抑制脑血栓形成的硫磷酸酯脱氧寡核苷酸的亲和性研究

目的为抑制脑血栓形成,合成与TF基因启动子区切应力反应元件(SSRE)形成三链DNA的硫代磷酸酯寡核苷酸(TFO).方法设计TFO序列14条,采用固相亚磷酰胺三酯固相法合成TFO.硫代磷酸酯修饰在TFO的3'末端进行.应用电泳迁移分析(EMSA)观察寡核苷酸和硫代脱氧寡核苷酸的亲和性.结果在设计合成的14条寡核苷酸中,与靶序列能形成三链DNA的TFO只有T21GTa、T14GTa和T15GTa,其Kd值分别为3.6×10-10、1.0×10-9和1.0×10-8(M),经硫代磷酸酯修饰后分别为2.3×10-9、3.8×10-9和1.5×10-8.结论硫代磷酸酯修饰的T21GTa-ps、T14GTa-ps和T15GTa-ps能够与TF基因启动子SSRE的3个位点形成三链DNA.     

抑制脑血栓形成的硫代磷酸酯脱氧寡核苷酸的亲和性研究

目的：为抑制脑血栓形成，合成与TF基因启动子区切应力反应元件（SSRE）形成三链DNA的硫代磷酸寡核苷酸（TFO）。方法：设计TFO序列14名，采用固相亚磷酰胺三酯固相法合成TFO。硫代磷酸酯修饰在TFO的3’末端进行。应用电泳迁移分析（EMSA）观察寡核苷酸和硫代脱氧寡核苷酸的亲和性。结果：在设计合成的14条寡核苷酸中，与靶序列能形成三链DNA的TFO只有T21GTa、T14GTa和T15GTa，其Kd值分别为3．6&;#215;10^-10、1．0&;#215;10^-9和1．0&;#215;10^-8(M)，经硫代磷酸酯修饰后分别为：2．3&;#215;10^-9、3．8&;#215;10^-9和1．5&;#215;10^-8。结论：硫代磷酸酯修饰的T21GTa-ps、T14GTa-ps和T15GTa-ps能够与TF基因启动子SSRE的3个位点形成三链DNA。     

诱骗寡核苷酸下调白血病细胞中组织因子高表达的研究

目的:探索针对诱导性组织因子(TF)高表达的靶向抑制的方法,探讨针对TF启动子-247～-230区域的诱骗寡核苷酸对U937-PR9细胞中PML/RARα融合蛋白诱导的TF表达是否存在竞争性抑制作用。方法:采用诱骗寡核苷酸技术,模拟TF启动子-247～-230的序列,人工合成该片段的双链DNA,转染U937-PR9细胞,通过流式细胞术监测转染效率并确定最佳电转浓度,通过实时PCR和酶联免疫吸附试验等技术,比较转染前后加Zn2+组与不加Zn2+组的TF表达水平变化。结果:流式细胞术显示,10μmol/L电转浓度下能够实现高效转染,标记的寡核苷酸在细胞内能够稳定存在72 h。未转染对照组中,加Zn2+诱导后TF表达明显上升;转染组中,加Zn2+诱导的TF表达水平与不加Zn2+组几乎没有差异。结论:特异性诱骗寡核苷酸对TF诱导性高表达存在靶向的竞争性抑制作用,该方法为急性早幼粒细胞白血病TF高表达引发的并发症提供了靶向治疗的新思路。     

NASBA和IS-NASBA的临床应用及研究进展

核酸序列依赖扩增(NASBA)是一种特异性等温扩增RNA的技术,是基于一个等温扩增酶系统,包括AMV逆转录酶(AMVRT)、RNaseH和T7RNA聚合酶。以RNA为模板,在AMV逆转录酶和引物Ⅰ的作用下合成cDNA,引物Ⅰ的5′端带有T7启动子序列,然后引物Ⅱ与cDNA链退火合成cDNA链的互补链,进而形成了含完整T7启动子序列的双链DNA。T7RNA聚合酶以此双链DNA为模扳,合成大量拷贝的RNA片段。原位NASBA(insituNASBA,ISNASBA)是结合原位杂交技术而发展起来的,主要用于分子病理学、分子形态学领域的杂交技术。     

