多器官功能障碍综合征大鼠肺组织血栓调节蛋白和基质金属蛋白酶-9的表达

Objective To determine the expressions of thrombomodulin (TM) and matrix metalloproteinase-9(MMP-9) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. Method Forty adult mule Sprague-Dawley (SD) rats were randomly divided into two groups,namely the normal control group and the MODS model group. The rats of model group were further divided into four subgroups as per different intervals (6 h, 12 h, 24 h and 48 h) ,and there were 8 rats in each groups. The animal models of MODS were estabhshed by two hits,the left eyeball of each model rat was removed to bleed to 2 mL/100g,and four hours later, lipopolysaccharide (LPS 5 mg/kg) was injected into intraperitoneal cavity of model rats. The same volume of saline was injected intraperitoneally into rats of control group instead of LPS. All rats were sacrificed at various intervals. The histological changes in lung tissue were observed by naked eye and light microscope. The expressions of TM and MMP-9 proteins were deteceted by using immunohistechemistry. One-way ANOVA was used for comparison among multiple group. Results (1)There were no histopathological changes in lung of rats of control group, and the lung injury was serious in MODS rots. (2) Compared with the rots of con-trol group, the expression of TM in lung tissue of MODS rats increased 6 hours after LPS, reached peak 12 hours later(P <0.01),and then decreased during 24～48 period.There was no significant difference in expression of TM between two groups 48 hours later. Compared with control group, the expressions of MMP-9 in lung tissue of MODS rats didn't significantly increase 6 ～ 48 hours after LPS (P < 0.01). Conclusions There are endothelium damage and extracellular matrix damage found in the lung tissue of rats at the early phase of MODS. TM and MMP-9 are good biomarkers of endothelium and extracellular matrix damage, and they can be used for diagnosing and es-timating the severity of injury lungs at the early phase of MODS.     

提高骨科临床实习教学质量应注意的几个问题

基于医学生在骨科临床实习中不利因素影响,从强调骨科基本操作和基础理论的学习,注重医德医风和心理素质的培养,建立有效的临床实习考评制度,加强临床教师队伍建设等几个方面出发,以培养出高质量、高素质的医学生,为我国医学事业的健康发展打下良好的基础。     

基质金属蛋白酶9在多器官功能障碍综合征大鼠肾组织的表达

炎性介质的大量释放导致内皮细胞损伤是引起多器官功能障碍综合征(MODS)的基础.有关器官内皮细胞激活和损伤在MODS病理过程中的作用研究较多~([1]),而细胞外基质在MODS中的变化及与其的关系目前还未检索到相关报道.因此,我们选择了降解细胞外基质主要成分Ⅳ型胶原的基质金属蛋白酶9(MMP-9)作为观察指标,并与血清MMP-9比较,探讨细胞外基质的变化在MODS肾损伤中的作用.     

构建及鉴定TACO特异性10-23DRz表达载体重组耻垢分枝杆菌

目的 构建一种可表达特异性10-23脱氧核酶(deoxyribozyme,DZ)的巨噬细胞靶向性细菌载体疫苗,并鉴定其在巨噬细胞表达10-23DZ的能力以及抑制靶基因表达的效果.方法 采用亚克隆方法将分枝杆菌复制子oriM克隆入10-23DRz真核表达载体pSDE01中,获取可在分枝杆菌和真核细胞中穿梭的重组质粒pSDE02.在前期研究基础上选择一有效的针对巨噬细胞taco基因的10-23DZ--DZ1,根据DZ1序列设计并制备DZ1的双链表达序列,插入pSDE02中获取重组质粒pDZM01.将pDZM01经电穿孔方法转化入耻垢分枝杆菌Ms1-2c株构建重组耻垢分枝杆菌rMS-DZ1.分别采用Ziehl-Heelson染色法和直接荧光观测法鉴定重组耻垢分枝杆菌的巨噬细胞靶向性.用rMS-DZ1感染巨噬细胞株RAW264.7,分别于感染后的24 h和48 h采用斑点杂交方法检测rMS-DZ1在巨噬细胞中表达DZ1的情况,再分别采取RT-PCR和免疫印迹方法检测巨噬细胞TACO表达的差异.结果 限制性内切酶酶切、菌落PCR及测序结果显示pSDE02、pDZM01和rMS-DZ1构建成功.rMS-DZ1感染RAW264.7细胞后24 h和48 h,经点杂交方法均检测到了DZ1的表达.半定量RT-PCR和免疫印迹检测结果显示,与未感染组比较,rMS-DZ1感染后24 h和48 h时巨噬细胞TACOmRNA分别下降了67.90%和57.14%,TACO蛋白水平分别下降了53.85%和68.92%.同时,表达对照寡脱氧核糖核苷酸DZ1U的重组耻垢分枝杆菌rMS-DZ1U感染RAW264.7细胞后,其TACOmRNA和TACO蛋白水平与未感染组比较均无明显差异.结论 本研究成功构建了一种可表达特异性10-23DZ的巨噬细胞靶向性重组耻垢分枝杆菌的载体,可在巨噬细胞内表达并抑制相应靶基因的表达,可作为研制治疗用疫苗的基础,这也是国内外首次报道可表达10-23DZ的细菌性载体.

