西达本胺对骨髓增生异常综合征患者骨髓间充质干细胞成骨分化的影响

目的:探索西达本胺对骨髓增生异常综合征(MDS)患者骨髓间充质干细胞(MSC)成骨分化的作用及机制。方法:分离培养正常对照者及MDS患者的骨髓MSC,CCK-8法检测西达本胺对MSC增殖活性的影响,组蛋白去乙酰化酶(HDAC)活性荧光检测试剂盒及Western blot检测西达本胺对MSC中HDAC的影响。对MSC成骨诱导分化,在d 3进行碱性磷酸酶活性检测,d 21进行茜素红染色观察钙结节形成情况,在d 7和21分别检测成骨相关早期及后期基因的mRNA表达变化。RT-PCR及Western blot分别检测西达本胺对MSC成骨关键转录因子Runt相关转录因子2(RUNX2)mRNA及蛋白表达的影响。结果:随着西达本胺浓度的增加,MSC增殖受到抑制,但低浓度(1μmol/L)西达本胺对MSC无显著的增殖抑制作用,但可显著抑制HDAC活性。在正常对照组及MDS患者的骨髓MSC中,西达本胺(1μmol/L)可以显著增加碱性磷酸酶活性、促进钙结节形成及上调成骨基因的mRNA表达,从而恢复MDS患者MSC较正常对照组MSC减弱的成骨分化能力。机制研究结果显示,西达本胺促成骨作用可能与RUNX2...     

BMP6对小鼠骨髓基质细胞定向成骨分化的影响

目的初步研究BMP6对小鼠骨髓基质细胞定向成骨分化的影响,探讨其作为定向诱导细胞成骨分化细胞因子的可能性。方法BMP6腺病毒感染骨髓基质细胞（BMSC）。染色和定量检测碱性磷酸酶（ALP）的表达;实时荧光定量聚合酶链反应检测骨桥素（OPN）和骨钙素（OC）的表达;荧光素酶报告基因实验检测BMP6对于转录因子Smad和成骨关键基因Runx2的活化作用。结果BMP6能诱导BMSC细胞ALP的表达,且具有剂量依赖性;BMP6刺激后,BMSC细胞晚期成骨基因OPN、OC的表达均有明显的增加;BMP6可以活化Smad和成骨关键基因Runx2。结论BMP6可以促进小鼠骨髓基质细胞定向成骨分化。     

1,25-二羟基维生素D3对人牙周膜干细胞体外成骨作用的机制研究

目的探索1,25-二羟基维生素D3[1,25(OH) 2D3]在人牙周膜干细胞体外骨向分化中的作用。方法利用流式细胞仪对人牙周膜干细胞(PDLSC)进行细胞膜抗原鉴定;实验分为三组:A组为PDLSC未诱导组,B组为PDLSC成骨诱导组,C组为1,25(OH) 2D3+PDLSC成骨诱导组。21 d后利用化学定量法检测三组钙含量及碱性磷酸酶(ALP)含量,实时荧光定量PCR检测成骨相关蛋白骨钙素(OCN)、RUNX2、Ⅰ型胶原(COLⅠ)和ALP mRNA表达。结果PDLSC高表达CD29和CD44,而低表达CD31和CD45;且21 d后三组细胞钙含量及ALP含量:C组 B组 A组;OCN、RUNX2、COLⅠ和ALP mRNA表达为C组 B组 A组。结论 1,25(OH) 2D3可促进PDLSC的体外骨向分化,并可加速PDLSC细胞基质的矿化。     

脉冲电磁场对骨髓间充质干细胞成骨成脂肪分化的影响

目的研究15Hz、1mT脉冲电磁场对骨髓间充质干细胞向成骨、成脂肪分化的影响。方法体外培养SD大鼠骨髓间充质干细胞，取第3代细胞，用15Hz、1mT脉冲电磁场刺激，按照碱性磷酸酶（ALP）检测试剂盒说明书步骤检测ALP活性，用半定量逆转录聚合酶链反应（RT—PCR）法检测骨髓间充质干细胞成骨、成脂肪指标的表达，油红O染色观察其成脂肪诱导分化情况。结果15Hz、1mT脉冲电磁场明显促进骨髓间充质干细胞ALP活性（P〈0．01）以及成骨蛋白（骨钙蛋白、骨桥蛋白）的mRNA表达；抑制脂肪素、脂肪细胞结合蛋白2（AP-2）等成脂肪转录因子的表达，抑制骨髓间充质干细胞向成脂肪分化。结论15Hz、1mT脉冲电磁场可促进骨髓间充质干细胞向成骨分化，抑制其向成脂肪分化。     

