马血清在脂肪干细胞成肌诱导中的作用

背景：利用 5-氮杂胞苷诱导脂肪干细胞成肌实验对培养技术以及细胞活性要求较高,诱导成肌时间较长。倘若某种添加剂可缩短诱导时间,将具有重要意义。目的：评估马血清在 5-氮杂胞苷诱导脂肪干细胞成肌细胞实验中的作用。方法：将第1代长满100 mm培养皿的脂肪干细胞传代至3个6孔板,实验组培养基中加入体积分数为5%马血清＋10 μmol/L 5-氮杂胞苷;对照组培养基中单纯添加 10 μmol/L 的 5-氮杂胞苷;空白组为单纯低糖 DMEM 培养基,余培养、传代及鉴定所需的条件相同。每日光镜观察记录形态,在培养的第 7,14,28,35 天行免疫荧光和流式细胞仪检测肌蛋白表达差异。结果与结论：免疫荧光检测成肌特异性胞质蛋白表达以及流式细胞检测相应蛋白表达率提示,加入马血清后的实验组脂肪干细胞的成肌速度和诱导成肌所需时间明显比单纯加入 5-氮杂胞苷的对照组更有优势。实验初步表明马血清在促进脂肪干细胞诱导成肌方面起着缩短诱导成肌所需时间的作用。     

马血清在脂肪干细胞成肌诱导中的作用

背景:利用 5-氮杂胞苷诱导脂肪干细胞成肌实验对培养技术以及细胞活性要求较高,诱导成肌时间较长。倘若某种添加剂可缩短诱导时间,将具有重要意义。目的:评估马血清在 5-氮杂胞苷诱导脂肪干细胞成肌细胞实验中的作用。方法:将第1代长满100 mm培养皿的脂肪干细胞传代至3个6孔板,实验组培养基中加入体积分数为5%马血清+10 μmol/L 5-氮杂胞苷;对照组培养基中单纯添加 10 μmol/L 的 5-氮杂胞苷;空白组为单纯低糖 DMEM 培养基,余培养、传代及鉴定所需的条件相同。每日光镜观察记录形态,在培养的第 7,14,28,35 天行免疫荧光和流式细胞仪检测肌蛋白表达差异。结果与结论:免疫荧光检测成肌特异性胞质蛋白表达以及流式细胞检测相应蛋白表达率提示,加入马血清后的实验组脂肪干细胞的成肌速度和诱导成肌所需时间明显比单纯加入 5-氮杂胞苷的对照组更有优势。实验初步表明马血清在促进脂肪干细胞诱导成肌方面起着缩短诱导成肌所需时间的作用。     

前列腺素E1对人类脂肪源性基质细胞体外增殖的影响

背景：近年来脂肪源性基质细胞成为研究与应用的热点之一,但前列腺素 E1 对脂肪源性基质细胞增殖的作用影响尚未见报道。目的：观察前列腺素 E1 对人类脂肪源性基质细胞体外增殖的影响。方法：从人正常脂肪组织分离培养脂肪源性基质细胞,用 5 种不同浓度的前列腺素 E1 处理培养的细胞,其中 3×10-6,6×10-6,9×10-6,12×10-6 mol/L 作为实验组,0×10-6 mol/L 作为对照组,观察经不同浓度前列腺素 E1 处理后细胞的增殖情况。结果与结论：不同浓度实验组的细胞数量与对照组相比均显著升高,差异有显著性意义（P〈0.05）,各实验组内相比,细胞数量增殖无显著性差异（P〉0.05）。提示前列腺素 E1 具有促进脂肪源性基质细胞的增殖作用,且前列腺素 E1 的影响效果无剂量依赖性。     

不同代次大鼠脂肪间充质干细胞增殖及成神经球的能力比较

背景：脂肪间充质干细胞是一组具有多向分化潜能的干细胞,体外在一定条件下可分化为神经干细胞。 目的：观察不同代次大鼠脂肪间充质干细胞体外培养增殖能力及诱导成神经球的潜力。 方法：取SD大鼠脂肪体外分离培养出脂肪间充质干细胞并传代扩增,分别在P3、P6、P10、P20时观察其形态学变化、测定增殖速度。流式细胞仪检测定不同代次细胞表型及细胞周期。将脂肪间充质干细胞诱导为神经球,计数成神经球率。 结果与结论：脂肪间充质干细胞主要呈长梭形,不同代次的脂肪间充质干细胞均具有很强的体外增殖能力;除P3细胞其他代次细胞均高表达CD29、CD44、CD73,低表达CD45、CD34。细胞周期中G0/G1期细胞所占比例P3为93.4%、P6为92.7%、P10为92.4%、P20为86.0%。P6、P10诱导成神经球率均显著高于P20（P〈0.05）,P3细胞较难诱导成神经球。提示以脂肪间充质干细胞作为种子细胞时,应注意选择适宜的扩增代数,以便在获得足够纯度细胞的同时保留细胞最佳的分化潜力。     

