双硫仑联合铜离子对人骨肉瘤细胞增殖与凋亡的影响

文题释义：双硫仑：是一种广泛用于治疗慢性酒精上瘾以及古柯碱的戒断药物,其为二硫代氨基甲酸盐,水溶性极差。早在20世纪中期,Jacobsen等发现双硫仑可有效抑制乙醛脱氢酶的活性,造成乙醛的饱和氧化反应,其本身和代谢产物可引起体内相关蛋白失活,使人体产生不适反应,从而达到抗酗酒目的。二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDTC)：白色至无色片状结晶,有吸湿性,易溶于水,溶于乙醇、甲醇、丙酮,不溶于乙醚和苯。水溶液呈碱性并逐渐分解,遇酸能分离出二硫化碳而使溶液混浊,熔点 94-102 ℃。最大吸收波长(乙醇中)257、290 nm(摩尔吸光系数1 200、13000)。低毒,半数致死量(大鼠,经口)2 830 mg/kg。研究显示,双硫仑在进入人体后会迅速转化为体内代谢物DDTC,Cu可与DDTC结合形成较稳定的DDTC-Cu复合物。背景：研究表明双硫仑本身具有抗肿瘤活性,可联合铜(Cu)离子在体内外水平对多种肿瘤发挥抑癌作用,但关于双硫仑对骨肉瘤细胞增殖和凋亡作用的影响尚未阐明。目的：探讨双硫仑-Cu在体内外水平对骨肉瘤增殖与凋亡能力的影响以及可能的作用机制。方法：实验方案经山西医科大学动物实验伦理委员会批准(批准号为2017LL077)。①体外实验：配置双硫仑在进入人体后的转换物二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDTC)和Cu离子的复合物DDTC-Cu(0.5,1,2,3和5 μmol/L),设置DDTC单药(5 μmol/L)、Cu单药(5 μmol/L)和空白对照组。药物处理人骨肉瘤细胞Saos-2细胞和MG-63细胞,CCK8法检测不同浓度DDTC-Cu对Saos-2细胞和MG-63细胞的增殖抑制作用;采用AnnexinV-FITC/PI双染法检测DDTC-Cu对Saos-2细胞凋亡水平变化;②体内实验：取4周龄BALB/c-nu/nu雌性裸鼠共10只,随机分为DDTC-Cu组和对照组。采用异位移植方法,在裸鼠右侧背部皮下注射Saos-2细胞和Matrigel混悬液(1∶1混合),注射量400 μL/只;接种2周后,对照组裸鼠腹腔注射地塞米松(0.5 mg/kg,隔日1次),DDTC-Cu组裸鼠腹腔注射地塞米松(0.5 mg/kg,隔日1次)和DDTC-Cu复合物(10 nmol/g,隔日1次);观察两组荷瘤小鼠移植瘤生长情况,绘制瘤体生长曲线。接种5周后麻醉下处死动物,完整取出瘤体,免疫组织化学检测瘤体石蜡切片组织中ki67蛋白的表达水平,Western blot检测瘤体组织中细胞增殖和凋亡蛋白的表达及JNK通路蛋白表达的变化。结果与结论：①体外实验结果：DDTC-Cu组对骨肉瘤细胞的增殖抑制作用明显高于其他3组;CCK-8实验结果显示DDTC-Cu对骨肉瘤细胞增殖抑制呈剂量依赖性,两株细胞的24 h药物半抑制浓度分别为0.337 μmol/L和0.487μmol/L;流式细胞学检测结果显示DDTC-Cu可呈剂量依赖性促进Saos-2细胞的凋亡;②体内实验结果：DDTC-Cu组裸鼠移植瘤的体积和质量均小于对照组;免疫组织化学结果显示,DDTC-Cu组的瘤体中ki-67蛋白表达水平较对照组降低;Westernblot检测结果显示,DDTC-Cu组瘤体蛋白中p-JNK和c-jun的表达水平均上调。结果提示,双硫仑联合Cu离子在体内外水平抑制人骨肉瘤细胞增殖并促进骨肉瘤细胞凋亡,其作用机制可能与JNK通路活化有关。ORCID: 0000-0003-4818-901X(徐朝健)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

