超声靶向微泡破裂联合PEI增强小鼠EGFP基因心肌转染

目的探讨超声靶向微泡破裂(UTMD)联合聚乙烯亚胺(PEI)增强BALB/c小鼠心肌绿色荧光蛋白基因(EG-FP)转染的可行性和应用价值。方法实验分为7组:PBS组、裸质粒组、质粒 超声辐照组(P US)、质粒 SonoVue 超声辐照组(P UTMD)、质粒 PEI组(P PEI)、质粒 PEI 超声辐照组(P PEI US)、质粒 PEI SonoVue 超声辐照组(P PEI UTMD)。由BALB/c小鼠尾静脉注入EGFP质粒和SonoVue微泡或PEI的复合物,处理4d后检测心肌基因表达效率及HE染色,并对超声辐照后的质粒完整性进行分析。结果电泳显示超声辐照不会损坏DNA或PEI/DNA复合物。非超声辐照时,EGFP只在心内膜下层表达;而超声辐照时,表达最强的位置为靠近探头的左室前壁;超声联合PEI时,EGFP的分布差异不明显。P PEI UTMD组的转染率最高,荧光强度最强。结论UTMD联合PEI可高效、靶向地将质粒DNA输送至心肌,这种非侵入性的技术在心脏基因治疗上很有前景,有望应用于迅速发展的心脏病基因疗法。     

超声靶向微泡破裂联合聚乙烯亚胺介导大鼠心肌母细胞Bax基因沉默

目的 探讨超声靶向微泡破裂(ultrasound-targeted microbubble destruction,UTMD)联合聚乙烯亚胺(polyethyleneimine,PEI)的方法转染大鼠心肌母细胞(H9c2细胞)以达到沉默Bax基因的效果.方法 构建靶向Bax基因的shRNA表达质粒,将其与微泡及PEI共同处理后加入H9e2细胞并予以超声辐照,实验分组为:①单纯质粒组;②Lipofectamine2000+质粒组;③UTMD+质粒组;④PEI+ UTMD+质粒组;⑤PEI+ SonoVue+质粒组(无超声辐照).采用荧光显微镜和FCM法评估基因转染效率,应用RT-PCR检测Bax mRNA水平的变化,Western Blot检测蛋白表达情况.结果 超声辐照对所构建的BaxshRNA表达质粒无破坏.H9c2细胞采用UTMD+ PEI处理后,转染率显著高于其他各组(P＜0.05).PT-PCR及Western Blot检测结果显示,PEI+UTMD+质粒组Bax mRNA及Bax蛋白表达抑制率分别为(71.41±4.91)％及(64.09±2.38)％,显著高于其余各组(P＜0.05).结论 超声靶向微泡破裂联合聚乙烯亚胺的方法能有效地沉默H9c2细胞Bax基因的表达.     

超声靶向微泡破坏联合聚乙烯亚胺增强裸鼠移植瘤基因输送的初步研究

目的 探讨超声靶向微泡破坏(UTMD)联合聚乙烯亚胺(PEI)增强裸鼠Hela细胞(人宫颈癌)移植瘤基因输送的可行性和应用价值.方法 分别将2种质粒DNA[红色荧光蛋白质粒(RFP)和荧光素酶质粒(pCMV-LUC)]与PEI以不同氮/磷酸盐比(N/P比)混合,利用凝胶阻滞实验对PEI/DNA复合物进行分析.经荷瘤裸鼠尾静脉分别注入PBS、质粒、质粒+Sono Vue微泡、PEI/DNA复合物+Sono Vue微泡,仅对一侧肿瘤行超声辐照(3 MHz、2 W/cm2、2 min、20%占空比),另一侧肿瘤作为对照,并对该转染方法 的靶向性进行分析.超声辐照3 d后处死动物,行RFP表达观察、荧光素酶活性检测和组织学检查.结果 琼脂糖凝胶电泳显示PEI可有效地缩合质粒DNA.与裸质粒组比较,UTMD(超声辐照+Sono Vue微泡)能明显提高RFP转染率.与非辐照对照侧比较,UTMD的运用使RFP表达明显增强,荧光素酶活性增加了14倍(P＜0.01).UTMD联合PEI可显著增强基因转染,受辐照移植瘤的荧光素酶活性增加了10倍(P＜0.01);与非联合PEI时比较,荧光素酶表达增加了111倍(P＜0.01).无论有否超声照射,裸鼠其他器官组织中均无明显的基因表达(P＞0.05),且未观察到明显的组织损伤.结论 UTMD联合PEI可显著增强报告基因在肿瘤组织的靶向传输和转染,是一种很有前景、新型而安全的体内基因输送方法.     

