE2-CREG-EGFP-N1重组质粒,其可携带目的基因在小鼠动脉内皮细胞中有效表达。     

慢性难愈合创面早期防控体系建设探索

北大医疗鲁中医院烧伤皮肤外科在烧伤患者逐渐减少,科室被压缩、合并的情况下,受付小兵院士"战时治烧伤,平时治创面"理念的启发,于2015年成立烧伤与创面修复中心,在淄博市开展了以慢性难愈合创面为主的创面诊疗工作,通过普及创面修复知识,开展居家照护、多学科诊疗、医护一体的工作模式,建立创面修复移动工作站,筹建精准扶贫基地,...     

组织工程学--21世纪面临的机遇与挑战

组织工程学是近20年来医学生命科学领域最具进展与挑战性的前沿科技领域之一,被誉为“一场意义深远的医学革命”、“再生医学的新时代”,标志着复制再生组织与器官的时代的来临!由于它具有修复创伤、重建功能、挽救生命、提高生存质量所特有的优势及巨大的社会、经济价值,而倍受国际学术界及各国政府的高度关注与支持。目前国际上组织工程皮肤、组织工程软骨已有产品面世,并得到美国FDA的批准,国际上已成立了近百家组织工程公司,我国多家大学、研究单位亦相继成立了组织工程研发中心或组织工程公司。目前不但可以构建组织工程化组织,而且复杂生命器官组织工程化构建亦已在国内外积极展开,同时,一些组织工程化组织已初步应用于临床并取得了可喜的临床疗效,显示出组织工程技术可喜的局面、巨大的生命力与宏伟的远景。然而,组织工程学是一门新兴学科,是跨领域、多学科参与合作的边缘学科,目前的工作仅仅是初始,还有大量基本科学问题尚待阐明,大量技术难题尚待攻克;光明与困难共存,机遇与挑战同在;应不失时机抓住机遇,开拓创新,迎接挑战,全力打造组织工程技术的新时代,使组织工程技术能够尽早造福于人类。     

丙型肝炎病毒E1E2蛋白原核表达载体的构建及表达产物的鉴定

目的 :构建丙型肝炎病毒 (HCV)包膜糖蛋白 (E1E2 )基因的原核表达载体 ,为研究HCVE1E2蛋白属性及基因免疫奠定基础。方法 :设计HCVE1E2区基因上、下游引物 ,分别引入BglⅡ及EcoRI酶切位点 ,以含有HCVH株基因序列的质粒pBRTM/HCV1- 30 11为模板 ,通过PCR扩增获得HCVE1E2区基因片段 ,将其克隆入原核表达载体pRSETA ,然后通过酶切和序列测定对其进行鉴定 ;将此表达载体转化入表达菌BL2 1(DE3)pLySs诱导表达 ,并采用SDS -PAGE对表达产物进行鉴定。结果 :经酶切及序列测定分析表明 ,成功地构建了重组原核表达载体pRSETA -HCVE1E2 ;SDS -PAGE显示目的蛋白大量表达 ,且呈非溶状态。结论 :HCVE1E2融合蛋白可以在大肠杆菌中大量表达 ,为HCVE1E2蛋白的制备及基因疫苗的研制奠定了基础。     

Correlates of Suicide in Building Industry Workers

Suicide within the construction industry in Queensland, Australia was reportedly high in a recent Royal Commission report. The current study examined the incidence and causes of suicide in this industry using psychological autopsy and focus group investigations. A total of 64 male suicides occurred over the seven-year period, representing a crude suicide rate of 40.3 per 100,000, significantly greater than the working age Australian male rate. Young employees were at excessive risk with separation/divorce, relationship problems, and untreated psychiatric conditions the major contributors. Focus groups emphasized the importance of work/home interface factors and industry-specific factors preceding suicide.     

Pacemaker leads

Present day pacemaker leads are far superior in every respect to those of the past. Modification of fixation characteristics has reduced displacement rates to 1% or less in most centers. Fracture of multifilar leads is a rarity. Biodegradation of polyurethane insulation appears to be an isolated problem specific to individual lead models and may be related to physical stresses incurred during manufacture or lead insertion. Recent evidence has incriminated an interaction of polyurethane with silver which arises from the drawn braised strand conductor substrate of those leads in which this problem has been noted. This may explain why the problem has been restricted to specific lead models of one manufacturer to date. Lack of uniformity of lead terminal size between manufacturers and even within the same manufacturer's product line continues to baffle this observer. Although past problems of lead displacement have been markedly reduced, the difficulty of removing chronic leads which have become septic appears to have worsened. Modification of existing leads to ensure that the interface between electrode tip and proximal shaft is unidiametric is essential.     

利用重构pc-MDR1质粒快速诱导肝癌细胞耐药

目的：从裸鼠原位耐药肿瘤组织中进行MDR1 cDNA的全克隆和pc-MDR1重组质粒的构建，并转染HepG2细胞，快速诱导其耐药，并筛选其抗性克隆。方法：设计单酶切位点引物，利甩长距离逆转录PcR(Long RT-PCR)技术，由HepG2耐药细胞扩增MDR1 cDNA，约3．8kb大小。将其插入至真核表达载体pcDNA3．0质粒中构建pc-MDR1诱导质粒，使其能够在真核细胞中表达p-gp蛋白。转染HepG2细胞，并用G418筛选出转染的细胞。结果：成功地扩增出3．8kb左右的MDR1 cDNA片段，经酶切鉴定、RT-PCR扩增特异片段和序列测序结果显示质粒构建初步成功，用G418筛选细胞耐药抗性克隆的时间约为14d。结论：从耐药肿瘤组织中进行MDR1 cDNA的全克隆和pc—MDR1诱导质粒的构建成功快速诱导了肝癌细胞发生耐药，为进一步研究肿瘤细胞的耐药机制奠定了很好的基础。     

颅面骨三维有限元模型的建立

选择条件合适的病员一人，采用ＣＴ技术获取其颅面骨（包括下颌骨）各层解剖轮廓图，然后选择节点，划分单元，在图形数字化仪上测量节点座标，对其编号后输入计算机，根据ＮＡＳＴＲＡＮ程序单元卡的填法连接各节点建立颅面骨的三维有限元模型，采用该模型，可以计算各种矫治力在颅面复合体产生的生物力学效果。     

显微外科特色护理专科建设及效果

目的探索显微外科特色护理专科建设的方法及效果。方法 2014~2015年显微外科创建特色护理专科,主要从科室管理、专科队伍建设、护理质量、专科护理技术和护理服务特色5个方面建设。比较特色护理专科建设前后科室护理不良事件发生率、护理服务满意度及护理科研成果。结果特色护理专科建设后,显微外科护理不良事件发生数显著低于建设前,患者对护理服务的满意度显著高于建设前,护士发表论文数、开展新技术新业务数、申报管理创新项目数及申请专利数显著多于建设前(P0.05,P0.01)。结论显微外科建设特色护理专科有效减少了科室护理不良事件发生,提高了患者的护理满意度及护理人员的科研产出。