双硫仑样反应知晓率的问卷调查分析

目的通过问卷的方式对双硫仑样反应知晓率进行调查,引起大家对双硫仑样反应的认识和关注。方法：通过2007年1月-2008年12月对本县县、乡两级医疗机构的医务人员和患者做双硫仑样反应知晓率的问卷调查,发放问卷12000份,收回有效答卷11836份,对数据进行统计处理和数据分析。结果：11836份调查表中,药剂人员知晓率最高（37.6％）,其次为医生（34．1％）,行管人员（25．4％）,护士（19％）,普通民众知晓率最低（6．4％）,平均13．6％。结论：医务人员和民众普遍对双硫仑样反应知之甚少,应加大其相应的健康宣教力度,预防双硫仑样反应的发生。     

18例双硫仑反应的急诊临床分析

目的：探讨双硫仑样反应的临床表现以及急诊救治要点。方法：对2010-2012年我院急诊科收治的18例双硫仑样反应患者的临床表现及救治情况进行回顾性分析。结果：18例患者经积极的治疗全部治愈出院。结论：双硫仑反应是临床常见的急诊,临床反应具有多样性,但经积极救治可痊愈。     

双硫仑样反应的发生与防治

目的介绍双硫仑样反应的有关知识,为临床安全用药提供参考。方法查阅《万方数据库医药信息系统》,选择代表性文献进行归纳与分析。结果能引发双硫仑样反应的药物主要有头孢菌素类、咪唑类、呋喃类抗菌素。结论双硫仑样反应在临床上很常见,应引起重视。     

双硫仑样反应的发生与防治

目的 介绍双硫仑样反应的有关知识,为临床安全用药提供参考.方法 查阅<万方数据库医药信息系统>,选择代表性文献进行归纳与分析.结果 能引发双硫仑样反应的药物主要有头孢菌素类、咪唑类、呋喃类抗菌素.结论 双硫仑样反应在临床上很常见,应引起重视.     

抗生素类药物致双硫仑样反应临床分析

目的 探讨引起双硫仑样反应的相关抗生素药物的种类、临床表现、治疗方法,引起临床医师的重视.方法 对我科收治的58例双硫仑样反应患者的成因、临床表现、治疗方法进行分析结合文献报导的422例完整病例的临床资料.结果 所有患者中男性占比例较高,可有不同程度的面红、心慌、胸闷、头晕、头痛、恶心、呕吐,严重者致休克、引发心血管事件等.入院后进行吸氧、补液、扩容纠正休克、静推地塞米松、纳洛酮等综合治疗,所有患者均痊愈出院.结论 应用抗生素期间或停药后一周内应避免饮酒或应用含乙醇的制品,以防出现双硫仑样反应.     

头孢类抗生素引起双硫仑样反应的观察

双硫仑样反应又称为戒酒样反应。是由于酒精中间代谢产物乙醛降解受阻,使乙醛在人体内蓄积而产生的中毒现象。头孢类抗生素因抗菌谱广,疗效肯定,在临床上应用广泛,患者在应用该类药品期间或停药后7天内饮酒或接受含有酒精的药物及饮料者可出现双硫仑样反应。主要表现为面色潮红,诉头痛、头晕、心慌、气促、呼吸困难、烦躁不安、恶心、呕吐及心前区疼痛,查体时可有血压下降、心率加速（可达120／min）。我院在两年来共成功抢救14例双硫仑样反应的患者,现将急救与护理体会总结如下。     

