构建阵挛性癫痫伴破碎红纤维综合征患者来源的诱导多能干细胞系

目的构建阵挛性癫痫伴破碎红纤维综合征(MERRF)患者来源的诱导多能干细胞(i PSCs)。方法采集MERRF患者外周血并分离PBMC,用含有4个重编程转录因子(OCT4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,获得患者来源的i PSCs;用细胞形态学、免疫荧光染色、拟胚体形成分化和RT-PCR对i PSCs的多能性进行鉴定;核型分析验证其安全性;焦磷酸测序检测患者来源的i PSCs中线粒体DNA(mt DNA)第8344位的突变比例。结果患者来源的i PSCs经过多能性验证实验证明其具有多能性;核型分析表明,i PSCs核型正常;建立的6个i PSCs细胞系中,mt DNA 8344位点的突变比例分别为68.24%、60.51%、45.95%、24.06%、62.11%和0。结论通过非整合重编程技术成功构建MERRF患者来源i PSCs细胞系,为进一步研究MERRF疾病提供多能干细胞资源。     

人骨髓间充质干细胞体外定向诱导分化为心肌样细胞的动作电位观察

目的 观察诱导分化为心肌样细胞的人骨髓间充质干细胞(hMSCs)是否可产生动作电位.方法 体外分离培养hMSCs,5-氮胞苷诱导向心肌细胞(CMs)分化.分别以体外分离培养的原代搏动的CMs和同代次的hMSCs为阳性对照和阴性对照,利用膜片钳技术观察动作电位.结果 在30个诱导分化成心肌样细胞的hMSCs中有6个细胞可诱发出动作电位;CMs具有典型的动作电位;未经诱导的hMSCs没有引出动作电位,5-氮胞苷诱导后细胞可以引出微弱的动作电位.结论 实验结果表明hMSCs经5-氮胞苷诱导后,在电学特性方面具有向CMs分化的潜能;与CMs具有的电生理特性相似,这些心肌样细胞具有兴奋性.     

正常与损伤条件下乳鼠原代心肌细胞培养上清液对人骨髓间充质干细胞向心肌样细胞的诱导作用

目的 体外观察正常及损伤状态下的心肌细胞(CMs)培养上清液诱导人骨髓间充质干细胞(hMSCs)向心肌样细胞分化的作用.方法 体外分离培养hMSCs及乳鼠原代CMs,制备正常搏动状态及损伤状态下的CMs培养上清液,分别对传代hMSCs培养诱导27～30 d.倒置显微镜观察两组的细胞形态,免疫细胞化学染色检测两组心肌细胞特异性标志物cTnI及Desmin的表达.结果 hMSCs经正常搏动CMs上清诱导培养后,只有少数细胞变宽大,部分细胞表达cTnI,但不表达Desmin;经损伤CMs上清诱导培养后,hMSCs变为宽大,大部分细胞表达cTnI,部分细胞表达Desmin.定量分析,正常与损伤状态下培养上清液诱导细胞表达cTnI、desmin的阳性百分比及阳性表达强度OD值均有非常显著性差异(P<0.01).结论 正常与损伤状态下CMs培养上清液均可诱导hMSCs向心肌样细胞分化,CMs损伤状态下的细胞培养上清液更有利于促进其分化.     

脂多糖刺激多能干细胞诱导人源心肌细胞与大鼠心肌细胞的体外模型比较

目的比较脂多糖(lipopolysaccharides,LPS)对多能干细胞诱导的人源心肌细胞(human induced pluripotent stem cell-derived cardiomyocytes,hiPS-CMs)及原代新生大鼠心肌细胞(cardiomyocytes,CMs)的影响。方法不同浓度LPS处理hiPS-CMs和原代新生大鼠CMs 24~48 h,通过实时无标记细胞分析技术(xCELLigence Real-Time Cell Analyser Cardio system RTCA)观察hiPS-CMs和原代新生大鼠CMs的电生理变化;qRT-PCR方法检测NPPB mRNA表达水平和qPCR阵列法检测炎性基因表达水平。结果不同浓度LPS刺激后,原代新生大鼠CMs的电生理变化为搏动频率增加和搏动幅度减少(P<0.01),NPPB mRNA表达水平增加(P<0.01)。在hiPS-CMs中,相对应的LPS浓度刺激未能引起搏动幅度和搏动频率显著变化(P>0.05),NPPB mRNA表达水平未显著增加(P>0.05)。进一步增加浓度(2.5μg/mL~40μg/mL),hiPS-CMs的搏动幅度和搏动频率仍未发生明显变化(P>0.05),NPPB mRNA在高浓度LPS(5μg/mL~40μg/mL)发生差异性改变(P<0.01)。最后,在炎症相关基因表达方面,原代新生大鼠CMs表现为C3、Gpnmb、Atf3、Il6r和Ly96基因水平上调至1.5倍,hiPS-CMs表现为AK4、TOLLIP、SPP1、FABP1、IL6R、LY96和C3基因水平上调至1.5倍。结论与原代新生大鼠CMs相比,hiPS-CMs受到LPS的损伤明显减轻,表现出不同的炎症基因表达模式。     