Abstract：

Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.     

腺苷脱氨酶对结核性脑膜炎诊断价值的Meta分析

目的 探讨脑脊液腺苷脱氨酶(ADA)检测在结核性脑膜炎(TBM)中的诊断价值.方法 检索1990年1月-2010年10月Cochrane图书馆、PubMed和万方数据库等数据库,按照Cochrane协作网推荐的诊断试验纳入标准筛选文献,采用QUADAS工具评价纳入文献质量,应用Meta-Disc 1.4软件对纳入研究进行综合定量评价并绘制综合受试者工作曲线(SROC).结果 共检索到相关文献149篇,排除134篇,符合纳入标准的15篇进入Meta分析,涉及研究对象1 642例.异质性检验提示存在异质性,按随机效应模型进行Meta分析,ADA诊断TBM的敏感度、特异度、阳性似然比、阴性似然比、诊断优势比的汇总值分别为0.73[95%CI(0.70,0.76)]、0.90[95%CI(0.88,0.91)]、7.79[95%CI(4.80,12.63)]、0.25[95%CI(0.17,0.35)]和36.01[95%CI(16.08,80.66)]SROC下面积AUC为0.8856(Se=0.0412),Q值为0.8162.结论 脑脊液ADA对TBM的临床诊断价值有较低的敏感度和较高的特异度,测定脑脊液ADA可作为诊断TBM的辅助诊断指标.     

腓肠神经营养血管蒂逆行岛状皮瓣修复足踝部软组织缺损

1999年6月至2010年12月,我们采用腓肠神经营养血管蒂逆行岛状皮瓣修复足踝部软组织缺损患者32例,获得了满意的疗效,现报告如下。1临床资料本组32例,男22例,女10例。年龄3～67岁,中位数36岁。均为足踝部软组织缺损患者。致伤原因:     

巨噬细胞极化及其在感染性疾病中的作用

巨噬细胞作为重要的固有免疫细胞和抗原提呈细胞,在机体抗感染免疫中发挥着重要作用.在不同的微环境下,巨噬细胞可发生表型和功能极化从而呈现出不同的功能,一般将经典途径和替代途径活化的巨噬细胞(M1型和M2型)作为巨噬细胞功能可塑性调变的两个极端.随着研究的深入,巨噬细胞极化类型的诱导及特点也逐渐被揭示,其在微生物感染免疫中的作用日益受到重视.     

结核分枝杆菌双组分调节系统Dos的研究进展

全球目前约有1/3人口感染了结核分枝杆菌(Mycobacterium tuberculosis,MTB).在这些感染者中,约有10%可能发展为活动性结核病,其余绝大多数为无症状的潜伏感染者.在潜伏感染者体内,MTB以一种代谢缓慢的休眠菌形式存在,当机体免疫力低下时,这种休眠菌可能转变为代谢活跃的细菌,引起活动性结核病.另一方面,在活动性结核病患者体内,在机体免疫或化疗药物的压力下,也会有一部分MTB以休眠形式存在.现有的抗结核药物大都是针对结核菌的繁殖阶段起作用,对引起潜伏感染的休眠菌作用效果甚弱.因此,为了杀灭残存的休眠菌,目前的结核病化疗方案至少需要维持化疗6个月以上,代价昂贵且效果不佳.研究表明,机体整体免疫状态和MTB所处的局部微环境对MTB存在状态以及毒力有重要影响.近年研究发现,MTB双组分调节系统(two-component system,2CS)与MTB应对外界环境刺激发生的代谢改变明显相关,研究该系统的调控机制,对寻找抗结核潜伏感染药物的分子靶位以及开发新型疫苗具有重要意义.本文就MTB 2CS Dos的研究进展进行综述.     

足部组织在手部组织缺损中的应用

目的 :总结足部组织修复手部组织缺损的临床应用及疗效观察。方法 :应用足部组织游离移植修复手部组织(皮肤、肌腱、血管、骨关节等 )缺损 3 8例 ,急诊Ⅰ期修复 2 2例 ,择期Ⅱ期修复 16例。①腕部掌侧组织缺损合并主干血管长段损伤 2例。②拇手指组织部分缺损 ,Ⅲ°以下拇指缺损拇趾甲皮瓣修复[1 ] 4例 ,第 2、3足趾部分或全部游离移植再造手第 1、2、3指 14例 ,单纯趾甲瓣移植修复第 2指指甲缺损 2例。③手部掌背侧皮肤缺损 ,虎口及手指处皮肤挛缩或缺损12例 :应用足背皮瓣及其复合组织瓣 3例 ,足背多叶型皮瓣 3例 ,趾蹼皮瓣 4例 ,趾侧方皮瓣 2例。④趾腹皮瓣 2例 ,骨关节移植 2例。结果 :需做血管吻合 3 6例 ,坏死 2例 ,其中因伤口感染坏死 1例 ,1例因为血管栓塞时间过长失去探查机会而坏死 ,成活率 97.2 %。手部功能及外形恢复均较为满意。供区 2例植皮部分坏死瘢痕愈合行走时疼痛。结论 :手部组织缺损选择与其功能及外观相似的足部组织修复 ,术后效果满意。