大黄素阻抑人乳腺癌MCF-7细胞增殖和细胞周期进程

 目的：探讨大黄素抗人乳腺癌MCF-7细胞增殖及其相关机制。方法：采用MTT法检测大黄素对MCF-7细胞的增殖抑制作用;流式细胞术分析周期分布和凋亡情况;原子力显微镜(AFM)观察细胞膜表面超微结构的变化。结果：大黄素呈剂量依赖性地抑制MCF-7细胞的增殖;大黄素能使MCF-7细胞阻滞在G0/G1期;Annexin V/PI双染法结果表明,大黄素对MCF-7细胞没有明显的促凋亡作用。AFM观察细胞膜超微结构,结果显示对照组细胞核区饱满,膜表面平坦光滑;大黄素作用48 h可致细胞核区坍塌萎缩,细胞表面颗粒密集,膜表面的平均粗糙度(Ra) 和均方粗糙度(Rq) 与对照组相比均有显著增加(P<005)。结论: 大黄素通过阻断MCF-7细胞的细胞周期进程及影响细胞膜超微结构而发挥抗肿瘤作用。     

红藻氨酸处理对星形胶质细胞超微结构影响的研究 --原子力显微镜及透射电镜观察

为了观察体外培养的大鼠海马星形胶质细胞(ASTs)在红藻氨酸(KA)作用下的超微结构变化,我们体外培养大鼠乳鼠海马ASTs,在KA不同浓度(25μmol/L、250μmol/L)及时间(10min、100min)处理条件下,采用原子力显微镜(AFM)扫描细胞表面超微结构改变,透射电镜(TEM)观察细胞内部超微结构变化。结果表明,与对照组相比,低浓度KA(25μmol/L)处理组的细胞表面超微结构无明显改变,但胞内出现空泡样变,溶酶体增多;高浓度KA(250μmol/L)处理组的细胞表面有“孔洞”、“裂隙”样改变,直径在数百纳米,这些表现在KA长时间处理组(100min)更加明显;TEM下可见溶酶体增多、胞核扭曲,在KA长时间处理组甚至出现了自噬小体。以上结果说明培养的ASTs经KA处理后能发生一些损伤性的超微形态结构变化,且具有剂量及时间依赖性,而AFM观察到的KA处理使细胞膜出现“孔洞”、“裂隙”样变化可能是不可逆性兴奋性神经毒性损伤的结构基础。     

DMSO诱导MCF-7细胞凋亡的研究

目的研究二甲基亚砜(DMSO)对MCF-7细胞凋亡的诱导作用。方法用不同浓度DMSO处理体外培养的MCF-7细胞,应用倒置光显微镜观察细胞形态学变化,用MTT比色法检测细胞存活率;Hoechst33258/PI荧光染色,用荧光显微镜分析凋亡细胞比率;琼脂糖凝胶电泳检测DNA梯状条带。结果在倒置光学显微镜下观察1%DMSO处理细胞12h后细胞形态发生变化。约有50%以上的细胞变圆,细胞内有多泡小体形成。随DMSO浓度增加和作用时间的延长,细胞存活率明显下降,经MTT检测其IC_(50)值为1%;荧光显微镜下可见60%以上细胞核染色质凝集,核碎裂等凋亡细胞的形态学变化;琼脂糖凝胶电泳呈现梯状条带(DNA ladder)。结论适当浓度的DMSO能够抑制乳腺癌细胞增殖并诱导其凋亡。     

抑制骨架蛋白对正常大鼠肾细胞力学性能的影响

目的确定正常大鼠肾细胞内骨架蛋白的变化对细胞力学性能的影响。方法通过微管吸吮技术对正常大鼠肾细胞用细胞松弛素D、秋水仙素和blebbistatin进行处理,然后分析细胞的杨氏模量和黏弹性的变化。结果经过骨架蛋白抑制剂的处理,细胞的弹性模量表现出显著的下降;与对照组相比,秋水仙素处理后细胞的黏弹性没有显著变化,而细胞松弛素D和blebbistatin处理后细胞的黏弹性显著降低。结论Blebbistatin对正常大鼠肾细胞力学特性的影响不依赖于细胞骨架的改变;抑制肌动蛋白聚合或抑制肌球蛋白IIATP酶的活性能够大幅降低细胞杨氏模量和黏弹性,微管蛋白是否正常聚合与细胞杨氏模量相关而对细胞黏弹性没有显著影响。     