纳米微泡介导GFP转染小鼠骨骼肌细胞的实验研究

目的 探讨两种纳米微泡(白蛋白外膜和磷脂外膜)增强基因转染小鼠骨骼肌的作用.方法 以正常C57B10小鼠胫前肌为研究对象,目的基因GFP与微泡混合注入小鼠胫前肌,一侧胫前肌经超声(1MHz脉冲波,脉冲重复频率为100 Hz,20％工作周期,空间峰值时间峰值声强为2 W/cm2,辐照作用时间30 s),另一侧胫前肌不经超声辐照.观察白蛋白纳米微泡及磷脂纳米微泡增强骨骼肌细胞GFP转染水平的作用.1周后处死小鼠,荧光显微镜观察发出绿色荧光者为GFP阳性肌纤维细胞,计数最大GFP阳性肌纤维细胞数,作为GFP基因转染效率指标.结果 ①白蛋白纳米微泡组和白蛋白纳米微泡+超声组最大GFP阳性肌纤维数显著高于阴性对照组(P＜0.05),显著低于阳性对照组(P＜0.05).②磷脂纳米微泡组与阴性对照组及阳性对照组比较,最大GFP阳性肌纤维数差异均无统计学意义(P＞0.05).磷脂纳米微泡+超声组最大GFP阳性肌纤维数较阴性对照组显著增高(P＜0.05);磷脂纳米微泡+超声组与阳性对照组最大GFP阳性肌纤维数差异无统计学意义(P＞0.05).结论 纳米微泡可增强基因转染骨骼肌细胞效率,白蛋白含氟化气体纳米微泡具有发展潜力.     

共聚物P85联合超声微泡介导基因转染的体外实验研究

目的 探讨共聚物P85与黏附增强型绿色荧光蛋白(enhanced green fluoresent protein,EGFP)质粒的微泡造影剂(microbubble,MB)结合后联合一定强度的超声(ultrasound,US)辐照,是否增强人肝癌细胞(HepG2)的质粒转染及表达.方法 以人肝癌细胞(HepG2)为研究对象,质粒DNA是可表达绿色荧光蛋白的pEGFP,添加微泡造影剂或共聚物P85后进行脉冲多普勒超声辐照(频率1 MHz,声强1 W/cm2,工作周期20%,时间20 s).24 h后台盼蓝染色评价细胞存活率,荧光显微镜和流式细胞仪评价细胞的基因转染率.结果 有超声辐照组的pEGFP转染率明显高于无超声辐照组(P＜0.01),有超声辐照组中pEGFP+30%MB+P85+US组转染效率(22.14±3.06)%优于单独使用微泡组(11.34±2.2)%或P85组(9.72±1.21)%(P＜0.01).结论 微泡造影剂与共聚物P85结合同时给予超声辐照可增强基因转染效率,在基因治疗上有待于进一步关注和研究.     

超声微泡造影剂介导小鼠骨骼肌基因转染实验研究

目的探讨微泡造影剂在超声作用下是否可增加小鼠骨骼肌基因转染效率.方法 40只昆明小鼠随机分为4组,每组10只,第一组:在胫前肌注射造影剂与绿色荧光蛋白(GFP)质粒的混合溶液;第二组:注射与第一组相同的混合溶液后立即加超声辐照;第三组:注射GFP;第四组:在注射GFP后立即用超声辐照.7天后取小鼠胫前肌观察绿色荧光蛋白的表达情况.结果第一组与第二组有较多GFP表达,部分肌纤维绿色荧光较明亮,部分较暗淡;第三组和第四组GFP表达量较少.第一组与其余各组间的差异有显著性意义,P＜0.05;第二组与其余各组间的差异有显著性意义,P＜0.05;第三组与第四组间的差异无显著性意义,P＞0.05.结论超声微泡造影剂在超声作用下可明显增强小鼠骨骼肌的基因转染效率;未加超声波作用时,直接肌注携基因的超声微泡造影剂亦可增加小鼠骨骼肌的基因转染效率.     