人脐血间充质干细胞向多巴胺能神经元分化的体外诱导

背景:细胞替代治疗帕金森病成为目前研究的一个热点,如何进行体外诱导获得足量的有效多巴胺能神经元是治疗该疾病的一种策略。目的:探讨人脐血间充质干细胞体外诱导分化为多巴胺能神经元的可行性。方法:将传至第5代人脐血间充质干细胞以5×106L-1接种,先用20μg/L表皮生长因子+20μg/L碱性成纤维细胞生长因子预诱导24h后,分为4组:对照组加入含体积分数为10%胎牛血清的DMEM/F12培养液,不加任何诱导液;其余3组分别加入100μmol/L抗坏血酸,1μmol/L全反式维甲酸,50μg/L胶质细胞源性神经营养因子单独或联合进行诱导。结果与结论:各诱导组均表达酪氨酸羟化酶、多巴胺转运体、多巴胺受体D2 mRNA。诱导分化后的细胞能表达神经元、星形胶质细胞特异性抗原。与对照组相比,各诱导组巢蛋白、神经元特异性烯醇化酶、胶质纤维酸性蛋白、酪氨酸羟化酶、多巴胺转运体、多巴胺受体D2阳性细胞率均明显升高(P<0.05);且全反式维甲酸+胶质细胞源性神经营养因子组和3种物质联合诱导组升高幅度高于抗坏血酸组(P<0.05),3种物质联合诱导组升高幅度高于全反式维甲酸+胶质细胞源性神经营养因子组(P<0.05)。结果提示抗坏血酸,全反式维甲酸,胶质细胞源性神经营养因子单独或联合均能促进人脐血间充质干细胞向多巴胺能神经元分化,3种物质联合应用效果最高。     

联合诱导兔脂肪源性基质细胞的成肌潜能*☆

背景：成肌种子细胞是构建工程化成肌复合物的基础,如何优化其扩增是过渡到临床应用的核心。目的：分析将 MyoD、转化生长因子β1及5-氮杂胞苷不同模式下联合诱导兔脂肪源性基质细胞体外成肌潜能的变化。方法：取兔腹部脂肪,采用胶原酶消化法分离获取脂肪源性基质细胞,分别以不同方式进行成肌细胞诱导：5-氮杂胞苷诱导组;MyoD+转化生长因子β1诱导;5-氮杂胞苷+ MyoD+转化生长因子β1联合诱导组;并设空白对照。于诱导第1,4,8,12,16,20,24,28天观察细胞形态学特点,行 MTT 比色法、流式细胞仪和免疫组织化学检测鉴定细胞,检测Ⅲ型胶原含量。结果与结论：联合诱导组与其他组相比,细胞生长迅速,16 d 增殖达高峰,20 d 肌管数量增多,体积增大,排列规则,呈结蛋白强阳性表达,细胞周期显示联合诱导组脂肪源性基质细胞的 DNA 复制期细胞比例增加,间隙期细胞减少,Ⅲ型胶原含量明显增高,差异有显著性意义。结果提示,多因子联合5-氮杂胞苷模式能有效促进脂肪源性基质细胞向成肌细胞定向分化,细胞增殖显著,是成肌细胞体外诱导的理想方法。     

前列腺素E1对人类脂肪源性基质细胞体外增殖的影响

背景:近年来脂肪源性基质细胞成为研究与应用的热点之一,但前列腺素 E1 对脂肪源性基质细胞增殖的作用影响尚未见报道。目的:观察前列腺素 E1 对人类脂肪源性基质细胞体外增殖的影响。方法:从人正常脂肪组织分离培养脂肪源性基质细胞,用 5 种不同浓度的前列腺素 E1 处理培养的细胞,其中 3×10-6,6×10-6,9×10-6,12×10-6 mol/L 作为实验组,0×10-6 mol/L 作为对照组,观察经不同浓度前列腺素 E1 处理后细胞的增殖情况。结果与结论:不同浓度实验组的细胞数量与对照组相比均显著升高,差异有显著性意义(P<0.05),各实验组内相比,细胞数量增殖无显著性差异(P>0.05)。提示前列腺素 E1 具有促进脂肪源性基质细胞的增殖作用,且前列腺素 E1 的影响效果无剂量依赖性。     

