异硫氰酸苯己酯降低U266细胞株p16基因甲基化的研究

本研究探讨异硫氰酸苯己酯对p16基因高甲基化的多发性骨髓瘤U266细胞株是否有去甲基化的作用。用异硫氰酸苯己酯0、5、10μmoL／L孵育U266细胞株，提取DNA备用。用亚硫酸氢盐处理正常人基因组DNA，并以此为模板进行PCR扩增。设计1组特殊荧光标记探针以构建1种检测p16基因启动子区3个CpG位点甲基化改变的芯片，特殊荧光标记探针包括1对非甲基化探针和甲基化探针，检测相邻的3个CpG位点甲基化的程度。采用基因芯片的方法，将完全未甲基化的DNA和完全甲基化的DNA混合后制成标准曲线，将U266细胞株样本处理后点在芯片上与标准曲线进行比较后得出定量检测的结果。结果表明：芯片上探针的可重复性和精确性很好，0、5、10μmol／L异硫氰酸苯己酯处理后的U266细胞株p16基因的甲基化程度分别为78．2％、61．7％、54．8％。结论：异硫氰酸苯己酯可以降低U266细胞株p16基因甲基化的程度。     

巢式甲基化特异性聚合酶链反应在p16基因启动子过甲基化检测中的应用

目的　介绍巢式甲基化特异性聚合酶链反应 (nMSP)法 ,探讨了最佳PCR扩增条件 ,并用该法分析新鲜癌组织、石蜡包埋组织及肿瘤患者血清中p16基因启动子的甲基化状态。方法　将基因组DNA变性成为单链 ,用亚硫酸氢盐修饰单链DNA ,将所有未甲基化的胞嘧啶被转变为尿嘧啶 ,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因特异引物 ,进行巢式聚合酶链反应扩增 ,最后经凝胶电泳检测目的片段。结果　在 3种类型的标本中都检测出了p16基因启动子甲基化。用甲基化特异性聚合酶链反应法 (MSP)和nMSP法分别检测 34例非小细胞肺癌 (NSCLC)患者血清 ,p16基因启动子甲基化阳性率分别为 5 3% (18/34)和 74 % (2 5 /34) ,nMSP法具有更高的灵敏度。结论　筑巢式甲基化特异性聚合酶链反应是一种灵敏度高、特异性强的甲基化检测方法 ,可广泛应用于不同类型标本基因启动子甲基化分析。     

错配杂交化学发光法检测亚甲基四氢叶酸还原酶基因多态性

目的 建立一种基于错配杂交及化学发光技术的检测亚甲基四氢叶酸还原酶（methylenetetrahydrofolate reducatase,MTHFR）基因多态性的新方法。方法 针对多态位点设计两条寡核苷酸探针,分别与野生型及突变型序列互补。将两条探针分别与含多态位点C677T的PCR扩增产物杂交,然后用化学发光法检测,根据两条探针的发光值之比确定样品的基因型。结果 用本法随机检测了50例DNA样品,结果3种基因型的发光值之比可以严格区分,其中野生型（C／C）15例,杂合型（C／T）28例,突变型（T／T）7例,与参考方法（PCR—RFLP）的检测结果完全一致。结论 本方法操作简便、检测快速而且费用低廉,可广泛应用于MTHFR基因突变及多态性检测。     

错配杂交化学发光法检测MTHFR基因多态性

目的建立一种基于错配杂交及化学发光技术检测亚甲基四氢叶酸还原酶（methylene tetrahydrofolate reduetase。MTHFR）基因多态性的新方法。方法针对多态位点设计两条寡核苷酸探针,分别与野生型及突变型序列互补。将2条探针分别与含多态位点C677T的PCR扩增产物杂交,根据2条探针的发光值之比确定样品的基因型。结果用本法随机检测50例DNA样品,结果3种基因型的发光值之比可以严格区分,其中野生型（C／C）15例,杂合型（C／T）28例,突变型（T／T）7例,与参考方法（PCR—RFLP）的检测结果完全一致。结论本方法操作简便、快速且费用低廉,可广泛应用于MTHFR基因突变及多态性检测。     

CpG岛甲基化异常的致癌作用

真核生物染色体DNA甲基化(DNA methylation)是基因表达调控的方式之一.DNA复制后,由DNA甲基转移酶(DNA methyltransferase,DNMTs)催化S-腺苷蛋氨酸的甲基转移到DNA分子中形成5-甲基胞嘧啶(5-methylcytosine),从而生成甲基化DNA.高等真核细胞通常是对DNA分子上5′-CpG-3′序列的胞嘧啶进行甲基化修饰.CpG二核苷酸在人类基因组中占10%,其中70%～80%呈甲基化状态,可称为甲基化CpG位点.非甲基化CpG二核苷酸区域仅占基因组的1%～2%,这些CpG二核苷酸以较大密度分布于基因5′端,称为CpG岛.CpG岛一般为0.5～2 Kb长,通常位于基因的5′端启动区,也可延伸至基因的外显子区[1].     