超声微泡靶向破坏转染shRNA沉默Survivin表达诱导裸鼠宫颈癌移植瘤凋亡的研究

目的 通过超声微泡靶向破坏(UTMD)对靶向存活素的短发夹状重组质粒(Survivin-shRNA)进行转染,探讨其凋亡诱导效应、增殖抑制作用及其安全性.方法 将荷人宫颈癌(Hela)裸鼠随机分为三组:质粒+超声辐照(P+US)组,注入质粒溶液后予以超声辐照;质粒+微泡+超声辐照(P+UTMD)组,注入质粒/微泡复合物后辐照;对照组,不予任何处理.对组织样本行组织学检查,采用免疫组化SABC法检测移植瘤增殖细胞核抗原(PCNA)、Survivin、Bcl-2、Bax、Caspase-3、Ki-67、核干细胞因子(NS)、p53蛋白在各组肿瘤标本中的表达.结果 P+UTMD组的PCNA、Ki-67、Bcl-2、Survivin及NS蛋白表达下降,而Bax、Caspase-3、p53蛋白表达明显增加,与对照组及P+US组比较差异均有统计学意义(P＜0.05).结论 UTMD联合Survivin-shRNA能有效沉默Survivin基因表达,具有抑制增殖和促分化的作用,并诱发细胞凋亡,无明显副作用,为癌症基因疗法提供一种高效、无创、有前景的新方法.     

超声联合声诺维辐照增强GFP基因在鼠胶质瘤C6细胞中的转染

目的 探讨超声联合声诺维微泡辐照对增强型绿色荧光蛋白基因(EGFP)在鼠胶质瘤C6细胞中的转染增效作用及安全性.方法 实验分为4组:单纯质粒组、质粒+微泡组、质粒+超声组和质粒+超声+微泡组.辐照条件为超声频率1 MHz,声强1 W/cm2,辐照时间30 s,占空比20%.按不同实验条件辐照后,应用激光共聚焦显微镜和流式细胞仪评估基因转染效率,台盼蓝染色法评估细胞活力.结果 经超声联合微泡辐照C6细胞后,GFP基因转染效率较其他组显著增加,差异有统计学意义(P<0.01),且各组均未发现显著的细胞损伤.结论 超声联合声诺维微泡辐照可显著增强GFP基因在鼠胶质瘤C6细胞的转染,为临床胶质瘤基因治疗提供了一种安全,有效的非病毒基因转染方法.     

几种共聚物对小鼠骨骼肌超声介导基因转染表达的影响

目的 探讨超声和共聚物在质粒DNA对小鼠骨骼肌基因转染中的影响.方法 应用三种水溶性不同的共聚物F127,L61和P85,与DNA混合后直接小鼠胫前肌注射,并超声辐照.超声频率为1 MHz,脉冲重复频率100 Hz,占空系数为20%,用3 W/cm2声强辐照1 min.实验1周后取出胫前肌并快速冰冻切片,荧光显微镜计数表达GFP的转染肌纤维数,苏木精和伊红染色评价肌肉损伤情况.结果 三种共聚物均可显著提高质粒DNA的基因转染(P＜0.01),P85的基因转染效率高于其他两种共聚物(P＜0.05).超声虽对单纯质粒、F127和L61组的基因转染无显著促进作用,但与P85有协同作用,可显著提高P85组的基因转染效率(P＜0.01).在给定超声剂量和共聚物浓度下,二者在提高质粒DNA基因转染的同时,并不显著增加对组织的损伤作用.结论 共聚物P85不但可显著提高质粒DNA在小鼠骨骼肌的基因转染,且与超声具有协同作用.     

超声辐照微泡促骨髓间充质干细胞归巢于大鼠肾组织实验研究

目的 观察超声辐照微泡对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)归巢于急性肾小管坏死模型肾组织的作用.方法 将制备成功的40只肾小管坏死模型大鼠随机分为5组(8只),即(1)单纯模型组(对照组);(2)0.5 W/cm2超声+微泡组(0.5 US+MB);(3)干细胞组(MSCs); (4) 0.5 W/cm2超声+微泡+干细胞组(0.5 US+ MB+MSCs);(5)1.0 W/cm2超声+微泡+干细胞组(1.0 US+ MB+ MSCs).7d后将各组大鼠处死,取材用于荧光显微镜观察MSCs在肾组织的分布情况,采用荧光定量PCR测定细胞间黏附分子1(ICAM-1)基因表达水平.结果 采用荧光显微镜观察,DAPI标记的MSCs细胞核(细胞核带蓝色荧光),1.0 US+ MB+ MSCs组肾组织骨髓干细胞数量明显多于0.5 US+ MB+ MSCs组及MSCs组(P＜0.05).0.5 US+MB组、0.5 US+ MB+MSCs组及1.0 US+ MB+MSCs组ICAM-1的基因水平明显高于对照组及MSCs组(P＜0.05).结论 1.0 W/cm2超声辐照微泡促进肾组织细胞ICAM-1表达增加,从而促进MSCs归巢于急性肾小管坏死模型的肾组织.     

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.