IL-1β预刺激骨髓间充质干细胞通过NF-κB通路调节其成骨分化潜能

目的:研究不同作用时间的IL-1β对骨髓间充质干细胞(BMMSC)成骨潜能的影响以及NF-κB等通路在其中的作用。方法:将正常人BMMSC在体外给予IL-1β处理1或7 d,再予成骨诱导分化后分别采用碱性磷酸酶(ALP)染色和茜素红染色(ARS)方法检测BMMSC成骨分化;采用real-time PCR法检测BMMSC中IGF-1、EphB4和OPG基因mRNA表达水平;细免疫组织化学染色方法检测BMMSC的BMP-2和p-Smad1/5/8蛋白表达情况;Western blot方法检测BMMSC中iκBα和p-iκBα蛋白的表达水平,并将IL-1β处理组的结果与对照组比较。结果:与对照组相比较,IL-1β处理组BMMSC的成骨分化潜能明显增强,但这一差异可被NF-κB抑制剂减弱;另外,处理组的iκBα蛋白水平的表达降低(P0.05),p-iκBα蛋白水平的表达增高(P0.05);与此同时,处理组BMMSC的BMP-2表达水平增高,但p-Smad1/5/8的表达水平无明显变化;IL-1β处理组BMMSC的IGF-1、EphB4、OPG mRNA表达水平上调(P0.05)。与成骨结果比较显示,IL-1β处理1 d组与对照组的差异,不如IL-1β处理7 d组明显。结论:骨髓微环境中高水平IL-1β长期作用于BMMSC,可以通过NF-κB通路而不是BMP-2/Smad通路对其成骨分化产生影响。     

人骨髓基质细胞在骨组织工程临床应用中的探讨

目的研究人骨髓基质干细胞经体外诱导表达成骨细胞表型,作为骨组织工程临床应用种子细胞的可行性。方法人骨髓中密度梯度法分离骨髓基质干细胞,诱导组予条件培养液,对照组予单纯DMEM培养液,分别培养至第3代细胞,计算各代倍增时间和3代总的扩增倍数,经相差显微镜观察,钙结节茜素红染色,四环素荧光,免疫荧光检测骨钙蛋白,成骨细胞转录因子RT-PCR检测Ⅰ型胶原,骨钙蛋白mRNA表达。结果诱导组骨髓基质干细胞(MSC)经体外诱导,三代平均扩增163.4倍后,形成钙结节,骨钙蛋白(OCN),成骨细胞转录因子(cbfa1)免疫荧光结果阳性,RT-PCR证实Ⅰ型胶原,骨钙蛋白mRNA的表达;对照组未能形成钙结节,无成骨细胞特征性蛋白OCN,cbfa1的表达。结论人MSC体外经条件培养液诱导培养三代后仍可表达成骨细胞表型,可以满足骨组织工程临床应用对种子细胞质和量的需要。     

人牙周膜干细胞成骨分化过程中P2X7受体mRNA的表达特点及作用研究

目的 P2X7受体对牙周膜干细胞的成骨效果的影响作用机制研究。方法将实验室原代培养获得的人牙周膜干细胞分为4组,分别为对照组、三磷酸腺苷组、成骨诱导液组、三磷酸腺苷+成骨诱导液组,检测4组RUNX2基因、OCN基因及P2X7受体mRNA表达水平。结果在成骨后1周和成骨后2周RUNX2和OCN mRNA表达比较,三磷酸腺苷+成骨诱导液组高于成骨诱导液组(P0.05)。成骨后1周的RUNX2和OCN mRNA表达情况,三磷酸腺苷+成骨诱导液组高于成骨后2周且差异具有显著性(P0.05)。在成骨后1周和成骨后2周三磷酸腺苷组和三磷酸腺苷+成骨诱导液组的P2X7受体mRNA表达均高于对照组和成骨诱导液组,且差异具有统计学意义(P0.05);在成骨后2周三磷酸腺苷组的P2X7受体mRNA表达高于三磷酸腺苷+成骨诱导液组,且差异具有统计学意义(P0.05);三磷酸腺苷组成骨后1周的P2X7受体mRNA表达高于成骨后2周,且差异具有统计学意义(P0.05)。结论 P2X7受体可以明显提高牙周膜干细胞的成骨效果,且三磷酸腺苷可以激活P2X7受体的表达。     

