治疗性超声介导体外基因转染的参数优化

目的 探讨不同治疗性超声(TUS)参数对基因转染的作用,优化TUS参数以实现高效率的转染,减少对细胞活力和质粒完整性的影响.方法 将质粒和SonoVue微泡加入培养的Hela细胞后行超声辐照,改变超声强度、占空比以及辐照时间等参数,比较不同的TUS辐照策略对细胞活力及红色荧光蛋白(DsRed)表达效率的影响,以确定最佳辐照参数,并对质粒完整性进行分析.结果 低超声强度(0.4 W/cm2、1.0 W/cm2)、低占空比(10%和20%)时,细胞存活率较高(>80%),辐照1 min和3min之间的细胞活力差异不明显(P>0.05).当增加超声强度(1.6 W/cm2、2.2 W/cm2)和占空比(50%)时,细胞活力显著下降(P<0.05).当20%占空比,1.0 W/cm2的TUS辐照3min时,可实现最高的转染率.质粒DNA结构的完整性不受优化的TUS参数影响.结论 TUS是一种有效的基因输送方法 ,优化的参数在无显著细胞死亡和DNA损伤的前提下增加转染,这种非侵袭性的基因转染方法 可能是临床基因疗法的一种有用工具.     

超声辐照和SonoVue微泡在介导hAng-1基因体外转染时的作用和量效关系探讨

目的 探讨超声辐照和SonoVue微泡分别使用和联用在介导hAng-1基因体外转染过程中的作用以及辐照强度和微泡浓度对转染效率和细胞活性的影响.方法 实验分四组A组:单纯超声辐照+质粒组;B组:微泡+质粒组;C组:超声辐照+微泡+质粒组和空白对照组D组. C组内转染参数分别设置为超声照射强度0.5、1.0 、1.5和2.0 W/cm~2,微泡浓度5%、10%、20%、30%和40%.将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡对293T细胞进行转染,48 h后检测各组基因转染效率和细胞存活率. 结果转染48 h后C组转染效率最高,荧光阳性细胞数最多,强度最大;A组转染效率很低,见少量荧光表达;B、D组无明显基因转染发生.随着超声照射强度和微泡浓度的增加,基因转染效率会逐步升高,具有统计学意义.微泡浓度大于20%、超声照射强度超过1.5 W/cm~2后基因转染效率不再升高甚至降低,细胞死亡率显著增高(P<0.01).结论 SonoVue微泡介导外源基因转染必须联合超声辐照才能获得较好的转染效率.对于hAng-1基因和SonoVue微泡,选择声强1.5 W/cm~2,微泡浓度20%是相对最佳转染条件.     

超声介导微泡破坏增强survivin短发夹状RNA质粒转染对基因表达及细胞凋亡的影响

目的 观察超声介导微泡破坏(UMMD)转染survivin短发夹状RNA质粒对宫颈癌细胞(Hela)survivin基因表达及细胞凋亡的影响.方法 构建靶向survivin基因的短发夹状RNA质粒(shRNA)真核表达质粒(pSIREN-S),将pSIREN-S和SonoVue微泡加入培养的Hela细胞,予以超声辐照(P+UMMD),或用脂质体法转染细胞(P+L),以空白对照(C)、pSIREN-S质粒(P)、超声辐照(US)、SonoVue(S)、pSIREN-S+SonoVue(P+S)、pSIREN-S+超声辐照(P+US)为对照,FITC-annexin V/FITC和7-AAD双染后分析细胞凋亡,RT-PCR和蛋白质印迹检测survivin mRNA及蛋白表达的变化.结果 酶切及测序分析证实pSIREN-S构建成功.P+L组的细胞凋亡率(31.58±3.12)%显著高于各对照组(P＜0.01),但仍低于P+UMMD组(43.86±4.44)%.P+UMMD组的mRNA及蛋白表达相对水平分别为(16.67±2.73)%和(21.33±3.55)%,显著高于其他各组(P＜0.01).结论 UMMD联合shRNA干扰技术能显著阻抑靶基因survivin的表达,有效诱导细胞凋亡.UMMD为以RNA干扰为基础的肿瘤基因治疗及研究提供新思路,有望成为一种高效的非病毒基因疗法.     