MMP-7反义寡核苷酸对KATOIII细胞MMP-7mRNA表达的影响

目的 观察MMP -7反义寡核苷酸对恶性肿瘤细胞MMP -7mRNA表达的影响 ,探讨MMP -7反义寡核苷酸的治疗意义。方法 将嵌合性硫代磷酸修饰的MMP -7反义寡核苷酸通过FuGENETM6导入低分化的胃癌细胞株KATOIII,采用RT-PCR检测该细胞表达情况。结果 ①FuGENETM6能提高转染效率 ,增强反义抑制作用。②MMP -7反义寡核苷酸转染的KATOIII细胞MMP-7mRNA表达量(MMP -7/β-actin光密度比值为0.305±0.02)明显低于对照组、PS -sODN及PS -mODN组(分别为1.590±0.012 ,1.140±0.03 ,1.508±0.017) ,P<0.05。 结论 MMP -7反义寡核苷酸可明显的降低肿瘤细胞的MMP -7mRNA表达水平     

成纤维细胞生长因子9影响成骨细胞核心结合因子α1基因表达的作用

目的:探讨成纤维细胞生长因子9(fibroblastgrowthfactor-9,FGF-9)对成骨细胞核心结合因子α1(Cbfa1)的调节作用及其与有丝分裂原激活蛋白激酶(mitogen-activatedproteinkinase,MAPK)信号传导通路的关系,从而为骨质疏松症等骨性疾病发病机制的研究奠定了理论基础。方法:在成功构建含小鼠Cbfa1基因调控序列的荧光素酶报告基因表达质粒的基础上,利用瞬时转染技术,同时应用RT-PCR方法检测FGF-9对Cbfa1基因启动子活性和mRNA表达的影响,以及MAPK信号传导通路在其中的作用。结果:MC3T3-E1细胞中,FGF-9(1×10-9～1×10-5mol/L)作用24h能够降低Cbfa1基因启动子活性(P<0.05)。加入PD98059(10μmol/L)后阻断了FGF-9所诱导的Cbfa1基因启动子活性的降低(P<0.01)。C2C12细胞中,1×10-5mol/L的FGF-9处理后亦可以降低小鼠Cbfa1基因启动子活性(P<0.05),且未见到PD98059的阻断作用(P<0.01)。另外,在MC3T3-E1细胞中,FGF-9(1×10-9mol/L)处理后Cbfa1基因mRNA水平于第4天开始下降,且持续至第6天。C2C12细胞中未见到FGF-9(1×10-7mol/L)对Cbfa1基因mRNA表达水平的刺激或抑制作用。结论:FGF-9部分通过MAPK信号传导通路抑制MC3T3-E1和C2C12细胞中小鼠Cbfa1基因启动子活性及mRNA的表达。     

介导人α-反义寡核苷酸慢病毒载体的构建

目的构建介导人α-反义寡核苷酸的慢病毒载体,为α-反义寡核苷酸对β-地中海贫血基因治疗的体内试验提供稳定的转染细胞载体。方法根据人α-反义寡核苷酸序列设计siRNA、合成DNA片段,通过双限制性内切酶消化和连接的方法构建pGCSIL-vshRNA-GFP载体质粒,接着该质粒转化感受态的大肠杆菌E.coliDH5α,通过PCR及基因测序鉴定阳性克隆,再经Lipofectamine 2000将pGCSIL-vshRNA-GFP,pHelper 1.0和pHelper 2.0三质粒系统共转染293 T细胞包装病毒,通过绿色荧光蛋白的表达测定收集的病毒滴度。结果重组质粒的外源基因PCR鉴定正确,基因测序结果与所需要的α-反义寡核苷酸序列完全一致,浓缩后病毒滴度为5×109TU/ml。结论成功构建介导人α-反义寡核苷酸的慢病毒载体。     