巢式MSP法检测恶性血液病细胞株p16基因启动子甲基化状态的研究

本研究应用改进的甲基化特异性PCR（MSP）法，即巢式甲基化特异性PCR法检测6种肿瘤细胞株p16基因启动子的甲基化状态效率，探讨其在筛选p16基因启动子高甲基化的肿瘤细胞株，及将其作为研究基因甲基化与表达关系的理想细胞模型中的应用。6种肿瘤细胞株基因组DNA经碱变性后以亚硫酸盐修饰，再用巢式甲基化特异性聚合酶链反应扩增，分析检测其p16启动子区CpG岛的甲基化状态。结果表明：CA46、U266都有不同程度的p16基因启动子区甲基化，而Molt4、K562、HL-60、Jurkat的p16基因启动子区均未甲基化。结论：用巢式甲基化特异性PCR可以准确的检测出恶性血液病细胞株p16基因的甲基化状态，方法简单，灵敏且重复性强，可以广泛用于筛选各种p16基因启动子区甲基化的恶性血液病细胞株以及恶性血液病诊断。     

巢式MSP检测砷剂诱导人多发性骨髓瘤U266细胞系p16基因去甲基化及转录

本研究探讨用高灵敏度的DNA甲基化检测方法及DNA克隆测序分析法检测三氧化二砷(As2O3)的去甲基化作用,并对其可能的去甲基化作用机制进行分析。采用巢式甲基特异性PCR法(nested-methylation specificPCR,n-MSP)、DNA克隆测序分析法检测As2O3作用前后U266细胞株p16基因甲基化状态,应用RT-PCR检测p16、DNA甲基转移酶1(DNMT1)、DNMT3A、DNMT3B基因mRNA的表达,以生长曲线、MTT法、集落形成实验检测As2O3对骨髓瘤细胞生长和增殖的抑制作用。利用流式细胞仪DNA含量分析法探讨As2O3对多发性骨髓瘤细胞系U266周期的影响。结果表明:①未处理组U266细胞基因组DNA的胞嘧啶保持不变,而经As2O3作用的U266细胞基因组DNA的胞嘧啶均已变为胸腺嘧啶,这说明U266细胞存在p16基因甲基化,As2O3作用后p16基因异常甲基化的现象被逆转;②未处理组细胞p16基因不表达,As2O3作用72小时后p16基因表达增强,0.5μmol/L组、1.0μmol/组和2.0μmol/组p16基因表达阳性条带灰度值与β-肌动蛋白比值分别为(0.22±0.10)、(0.59±0.11)、(0.68±0.09),阳性对照灰度比值为(0.77±0.13),差异有非常显著的统计学意义(P<0.01);③与未处理组相比,As2O3作用72小时后甲基转移酶(DNMT)1、DNMT3A、DNMT3B的表达下降并呈浓度依赖性;④与对照组相比,3组不同浓度As2O3均能明显抑制骨髓瘤细胞生长,G0-G1期细胞增加。结论:As2O3可能通过抑制甲基转移酶(DNMT1)、DNMT3A、DNMT3B和(或)直接对p16基因去甲基化,使p16基因表达上调,恢复其活性,从而实现其对细胞周期的调控功能,将细胞阻滞于G0-G1期,抑制骨髓瘤细胞的增长。     

甲基化CpG结合蛋白2在肿瘤发生中的作用研究

表观遗传学主要从基因组修饰的角度来研究基因表达的调控机制,其中最具代表性的就是DNA甲基化,这也是目前胚胎学、肿瘤生物学关注热点。在哺乳动物中,DNA甲基化主要发生在二核苷酸胞嘧啶（CpG）第五位碳原子上,即5-甲基胞嘧啶（5-mC）。甲基化的CpG岛可产生阳性信号导致相关基因沉默。在不同组织或细胞不同发育阶段,基因组DNA上各CpG位点甲基化状态差异构成了基因组DNA甲基化谱,异常DNA甲基化会导致肿瘤发生。     

亚硫酸氢盐测序法检测上皮钙黏蛋白甲基化的优化探讨

目的探讨改进的亚硫酸氢盐测序法在上皮钙黏蛋白甲基化检测中的可行性。方法提取雄性F344大鼠肾脏组织基因组DNA,亚硫酸氢盐化学转化,通过PCR扩增上皮钙黏蛋白基因启动子区特定的序列,以蓝白克隆斑筛选和测序。结果雄性F344大鼠肾脏上皮钙黏蛋白基因启动子区CpG岛的26个CpG位点均未发生甲基化。结论改进后亚硫酸氢盐测序法能明显减少非特异性扩增,提高PCR效率,更适于基因甲基化状态的检测。     

毛细管电泳技术与分子信标技术结合检测基因

目的：研究毛细管电泳技术和分子信标技术结合进行基因检测，建立一种简便、敏感、特异和快速自动化的基因检测的新方法学。方法：提取胃癌患者的癌组织及癌旁组织DNA，PCR扩增p16基因外显于2，进行琼脂糖电泳检测，SSCP分析，PCR扩增产物与自行设计探针杂交后毛细管电泳检测。结果：有p16外湿子2 PCR扩增产物的癌组织与分子信标探针液相杂交后毛细管电泳仪检测可见检测峰。结论：毛细管电泳技术和分子信标技术结合检测基因产物是在临床及基础研究中可作为一种基因产物检测的实用手段。     

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.