超声联合聚乙烯亚胺增强MCF-7癌细胞基因表达的转染参数优化

目的 探讨超声辐照并超声造影剂联合聚乙烯亚胺(PEI)增强MCF-7乳腺癌细胞质粒DNA转染的最优条件及协同作用.方法 制备PEI/荧光素酶质粒(pCMV-luciferase-GL3)复合物,用于MCF-7癌细胞基因转染,超声辐照前添加超声造影剂SonoVue.通过荧光素酶活性和细胞存活率的测定,对超声辐照参数进行优化,对质粒浓度、孵育时间、血清、溶媒类型、培养基体积等因素进行分析.结果 细胞活力和荧光素酶活性均受超声强度、辐照时间和占空比的影响,适当条件的超声辐照可促进PEI/DNA复合物渗透入胞内,从而提高质粒DNA的转染率.最优超声辐照条件为1 w/cm2,10%占空比,辐照3 min.超声辐照并超声造影剂联合PEI的转染效率显著高于单纯超声辐照和PEI转染(P<0.01).在超声辐照前将细胞与PEI/DNA复合物共孵育2h时,荧光素酶活性显著增强(P<0.01).此外,血清、培养基体积和溶媒类型也对转染效率有影响.结论 优化的超声和转染参数能显著提高MCF-7癌细胞的基因表达效率.超声辐照并超声造影剂联合PEI对DNA转染效率有协同作用,是一种增强质粒DNA基因表达简单而有应用前景的方法.     

不同转染方法对荧光蛋白质粒转染人胚肾293T细胞影响的实验研究

目的:探讨荧光蛋白质粒转染人胚肾293T细胞的最佳转染方法.方法:以脂质体转染法、超声辐照法、微泡造影剂SonoVue联合超声辐照及脂质体中加入微泡造影剂SonoVue并予以超声辐照4种转染方法,将含有绿色荧光蛋白报告基因的真核表达质粒pEGFP-N1转染人胚肾293T细胞,48 h后以荧光显微镜观察293T细胞中的绿色荧光蛋白表达情况并用流式细胞仪测算转染率.结果:脂质体 SonoVue 超声辐照法的转染效果最好(23.10±2.11%).脂质体 SonoVue 超声辐照法的转染率分别与脂质体转染法、超声辐照法、微泡造影剂SonoVue联合超声辐照法的转染率比较均有显著差异(P<0.01).结论:脂质体中加入微泡造影剂SonoVue并予以超声辐照能显著提高荧光蛋白质粒在人胚肾293T细胞中的转染效果.     

超声辐照SonoVue增强脂质体介导pEGFP-N1转染乳腺癌细胞

目的 探讨超声辐照SonoVue介导增强型绿色荧光蛋白报告基因质粒(pEGFP-N1)转染乳腺癌MCF-7细胞的最优条件以及超声联合微泡造影剂增强脂质体转染的效果.方法 体外培养人乳腺癌MCF-7细胞,按照不同的辐照条件分组:不处理组,超声辐照十质粒组,微泡浓效组,超声声张组,辐照时间组及工作周期组.分别进行超声辐照,辐照后以MTT法测细胞存活度,流式细胞仪测pEGFP-N1瞬时转染率,取得最佳的辐照条件.重新准备细胞后先以脂质体转染细胞,2 h后加入超声微泡并以最佳条件辐照,用同样的方法测细胞存活率及转染率.结果 20%的造影剂浓度、1.5 W/cm2的声强、90 s的辐照时间、20%的工作周期(占空比)为超声辐照SonoVue介导pEGFP-N1转染MCF-7细胞的最优条件,转染率为(14.21±2.55)%,细胞存活率为(84.60±2.85)%.在增强脂质体转染的实验中,超声微泡脂质体组的转染率最高(22.15±2.69)%,与其他各组相比差异有统计学意义(P<0.05),此时细胞存活率为(80.13±3.61)%.结论 20%的造影剂浓度、1.5 W/cm2的声强、90 s的辐照时间、20%的工作周期是MCF-7细胞的最佳转染条件,超声联合微泡能够增强脂质体转染的效果.     