错配杂交化学发光法定量检测p16基因过甲基化

目的建立一种基于错配杂交化学发光检测p16基因启动子区过甲基化的定量分析方法。方法用亚硫酸氢钠修饰基因组DNA,所有未甲基化的胞嘧啶都被转变为尿嘧啶,而甲基化的胞嘧啶则不发生变化。设计合成1对不含CpG位点的引物,同时扩增甲基化或非甲基化目的DNA片段,用2条分别与甲基化及非甲基化CpG位点互补的寡核苷酸探针与扩增产物进行杂交及化学发光检测,通过探针杂交信号强度比确定样品DNA中甲基化的p16基因的比例。结果错配杂交化学发光法具有较好的精密度(CV=5·2%~6·4%)和灵敏度(2·5×10-4pmol),检测已知混合样品的p16基因甲基化率分别为0%、23%、52%、70%及93%,与实际结果一致。结论本研究建立的错配杂交化学发光法是一种检测快速、操作简便的p16基因甲基化的定量检测方法。     

尾加压素对人单核巨噬细胞DNA、RNA及蛋白质合成的影响

目的:观察新近发现最强的缩血管活性肽之一的尾加压素对体外培养的人单核巨噬细胞DNA,RNA及蛋白质的合成过程的影响。方法:实验于2004-10/12在南方医科大学心内科实验室及广州军区总医院中心实验室完成。取健康自愿者50mL新鲜静脉血,用聚乙酰胺吡咯烷酮包被的硅胶颗粒液(d=1.130)分离单核细胞。采用24孔塑料培养板,每孔加入有巨噬细胞特性、浓度为2×105个/mL细胞0.5mL,随机分成4组。分别加入1×10-8,1×10-9,1×10-10,0mmol/L尾加压素,每组再随机分成3小组,分别加入3H-Leu,3H-TdR,3H-UR3.7×1010Bq/mL。培养7d,分别于第1、4、7天测定样品3H-Leu,3H-TdR,3H-UR的放射性,并分别代表蛋白质、RNA、DNA的合成状态。细胞蛋白含量测定:将细胞培养于8孔塑料培养板中,随机分成4组,培养至第3天分别加入1×10-8,1×10-9,1×10-10,0mmol/L尾加压素,继续培养4d,收集细胞。Lowery法常规测细胞蛋白含量。结果:①在RPMI1640+300mL/L自体血清中,细胞生长、成熟、分裂状态良好,细胞合成DNA、RNA、蛋白质迅速,尤以3H-UR掺入作用较强。②成熟的人单核巨噬细胞与1×10-8,1×10-9,1×10-10,0mmol/L浓度范围的尾加压素孵育24h,未见细胞有形态学改变。1×10-10mmol/L浓度尾加压素在24h时即对3H-UR,3H-TdR掺入有抑制作用,随浓度升高和时间延长,作用均加强;且在1×10-9,1×10-8mmol/L浓度下对RNA作用较DNA强(0.608±0.005,0.730±0.007,t=18.8,P<0.01;0.500±0.007,0.571±0.004,t=10.4,P<0.01)。③加入1×10-8,1×10-9mmol/L浓度尾加压素与未加入尾加压素组比较对蛋白质的合成有抑制作用[(22.4±0.707)×10-3,(32.5±0.634)×10-3dpm,t=23.8,P<0.01;(27.35±0.636)×10-3,(32.5±0.634)×10-3dpm,t=12.8,P<0.01],但对蛋白质合成的抑制作用明显低于对DNA,RNA合成的抑制作用(0.688±0.003,0.571±0.004,0.500±0.007,t=12.3,17.5,P<0.001;0.840±0.007,0.730±0.007,0.608±0.005,t=9.6,31.2,P<0.001)。④1×10-8mmol/L尾加压素与成熟人单核巨噬细胞共同作用4d,与未加入尾加压素组比较,细胞的蛋白质含量显著降低(0.225±0.012,0.319±0.027,t=5.76,P<0.001),并呈浓度依赖性。结论:尾加压素在1×(10-10～10-8)mmol/L浓度范围内对单核巨噬细胞DNA,RNA合成有明显抑制作用,对RNA较强,对蛋白质作用较弱;其与成熟过程中人单核巨噬细胞较长时间孵育时,对蛋白质合成抑制作用变明显。尾加压素抑制单核巨噬细胞DNA,RNA,蛋白质合成代谢,从而影响细胞生长、分裂增殖及免疫吞噬等功能。     

