超声辐照微泡介导脂质体小干扰RNA转染视网膜色素上皮细胞

目的 探讨超声靶向破坏超声微泡介导脂质体小干扰RNA(siRNA)转染视网膜色素上皮(RPE)细胞的价值. 方法将人及大鼠RPE细胞隔孔接种于24孔板中(2×10~5/孔、1×10~5/孔),分5组处理:siRNA+超声(US)、siRNA+微泡(MBs)+US、siRNA+脂质体(L)、siRNA+L+US、siRNA+L+MBs+US.12小时后,荧光显微镜观察并应用流式细胞仪测定阳性细胞比例. 结果在无脂质体的条件下,超声或超声联合微泡不能促进siRNA转染人及大鼠RPE细胞.siRNA+L+US组siRNA转染人RPE细胞效率最高.siRNA+L+US+MB组siRNA转染人及大鼠RPE细胞的效率显著低于siRNA+L+US组. 结论超声辐照可促进脂质体介导的siRNA转染人RPE细胞.     

不同重组病毒载体转染大鼠骨间充质干细胞的效率及其基因表达

背景:利用基因工程细胞进行替代治疗和基因治疗中合适的供体细胞、靶细胞以及相应载体是细胞和基因治疗最为困难的部分.目的:检测重组腺病毒Ad5和Ad5F35、腺相关病毒rAAV1/2和rAAV2、慢病毒LV对大鼠骨髓间充质干细胞的感染效率和外源基因表达水平,拟筛选能够高效转导骨髓间充质干细胞的载体.设计、时间及地点:细胞基因工程对照观察,于2006-10/2007-03在上海市第一人民医院中心实验室完成.材料:清洁级SD大鼠10只用于制备骨髓间充质干细胞.所有重组病毒均携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因,AdS由本实验室制备,Ad5F35由解放军第二军医大学钱其军教授惠赠,rAAV2及rAAV1/2为本元正阳基因技术有限公司产品,LV由中科院上海生化与细胞生物学研究所郭礼和教授惠赠.方法:采用淋巴细胞分离液密度梯度离心法体外分离培养大鼠骨髓间充质干细胞,传至第4代用于实验.按1×105/孔接种于24孔细胞培养板中,1 d后细胞贴壁,设立5组:第1组向细胞培养液中加入10,100,1 000 MOI Ad5-EGFP,第2组加入10,100,1 000 MOIAdSF35-EGFP,第3组加入1×104,1×105vg rAAV1/2-EGFP,第4组加入1×104,1×105vg rAAV2-EGFP,第5组加入30 TU LV-EGFP.Ad病毒感染2d,rAAV及LV病毒感染6d.主要观察指标:EGFP阳性表达及荧光强度.结果:Ad3-EGFP感染24 h后镜下可见EGFP阳性细胞,随着病毒用量的增加EGFP阳性细胞数目增多,荧光亮度增强,12d后阳性细胞开始逐渐减少,荧光亮度减弱.AdSF35-EGFP感染情况与Ad5-EGFP基本相似,但EGFP阳性细胞数和荧光亮度均增加.rAAV1/2-EGFP或rAAV2-EGFP感染6 d后,EGFP阳性细胞数和荧光亮度均较弱.LV-EGFP感染第2天即可见少量EGFP阳性细胞,随着时间延长EGFP阳性细胞数目逐渐增多,至第6天表达EGFP荧光的细胞数量及亮度不再有明显变化.结论:腺病毒Ad5、AdSF35与慢病毒LV能够有效感染体外培养的骨髓间充质干细胞,并表达外源基因,感染效率与病毒用量之间存在量效关系.腺相关病毒rAAV1/2和rAAV2体外感染效果不佳.     

全氟丙烷人血白蛋白微球注射液辅助3D-HyCoSy检查在不孕患者输卵管通畅性评价中的应用价值

目的:研究全氟丙烷人血白蛋白微球注射液辅助实时三维子宫输卵管超声造影(3D-HyCoSy)应用于不孕患者输卵管通畅性评价的价值。方法:选取我院不孕患者76例(2018年2月-2019年2月),均行3D-HyCoSy检查,造影剂选择全氟丙烷人血白蛋白微球注射液,观察输卵管通畅性结果、影像学表现及不良反应。结果:76例不孕患者中45例输卵管双侧通畅,其中24例为原发不孕患者,21例为继发不孕患者;9例输卵管双侧堵塞,其中6例为原发不孕患者,3例为继发不孕患者;22例输卵管一侧通而不畅,其中9例为原发不孕患者,13例为继发不孕患者。结论:全氟丙烷人血白蛋白微球注射液辅助3D-HyCoSy检查不孕患者输卵管通畅性显影效果显著,能指导临床治疗方案的确定。     