血小板源性生长因子受体α参与成纤维生长因子对辐射诱导人骨髓间充质干细胞损伤的修复作用

目的:探讨碱性成纤维生长因子(basic fibroblast growth factor,b FGF)对辐射诱导人骨髓间充质干细胞(human bone marrow mesenchymal stem cell,h BMMSC)增殖、成骨分化损伤后的修复作用及所涉及的可能机制。方法:h BMMSC经0、6、12 Gy X射线辐照后,流式细胞术、cell counting kit-8(CCK-8)、Western blot法和茜素红染色法检测辐射对h BMMSC凋亡、增殖、成骨分化的影响;0、1、5、10、20 ng/ml浓度b FGF分别作用于辐射h BM M SC,探索抗辐射保护作用最佳浓度;Western blot法检测血小板源性生长因子受体α(platelet derived grow th factor receptor alpha,PDGFRα)蛋白表达动态变化。结果:辐射后h BM M SC增殖、成骨分化能力下降,b FGF能够修复h BMMSC增殖、成骨分化损伤(P0.05),5 ng/ml浓度作用最显著;仅辐射后早期(24 h)12 Gy组h BMMSC凋亡高于0 Gy组(P0.05),其余时间点辐射组与未辐照组细胞凋亡无差异(P0.05)。辐射后h BMMSC血小板源性生长因子受体-α表达明显下调,b FGF作用后PDGFRα表达上调。结论:辐射对h BMMSC凋亡无显著影响,b FGF对h BMMSC辐射损伤有修复作用,促进其增殖和成骨分化功能的恢复;h BMMSC增殖、成骨分化损伤与辐射诱导PDGFRα表达下调相关。PDGFRα参与b FGF对h BMMSC辐射损伤的修复。     

辛伐他汀对幼鼠骨发育及骨髓基质干细胞成骨分化的影响

背景:辛伐他汀作为常用降脂类药物,表现出一定的促成骨分化和骨形成作用,但目前的研究结果尚有争论,尤其对幼鼠骨发育的影响并无报道.目的:课题创新性设计观察辛伐他汀对幼鼠骨小梁骨形成相关因子表达以及骨髓基质干细胞成骨分化的影响.方法:1周龄雄性SD大鼠20只,随机分为实验组和对照组,每组10只.实验组皮下注射辛伐他汀[5 mg/(kg·d)]2周,对照组给与安慰剂2周.采用免疫组织化学方法检测胫骨上端骨小梁成骨相关因子骨形态发生蛋白2、基质金属蛋白酶13、血管内皮生长因子等因子的表达情况;取双侧股骨骨髓基质干细胞向成骨方向诱导培养.培养14 d分别进行碱性磷酸酶染色,21 d采用Real-time PCR法检测骨形态发生蛋白2、RUNX2、Osterix、MSX2、DLX3、DLX5等成骨过程中相关因子mRNA的表达,28 d进行von Kossa染色.结果与结论:①实验组和对照组胫骨上端骨小梁骨形态发生蛋白2、基质金属蛋白酶13、血管内皮生长因子等因子的表达差异无显著性意义.②两组碱性磷酸酶染色阳性细胞(胞浆蓝黑色)比例均在30％左右,差异无显著性意义(P＞0．05).③骨髓基质干细胞向成骨细胞诱导分化过程中相关因子骨形态发生蛋白2、RUNX2、Osterix、DLX3、DLX5、MSX2 mRNA表达水平差异无显著性意义.④von Kossa染色可见大小不等染为棕黑色的钙化点,其大小和密度两组比较差异无显著性意义.结果表明,皮下注射辛伐他汀[5 mg/(kg·d)]2周不能显著影响幼鼠骨小梁骨形成相关因子表达以及骨髓基质干细胞成骨分化.     

MicroRNA-20a通过调节CKIP-1促进小鼠C3H/10T1/2成骨分化

目的:研究microRNA-20a(miR-20a)对小鼠C3H/10T1/2细胞成骨分化作用的影响及其调控机制。方法:对贴壁培养的小鼠C3H/10T1/2细胞成骨诱导不同时间,ALP染色及应用qRT-PCR验证其诱导结果,同时观察MiR-20a在诱导过程中表达变化;细胞转染MiR-20a mimics,CKIP-1 siRNA,在荧光显微镜下观察转染效果,应用qRT-PCR测定过表达及干扰效率;qRT-PCR检测过表达MiR-20a及干扰CKIP-1对细胞成骨分化的影响及过表达MiR-20a对CKIP-1基因表达的影响。结果:成骨标记基因ALP、OSX、OCN、BSP随小鼠C3H/10T1/2细胞诱导过程逐渐升高,且成骨诱导分化过程中MiR-20a表达上调。过表达MiR-20a促进C3H/10T1/2细胞成骨分化,成骨标记基因碱性磷酸酶(ALP)、RUNX2、OSX、0CN、BSP显著高于对照组,并且抑制CKIP-1的表达;干扰CKIP-1能促进细胞成骨标记基因表达。结论:MiR-20a可能通过下调骨形成负调节因子CKIP-1的表达促进C3H/10T1/2细胞成骨分化。     

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.