原发性肾病综合征血三系细胞骤降1例

1　病例报告　　男 ,70岁。全身水肿 5个月伴皮肤紫癜 2 0 d余入院。5个月前在农村医院被诊断为原发性肾病综合征 ,服用雷公藤根等中草药进行治疗至今。半个月前因疲劳 ,再次诱发水肿 ,于 2 0 0 4 -0 6 - 10来我院就诊。 2 0 0 4 - 0 5 - 30～ 2 0 0 4 - 0 7- 0 6期间 ,共查血 7次(血三系细胞检查 ) ,结果为 :2 0 0 4 - 0 5 - 30 RBC4 .30× 10 1 2 / L,WBC8.0× 10 9/ L,PL T190× 10 9/ L;2 0 0 4 - 0 6 - 11RBC3.2 8×10 1 2 / L ,WBC1.4× 10 9/ L ,PL T5 0× 10 9/ L ;而后 5次检查血三系细胞呈逐渐上升趋势 ,2 0 0 4 - 0 7-…     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.     

Interprofessional approaches to creating safe,high quality health care

Three supplementary perspectives are presented arguing that interprofessional collaboration is both necessary and desirable. Nonetheless, there are often too many serious intra-professional barriers and obstacles to interprofessional collaboration to make it successful. Some of these barriers, it is argued and illustrated, are found in the multiple ways in which professional identity is tacitly acquired and embodied in the practitioners' habitual, everyday practice. The paper then explores ways in which reflection, especially Second order reflection, can help to elucidate and overcome these obstacles, as well as increasing professional adaptability and competence.     

Systematic Reviews Published in the January 2011 Issue of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD and monthly online. The January 2011 issue (first quarterly DVD for 2011) contains 4515 complete reviews, 1985 protocols for reviews in production, and 13,521 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 641,000 randomized controlled trials, and 14,018 cited papers in the Cochrane methodology register. The health technology assessment database contains over 9300 citations. One hundred and seven new reviews have been published in the last 3 months, of which five have potential relevance for practitioners in pain and palliative medicine.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

Prise en charge des entorses externes de cheville : étude clinique préliminaire sur l’efficacité du dispositif Myolux™

Ankle sprains are the most common injury of the musculoskeletal system and are associated with significant societal and economic impacts. It has been proven that classical therapeutic strategies may not be effective in preventing recurrent injuries: the recurrence rates reported in the literature can reach 73%. In order to provide an effective rehabilitation solution, a destabilizing orthosis was developed. This device is equipped with a mechanical articulator reproducing the subtalar mechanics and placed under the heel. In this paper, we present the main results of a preliminary clinical study conducted between 2004 and 2007. All subjects included in this study were treated with the abovementioned orthosis during 10 rehabilitation sessions of 30 minutes each. Data show a relatively low recurrence rate of 12% for the overall population. Moreover, it's of primary importance to note that this satisfactory ratio is largely reduced (3% of recurrence rate) for the 29 patients who performed one training session per month after the 10th initial rehabilitation sessions. Hence, the destabilizing orthosis appears to be an effective solution to prevent recurrent ankle sprains. However, joint protection requires long-term and regular training sessions. This result has motivated the development of a similar device allowing patients to perform training sessions at home. Finally, data obtained in this study are promising awaiting the final results of the comparative, multicentric and independent clinical trials currently managed by the Hospices Civils de Lyon.