全氟丙烷人血白蛋白微球注射液在实时三维子宫输卵管超声造影评价输卵管通畅性中的应用

目的全氟丙烷人血白蛋白微球注射液在实时三维子宫输卵管超声造影评价输卵管通畅性中的应用价值。方法选取在我院就诊的女性不孕症患者2 340例进行实时三维子宫输卵管超声造影检查,造影剂选用全氟丙烷人血白蛋白微球注射液,记录实时三维子宫输卵管超声造影检查结果,随访观察6～12个月,统计分析其妊娠结局。结果 2 340例患者中,47例因插管失败无法进行造影检查,检查成功率97.99%,其余2 293例患者均顺利完成实时三维子宫输卵管超声造影检查,共检查输卵管4 653条,其中输卵管通畅的共3 360条(72.21%);输卵管通而不畅共447条(9.61%);一侧输卵管阻塞共602条(12.94%);双侧输卵管阻塞共244条(5.24%)。对顺利完成造影检查的2 293例患者进行随访,其中321例(14.00%)患者在术后3个月自然怀孕,46例(2.01%)患者在术后6个月内自然怀孕。结论全氟丙烷人血白蛋白微球注射液作为二代造影剂,在子宫输卵管实时造影中的应用具有安全性高、诊断准确率高、不良反应小的特点,值得在临床检查中推广。     

超声辐照声诺维与重组腺相关病毒经不同途径转染大鼠视网膜的实验研究

目的 探讨超声辐照微泡造影剂声诺维(SonoVue)能否增强rAAV2-EGFP转染视网膜的效率.方法 62只Wistar大鼠进行玻璃体腔(10只)及视网膜下(52只)注射.实验分组:病毒+生理盐水、病毒+微泡、病毒+生理盐水+超声辐照和病毒+微泡+超卢辐照.第4、7、35、49、120 d分别用荧光体视镜观察,利用软件进行定量分析,并进行组织切片光镜检查.结果 玻璃体腔注射组持续观察2个月未见荧光;视网膜下注射组的超声辐照微泡组在第4、7、35 d较其他组转染效率高,超声辐照微泡造影剂对眼底组织无明显损伤.结论 超声辐照SonoVue可在体内促进rAAV2-EGFP转染大鼠视网膜.     

全氟丙烷人血白蛋白微球注射剂左心超声造影增强左心室内膜分辨的Ⅲ期多中心临床研究报告

目的由6家医院进行多中心研究,评价南方医科大学南方医院研制的全氟丙烷人血白蛋白微球注射剂经静脉注射后增强左心室内膜边界识别的效果和安全性。方法采用自身注射前后对照方法,对符合纳入标准的研究对象分别经静脉注入稀释的全氟丙烷人血白蛋白微球注射剂0.01ml/kg,由A和B研究者分别独立进行给药前、后造影效果评判比较。结果6家医院的303例受试者,单次静脉注射全氟丙烷人血白蛋白微球注射剂0.01ml/kg。A研究者评价结果左心室显影Ⅱ级占0.7%,Ⅲ级占99.3%;B研究者评价结果左心室显影Ⅱ级占0.3%,Ⅲ级占99.7%。两组研究者分别独立评价的造影有效率均为100%,差异无统计学意义(P>0.05)。各研究中心间用药后左心室显影等级差异亦无统计学意义(P>0.05)。A和B研究者评价左心室可清晰识别的节段数均显著增加,A研究者由(2.68±0.95)节段增加至(5.99±0.10)节段,B研究者由(2.82±1.03)节段增加至(5.99±0.11)节段(P<0.01)。两组研究者分别独立评价的结果差异无统计学意义(P>0.05),造影后内膜节段显示平均增强率为99.7%。全氟丙烷人血白蛋白微球注射剂对血压、心率、呼吸等生命指征无明显临床意义的影响。心电图无明显改变。血尿常规、肝肾功能等化验检查结果的变化亦在正常范围内。仅1例出现不良反应,为轻度恶心、腹泻,发生率为0.33%(1/303),提示该造影剂临床应用安全。结论全氟丙烷人血白蛋白微球注射剂经静脉注射后左心室内膜的分辨效果显著增强,明显提高对室壁节段性运动状态的判定。该造影剂不良反应少,且轻微,适合在临床上应用。     

超声联合微泡增强脂质体介导基因转染RPE细胞的实验研究

目的 比较声诺维联合超声与脂质体法介导基因转染RPE细胞的转染率和安全性,探讨超声联合微泡增强脂质体转染率的可行性.方法 RPE细胞培养在24孔板,质粒用量2 μg/孔,分为对照组、超声辐照组、超声联合微泡组(包括2 W/cm2和3 W/cm2两个亚组)、脂质体组、脂质体+超声+微泡组5组,每组10孔细胞.荧光显微镜观察基因转染情况,流式细胞仪检测基因转染率,Annexin Ⅴ-FITC和PI双染流式细胞法检测凋亡率.结果 单纯超声辐照组基因转染率为(1.06±0.13)%,超声联合微泡组分别为(15.81±1.70)%和(20.86±2.63)%,脂质体组为(30.06±3.49)%,明显高于超声联合微泡组(P<0.05).脂质体+超声+微泡组为(44.51±1.28)%,明显高于脂质体组(P<0.05).结论 脂质体介导EGFP转染RPE细胞的效率高于超声联合微泡的转染率.超声联合微泡能明显提高脂质体的转染率.     