地塞米松在家兔脂肪基质干细胞定向诱导为成骨细胞的作用

目的:观察不同浓度地塞米松对家兔脂肪基质干细胞生长、增殖及向成骨细胞分化的影响。方法:实验于2006-11/2007-01在南方医科大学组织工程研究中心完成。实验材料:成年新西兰大白兔购于南方医科大学实验动物中心(机构许可证号SYXK(粤)2005-0056)。实验方法:①脂肪基质干细胞获取:取成年新西兰大白兔腹股沟皮下脂肪组织一二毫升,分离培养脂肪基质干细胞,并按1∶3进行传代培养。选取生长良好的P3代细胞进行免疫组织化学染色测定CD44抗原表达。并于第3,7,14天观察细胞增殖情况。②脂肪基质干细胞成骨诱导:成骨诱导液由不同浓度地塞米松、50mg/L的维生素C、10mmol/L的β-磷酸甘油和DMEM完全培养基组成。实验分6组:0mol/L地塞米松组、10-8mol/L地塞米松组、10-7mol/L地塞米松组、10-6mol/L地塞米松组、10-5mol/L地塞米松组、对照组。选择生长良好P3代兔脂肪基质干细胞,用0.25%胰酶消化3～5min,制成单细胞悬液,调整细胞密度为1×104,接种于25cm2的培养瓶中,在体积分数为0.05的CO2饱和湿度、37℃恒温培养箱培养。24h后所有诱导组更换为成骨诱导培养液,进行成骨细胞诱导分化处理,记为诱导第0天,对照组换DMEM完全培养液。诱导期间,每隔二三天换1次成骨诱导液,对照组换1次常规DMEM完全培养液。③第7,14,21天茜素红染色鉴定成骨细胞特性,观察不同浓度地塞米松对兔脂肪基质干细胞的生长、增殖及向成骨细胞分化的影响。结果:①地塞米松对脂肪基质干细胞增殖的影响:与对照组相比较,10-6,10-5mol/L组细胞数目明显减少(t=2.8623,3.4245,3.5203,3.9594,2.2641,2.7012,P<0.05)。②地塞米松对脂肪基质干细胞成骨分化的影响:光镜下观察整个诱导分化期,对照组未观察到钙化结节形成,0mol/L地塞米松组只观察到个别钙化结节形成。在诱导第14天,10-5,10-6,10-7,10-8mol/L地塞米松组均可观察到少数钙化结节形成,10-5,10-6,10-7mol/L组间钙化结节数差异无显著性意义(t=0.5571,0.5929,1.1142,P>0.05),但钙化结节数目明显多于10-8mol/L组(t=5.4960,6.2811,6.3640,P<0.05)。在诱导第21天,10-5,10-6,10-7mol/L组均可观察到大量钙化结节形成,均明显多于10-8mol/L组(t=6.2703,6.4065,5.6349,P<0.05)。结论:>10-7mol/L高浓度地塞米松对脂肪基质干细胞的生长、增殖有明显抑制作用;在其他诱导条件一致的情况下,10-7mol/L地塞米松最有利于脂肪基质干细胞向成骨细胞分化。     

人脂肪间充质干细胞生物学特征及体外向类角质细胞的分化

目的：在体外不同培养条件下，脂肪来源问充质干细胞可以向成骨细胞、成软骨细胞、神经细胞、内皮细胞、肌肉细胞、脂肪细胞和肝脏细胞等不同胚层来源的细胞分化。观察脂肪间充质干细胞在体外培养并向类角质细胞分化的能力。方法：实验于2005—06／2006—03在解放军军事医学科学院完成。实验方法：取20-45岁健康女性（已取得患者同意）脂肪组织行脂肪抽吸术后的脂肪颗粒。参照Zuk等方法分离脂肪问充质干细胞。取培养的第3代细胞，采用流式细胞仪鉴定细胞表面分子及细胞周期。取状态良好、融合至瓶底80％的第20代细胞，在5mg／L的秋水仙素处理后采集分裂细胞，以Giemsa染色进行核型分析。取第3代细胞以5μm维甲酸和5μg／L表皮细胞生长因子培养基向角质细胞诱导分化。对照组用含体积分数为0．05胎牛血清的L-DNMEM培养基。实验评估：采用倒置显微镜观察细胞形态，免疫组织化学方法检测角质细胞的标志物CK19。结果：①脂肪来源问充质干细胞表面分子CD29、CD90、CD44呈阳性，而CD34呈阴性。②细胞增殖曲线呈S型，接种的前3d细胞处于滞留期，3d后进入对数生长期，6d后进入平台期，细胞的群体倍增时间约为29h。处于对数生长期的细胞80％以上处于G0和G1期。③细胞染色体核型分析显示，染色体数目2n=46，核型46，xx，为女性正常核型。④对照组细胞贴壁后，呈成纤维细胞样，细胞呈长梭形，排列成漩涡状，贴壁较好。诱导组从诱导的第3天开始，细胞逐渐变成不规则，诱导第10天出现铺路石改变，形似角质细胞。⑤免疫组织化学方法检测显示，部分细胞专一地对CK19抗体呈阳性反应，有棕色染色。结论：可从人脂肪抽吸物中分离出间充质干细胞，脂肪间充质干细胞在体外一定条件下可向类角质细胞分化。     