缺氧诱导因子-1对心肌细胞缺氧/复氧损伤的调节作用

目的 观察缺氧诱导因子-1α(hypoxia-inducible factor-1,HIF-1α)的活性与靶基因表达的变化,探讨其对体外培养的心肌细胞缺氧/复氧(hypoxia- reoxygenation,H/R)损伤的调节作用.方法 取SD新生大鼠(1～3日龄)分离培养心肌细胞;在培养的第3天将心肌细胞随机分为3组(每组重复6 次):正常对照组、缺氧/复氧组(H/R组)、甲基乙二酰基甘氨酸组(DMOG+H/R组);建立缺氧/复氧损伤模型,以脯氨酸羟化酶的抑制剂DMOG进行干预;观察各组心肌细胞的搏动频率;应用酶免疫法检测各组心肌细胞培养上清中的肌钙蛋白I(cTnI)含量;采用RT-PCR法检测各组HIF-1α及下游因子血红素氧合酶-1(HO-1)、血管内皮生长因子(VEGF)、葡萄糖转运蛋白-1(GLUT-1)mRNA水平.结果 与正常对照组相比,H/R组、DMOG+H/R组心肌细胞的搏动频率均明显减少(P﹤0.01),而DMOG+ H/R组心肌细胞的搏动频率又较H/R组明显升高(P﹤0.01);与正常对照组相比,H/R组、DMOG+H/R组的cTnI含量明显增高(P﹤0.01),且HIF-1α、HO-1、VEGF、GLUT-1 mRNA水平亦明显增高(P﹤0.01或P﹤0.05);与H/R组相比,DMOG+H/R组心肌细胞HIF-1α、HO-1、VEGF、GLUT-1 mRNA水平明显增高,而cTnI含量则明显降低(P﹤0.05).结论 HIF-1的稳定表达对心肌细胞缺氧/复氧损伤具有保护作用.     

成纤维细胞影响心肌细胞搏动频率的观察

目的:观察影响心肌细胞搏动频率的各种因素并探讨其作用。方法:实验于2003-05/2005-09于泰山医学院生化教研室、中心实验室完成。取新生的Wistar大鼠100只,无菌的取出心脏,培养不同浓度(0.5×108L-1,1.0×108L-1,1.5×108L-1,2.0×108L-1)的心肌细胞和成纤维细胞,分别在培养的第2,3,4天,利用Recorder-8050磁带式录像机观察并记录心肌细胞的搏动频率。同时将培养的心肌细胞分别用不同浓度的成纤维细胞培养液(80%,50%,20%)和不同时间间隔的成纤维细胞培养液(2,4,6d)进行处理,在培养的第2,3,4天观察心肌细胞的搏动频率。最后采用内皮素受体阻断剂BQ123(1.0×10-6mmol/L)处理心肌细胞,观察并分析内皮素能否影响心肌细胞的搏动频率。结果:①心肌细胞单独培养时,其浓度越大,培养时间越长,搏动频率越高。②低浓度组(0.5×108L-1)的搏动频率在96h达高峰,高浓度组(2.0×108L-1)的搏动频率在96h达高峰,两者之间有显著性差异(88.9±5.8次/min,144.2±6.2次/min,P<0.05)。③用成纤维细胞培养液培养心肌细胞,发现与对照组相比,第2天收集的培养液在48h时的作用最强(116.6±6.6次/min,89.7±4.2次/min,P<0.05);高浓度组在48h时的作用也最强,与对照组有显著性差异(100.3±7.9次/min,85.2±6.1次/min,P<0.05)。④BQ123能显著降低成纤维细胞培养液诱发的增加细胞搏动频率作用(113.9±6.5次/min,95.1±5.次/min,P<0.01)。结论:心肌细胞的搏动频率受多种因素的影响,是多种因素综合作用的结果。细胞的浓度、培养时间都能影响其频率;成纤维细胞培养液也能调节心肌细胞的频率;而内皮素可能是其发挥作用的关键物质之一。     

脱落细胞线粒体基因A1555G突变在诊断线粒体耳聋中的应用

目的用PCR-RFLP技术检测脱落细胞(尿液沉渣细胞和颊黏膜细胞)线粒体基因A1555G突变,探讨脱落细胞用于诊断线粒体基因突变相关耳聋的可行性。方法收集福建省某特殊教育学校126名聋哑学生和1个线粒体基因A1555G突变的遗传性耳聋家系6例患者外周血及尿液沉渣细胞和颊黏膜细胞,用PCR-RFLP技术筛查患者是否携带线粒体DNA A1555G突变,并与测序法进行比较。结果尿液沉渣细胞检测线粒体DNA A1555G突变的检出率最高(9/132),颊黏膜细胞和外周血细胞均为7/132。PCR-RFLP筛查结果与直接测序法基本相符。结论脱落细胞适用于耳聋相关线粒体DNA A1555G突变检测。     