超声介导下重组腺相关病毒载体转染心肌的最佳滴度的实验研究

目的 探讨超声促进携带增强型绿色荧光蛋白报告基因(EGFP)的重组腺相关病毒载体2(rAAV2)心肌转染过程中rAAV2的最佳滴度.方法 健康成年SD大鼠21只随机分为实验组与对照组.实验组给予连接于SonoVue微泡表面的rAAV2-EGFP尾静脉注入,滴度为1.5×109～3.0×101111 vg/ml(virus genome/ml),超声爆破造影剂,促进rAAV2-EGFP心肌内转染.对照组只给予尾静脉注射造影剂并超声爆破(无rAAV2-EGFP).2周后处死大鼠并制作心肌组织冰冻切片.荧光显微镜下检测荧光蛋白的表达量,代表rAAV2转染率.结果 随着注入的rAAV2滴度的增加,心肌内荧光表达量亦增加,至滴度达到1.5×1011vg/ml时荧光的表达量显著增加(P<0.01).结论 促进心肌组织转染的rAAV2的最佳滴度为1.5×1011vg/ml.     

声诺维联合超声介导视网膜色素上皮细胞基因转染的辐照条件优化研究

目的探讨微泡造影剂声诺维联合超声介导绿色荧光蛋白质粒转染人视网膜色素上皮(RPE)细胞的最佳辐照条件。方法人RPE细胞培养在24孔板,质粒用量为2μg/孔,分为不处理组、质粒 超声辐照组、造影剂浓度组、超声声强组、辐照时间组、声波形式组6组,每组5个细胞孔数,超声探头紧贴培养板底部辐照,72h后流式细胞仪测基因转染率,四甲基偶氮唑蓝法(MTT)测细胞活性。结果处理组转染率均大于不处理组(P<0.05)。加入浓度为20%的造影剂,以1W/cm2,50Hz、100Hz的脉冲波和连续波分别辐照1min,细胞生存率逐渐下降。20%的造影剂,50Hz的脉冲波,声强为3W/cm2,辐照1min组转染率最高(20.88±3.74)%,而细胞生存率为82.57%。20%的造影剂,50Hz的脉冲波,声强为2W/cm2,辐照1min组转染率为(15.73±2.52)%,细胞生存率为91.32%。结论20%的造影剂,2W/cm2,50Hz脉冲波辐照1min是RPE细胞基因传染的最佳辐照条件。     

超声靶向辐照微泡造影剂介导重组腺相关病毒转染肾癌细胞

目的 以肾癌细胞为研究对象,探索超声靶向破坏微泡(UTMD)技术对重组腺相关病毒(rAAV)转染率的影响。方法 应用不同感染复数(MOI)的rAAV2转染786-0细胞观察转染率。用不同条件UTMD预辐照rAAV2,观察病毒活性。以不同条件UTMD联合rAAV2转染人肾透明细胞癌(786-0)细胞,测定转染率及细胞生存率,RT-PCR检测细胞内病毒载体基因拷贝数。结果 1×10⁴~1×10⁶ MOI的rAAV2在786-0细胞的转染率为(17.28±2.44)%。UTMD声强≤2.0 W/cm²,时间≤120 s,微泡体积比≤40％,频率1 MHz,占空比(DC)50%,脉冲重复频率(PRF)100 Hz时,UTMD辐照不影响rAAV2的转染活性。在不影响细胞生存率及rAAV2活性的参数下(声强1 W/cm²,时间60 s,微泡体积比20％,频率1 MHz,DC 50%,PRF 100 Hz),UTMD介导rAAV2组细胞的转染率较单纯rAAV组提高2~3倍,并在5天内稳定维持提高效应;实时PCR显示,UTMD介导rAAV2组细胞内病毒载体量较单纯rAAV组提高近9倍。结论 UTMD可安全、有效且稳定地提高rAAV2在肾癌细胞的转染率。     

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.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

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.     

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.     

手转胎头术失败的原因与分娩结局临床分析

目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4％(55/72)]明显高于成功组[31.7％(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3％(42/72)]高于成功组[38.1％ (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9％ (28/72)]高于成功组[23.8％(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大＜6 cm、胎头位于坐骨棘上及宫口开大8～10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1％(44/72)]高于成功组[38.9％(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2％(52/72)]明显高于成功组[34.1 ％(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.