碱性成纤维细胞生长因子影响脂肪干细胞的血管内皮迁移

背景：良好血运机制的建立是工程化组织成功植入的关键。 目的：观察外源性碱性成纤维细胞生长因子对植入小鼠皮下的人脂肪来源干细胞-透明质酸钠复合物中人脂肪来源干细胞向血管内皮迁移情况的影响。 方法：由吸脂术所得的脂肪组织中分离提取人脂肪来源干细胞进行培养传代,取第3代人脂肪来源干细胞进行cm-dil荧光标记后制成 5×10^9 L-1的细胞悬液,碱性成纤维细胞生长因子配成2 mg/L的工作液。将由0.25 mL透明质酸钠凝胶、0.2 mL细胞悬液和0.05 mL工作液/DMEM混合制成的碱性成纤维细胞生长因子组/对照组移植物分别植入小鼠背部左右两侧皮下作自身对照,6周后取材,行苏木精-伊红染色和免疫荧光标记血管内皮细胞进行观察分析。 结果与结论：植入处未出现结节、坏死及液化,取材时无凝胶残留。苏木精-伊红染色见标本性质多为脂肪组织及疏松结缔组织。荧光显微镜下仍可见标记人脂肪来源干细胞的cm-dil荧光,其与标记小鼠血管内皮的FITC荧光重合数碱性成纤维细胞生长因子组多于对照组（P〈0.05）。提示碱性成纤维细胞生长因子促进透明质酸钠支架中的人脂肪来源干细胞向血管内皮迁移、分化。     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.     

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.     

Interprofessional approaches to creating safe,high quality health care

Three supplementary perspectives are presented arguing that interprofessional collaboration is both necessary and desirable. Nonetheless, there are often too many serious intra-professional barriers and obstacles to interprofessional collaboration to make it successful. Some of these barriers, it is argued and illustrated, are found in the multiple ways in which professional identity is tacitly acquired and embodied in the practitioners' habitual, everyday practice. The paper then explores ways in which reflection, especially Second order reflection, can help to elucidate and overcome these obstacles, as well as increasing professional adaptability and competence.     

Systematic Reviews Published in the January 2011 Issue of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD and monthly online. The January 2011 issue (first quarterly DVD for 2011) contains 4515 complete reviews, 1985 protocols for reviews in production, and 13,521 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 641,000 randomized controlled trials, and 14,018 cited papers in the Cochrane methodology register. The health technology assessment database contains over 9300 citations. One hundred and seven new reviews have been published in the last 3 months, of which five have potential relevance for practitioners in pain and palliative medicine.     

Modified micro-superior percutaneously-assisted total hip: early experiences & case reports

Because of the extensile nature and familiarity of the standard posterior-lateral approach to the hip, a family of "micro-posterior" approaches has been developed. This family includes the Percutaneously-Assisted Total Hip (PATH) approach, the Supercapsular (SuperCap) approach and a newer hybrid approach, the Supercapsular Percutaneously-Assisted Total Hip (SuperPATH) approach. Such approaches should ideally provide a continuum for the surgeon: from a "micro" (external rotator sparing) posterior approach, to a "mini" (external rotator sacrificing) posterior approach, to a standard posterior approach. This could keep a surgeon within his comfort zone during the learning curve of the procedure, while leaving options for complicated reconstructions for the more practiced micro-posterior surgeons. This paper details one author's experiences utilizing this combined approach, as well as permutations of this entire micro-posterior family of approaches as applied to more complex hip reconstructions.     

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.     

Structure and function of "metalloantibiotics"

Although most antibiotics do not need metal ions for their biological activities, there are a number of antibiotics that require metal ions to function properly, such as bleomycin (BLM), streptonigrin (SN), and bacitracin. The coordinated metal ions in these antibiotics play an important role in maintaining proper structure and/or function of these antibiotics. Removal of the metal ions from these antibiotics can cause changes in structure and/or function of these antibiotics. Similar to the case of "metalloproteins," these antibiotics are dubbed "metalloantibiotics" which are the title subjects of this review. Metalloantibiotics can interact with several different kinds of biomolecules, including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities. In addition to the microbial-originated metalloantibiotics, many metalloantibiotic derivatives and metal complexes of synthetic ligands also show antibacterial, antiviral, and anti-neoplastic activities which are also briefly discussed to provide a broad sense of the term "metalloantibiotics."