超声介导SonoVue微泡破裂对hAng-1基因完整性和表达效率的影响

目的 探讨超声联合SonoVue微泡介导hAng-1基因转染293T细胞的转染效率及基因完整性和表达状况.方法 构建eGFP-C3-hAn-1质粒,根据不同实验组的设计,应用相应的微泡联合超声辐照条件进行293T细胞的eGFP-C3-hAng-1基因转染,转染后48 h,以荧光显微镜观察到绿色荧光为转染成功标志;流式细胞术检测基因转染阳性细胞率,台盼蓝染色检测细胞生存率;RT-PCR和Western blot技术检测hAng-1基因的mRNA和蛋白表达;琼脂糖凝胶电泳检测经超声辐射后质粒的完整性.结果 ①微泡浓度为20%,DNA浓度为15 mg/L时进行基因转染可获较好转染效率和细胞生存率;②转染体系中血清的存在并不影响转染效率和细胞生存率;③最适转染条件下的超声辐照剂量不会影响DNA的完整性,且转染后的基因可正常表达mRNA并翻译目的蛋白.结论 微泡联合超声辐照能够介导体外细胞治疗性基因的转染,血清的存在并不影响基因的转染,转染后的基因能顺利地表达并具有正常功能.     

载自杀基因的靶向微泡联合超声抑制视网膜母细胞瘤的实验研究

目的 探讨载自杀基因的靶向微泡联合超声对视网膜母细胞瘤（RB）的抑制作用。方法 制备携带单纯疱疹病毒Ⅰ型胸苷激酶（HSV1-tk）质粒和VEGFR2抗体的靶向微泡,分为空白对照组、细胞+质粒组、细胞+质粒+SonoVue组、细胞+靶向微泡组、细胞+质粒+超声辐照组、细胞+质粒+SonoVue+超声辐照组和细胞+靶向微泡+超声辐照组进行实验。荧光显微镜观察基因转染情况,流式细胞仪检测转染率,加入丙氧鸟苷（GCV）后,检测细胞的抑制率。结果 细胞+靶向微泡+超声辐照组的转染率为（24.78±1.04）%,较细胞+质粒+SonoVue+超声辐照组（14.31±0.69）%高。随着GCV浓度的增加,培养时间的延长,各组的抑制率逐渐升高。当GCV浓度在100 mg/l,培养96h后,细胞+靶向微泡+超声辐照组对RB细胞的抑制率达（92.91±1.71）%。结论 在加入GCV后,携带HSV1–tk的靶向微泡联合超声能有效的抑制RB细胞。     

超声微泡破裂法联合阳离子脂质体介导基因转染的实验研究

目的 探讨超声微泡破裂法联合阳离子脂质体(cationic liposome,CL)介导绿色荧光蛋白质粒在肝癌细胞(HepG2)基因转染的可行性,并探索最佳转染条件.方法 依次采用培养液中是否含有血清和不同的CL浓度、不同超声辐照时间点、不同纳米级脂质微泡造影剂(nano-liposomal bubble,NB)浓度等处理因素进行细胞基因转染.荧光显微镜和流式细胞仪检测基因转染效率,CCK-8法检测细胞活性,以获得优化的转染参数.结果 血清能降低CL的细胞毒性,但对基因转染效率无明显影响,CL与质粒DNA质量比4∶1时可以达到相对高效低毒的转染效果,转染率(17.71±0.79)％,存活率(91.28±0.76)％.CL联合1h时间点辐照超声可以提高转染率至(24.85±0.78)％(P＜0.01),加入10％的NB可进一步提高转染率至(32.47±4.01)％(P＜0.05).结论 超声微泡破裂法可以有效增强CL介导的基因转染,联合应用为基因治疗提供了新思路.     

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。     

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.     

Clinical Pharmacokinetics of Morphine

Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.