成纤维细胞影响心肌细胞搏动频率的观察

目的：观察影响心肌细胞搏动频率的各种因素并探讨其作用。 方法：实验于2003-05／2005—09于泰山医学院生化教研室、中心实验室完成。取新生的Wistar大鼠100只，无菌的取出心脏，培养不同浓度（0.5&;#215;10^5 cells/mL, 1.0&;#215;10^5 cells/mL, 1.5&;#215;10^5 cells/mL, 2.0&;#215;10^5 cells/mL）的心肌细胞和成纤维细胞，分别在培养的第2，3，4天，利用Recorder-8050磁带式录像机观察并记录心肌细胞的搏动频率。同时将培养的心肌细胞分别用不同浓度的成纤维细胞培养液（80％，50％，20％）和不同时间间隔的成纤维细胞培养液（2，4，6d）进行处理，在培养的第2，3，4天观察心肌细胞的搏动频率。最后采用内皮素受体阻断剂BQ123（1．0&;#215;10^-6 mmol／L）处理心肌细胞，观察并分析内皮素能否影响心肌细胞的搏动频率。结果：①心肌细胞单独培养时，其浓度越大，培养时间越长，搏动频率越高。②低浓度组（0．5&;#215;10^8 L^-1）的搏动频率在96h达高峰，高浓度组（2．0&;#215;10^8 L^-1）的搏动频率在96h达高峰，两者之间有显著性差异（88．9&;#177;5．8次／min．144.2&;#177;62次／min，P〈0．05）。③用成纤维细胞培养液培养心肌细胞，发现与对照组相比，第2天收集的培养液在48h时的作用最强（116．6&;#177;6．6次／min．89．7&;#177;4．2次／min，P〈0．05）；高浓度组在48h时的作用也最强，与对照组有显著性差异（100．3&;#177;7．9次／min，85．2&;#177;6．1次／min，P〈0．05）。④BQ123能显著降低成纤维细胞培养液诱发的增加细胞搏动频率作用（113．9&;#177;6．5次／min，95．1&;#177;5．次／min，P〈0．01）。 结论：心肌细胞的搏动频率受多种因素的影响，是多种因素综合作用的结果。细胞的浓度、培养时间都能影响其频率；成纤维细胞培养液也能调节心肌细胞的频率；而内皮素可能是其发挥作用的关键物质之一。     

用非整合重编程技术构建人外周血单个核细胞来源的诱导多能干细胞

目的用非整合重编程技术构建人外周血单个核细胞(PBMC)来源诱导多能干细胞(i PSC)。方法收集健康人外周血标本,分离PBMC,用含4个重编程转录因子(OCT3/4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,并用无饲养层细胞的培养体系培育获得i PSC。通过细胞形态学、碱性磷酸酶(AP)染色、免疫荧光染色、拟胚体形成和分化实验对i PSC细胞进行多能性验证;用核型分析和仙台病毒基因组RNA检测对其进行安全性验证。结果在仙台病毒感染后16 d后,PBMC出现i PSC样克隆。i PSC可进行传代培养,且经多能性验证实验证明其具有多能性;安全性验证结果表明,i PSC核型正常,外源性病毒基因组RNA可被完全清除。结论建立了对PBMC进行非整合重编程的诱导体系,并获得了具有多能性、核型正常且不含外源基因的i PSC。     

9型腺相关病毒转染乳鼠心肌细胞的可行性及安全性

背景：9型腺相关病毒对心肌细胞具有更好的靶向转染,是目前研究基因治疗心脏疾病的理想载体。目的：探索9型腺相关病毒体外转染乳鼠心肌细胞的可行性及对乳鼠心肌细胞的毒性评估。方法：分离培养原代乳鼠心肌细胞,以携带荧光蛋白基因的9型腺相关病毒为载体,将分离纯化的乳鼠心肌细胞分为正常培养组、无病毒转染组、病毒转染组。结果与结论：第1周细胞搏动频率及百分率正常培养组和病毒转染组与无病毒转染组比较,差异无显著性意义（P〉0.05）,但正常培养组高于病毒转染组（P〈0.05）;3周后各组比较,差异无显著性意义（P〉0.05）。经9型腺相关病毒转染的心肌细胞24h开始有表达,4~6d荧光最强,3组细胞72h内不同时间点还原比率均接近于1.0。说明9型腺相关病毒能成功有效地转染乳鼠心肌细胞,对乳鼠心肌细胞无明显毒性作用。     

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.     

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.