体外循环30min对红细胞膜的表面结构及力学特性的影响

目的:应用原子力显微镜(AFM)观察和分析体外循环(又称心肺分流术,CPB)30 min对红细胞膜表面的超微结构和生物力学性能的影响。方法:择期行体外循环心脏手术患者10例,分别取手术前和体外循环转机30 min的中心静脉血各2 m L,分为对照(CON)组和CPB组,肝素抗凝。正立荧光显微镜观察计数2组非圆红细胞;AFM观察红细胞膜表面超微结构并进行力曲线测定。结果:与CON组比较,CPB组非圆红细胞比例差异无统计学意义;与CON组比较,CPB组细胞膜表面隆起呈紊乱排列,凹凸性增强,均匀度下降,膜表面颗粒分布有差异(P0.05),平均粗糙度(Ra)和均方根粗糙度(Rq)增大(P0.05),细胞膜黏附力升高(P0.05),但膜形变恢复力和力曲线斜率差异无统计学意义。结论:体外循环转机30 min可以引起红细胞膜的表面形貌和超微结构改变,黏附性增大。     

Mapping the CXCR4 receptor on breast cancer cells

The CXCR4 receptor triggers cell migration and, in breast cancer, promotes metastasis. To date, the dynamic assembly of CXCR4 on the cell surface as a mediator of receptor binding is not well characterized. The objective of this work is to quantify the density, spatial organization, and magnitude of binding of the CXCR4 receptor on live metastatic breast cancer (MBC) cells. We measured the Young's modulus, the CXCR4 surface density, and CXCR4 unbinding force on MBC cells by atomic force microscopy. We conclude that the CXCR4 density, spatial organization, and matrix stiffness are paramount to achieve strong binding.     

原子力显微镜观察青蒿琥酯对人胃癌细胞株SGC-7901膜表面形貌的影响

目的: 利用原子力显微镜在纳米级分辨率下观察药物对肿瘤细胞膜表面形貌结构的影响。方法: 采用不同浓度的抗肿瘤药物青蒿琥酯作用于人胃癌细胞株SGC-7901,24 h后运用原子力显微镜观察细胞膜表面形貌、三维结构及超微结构的变化。流式细胞术检测细胞凋亡情况。结果: 对照组细胞核区饱满,膜表面平坦光滑;加药组细胞核区塌陷,萎缩,膜表面形成了孔洞和凹陷;且随青蒿琥酯浓度的增加,孔洞直径加大,数量增多。超微结构观察到对照组细胞膜表面颗粒较密集,有的呈条索状,有的聚集成团,堆积较为紧凑;而加药组细胞膜表面有陷窝,颗粒稀少,分布较疏松。膜超微结构参数分析显示,加药组细胞核区高度降低,膜微区的平均粗糙度 (Ra) 和均方粗糙度 (Rq) 均降低。经流式细胞术分析,加药组细胞凋亡率随青蒿琥酯浓度的增加而增大,与对照组相比均有显著差异。结论: 本实验结果不仅有助于寻找细胞特异性的形貌学改变,也为揭示抗癌药物的作用机制提供微观形态学参考。     

Atomic force microscopic measurement of the mechanical properties of intact endothelial cells in fresh arteries

Mechanical properties of living endothelial cells in the abdominal aortas and in the medial and lateral wall of aortic bifurcations obtained from rabbits were determined by means of an atomic force microscope (AFM), focusing on the locational differences. Force (F)-indentation (delta) curves of the cells were expressed by an exponential function: F = a(exp(b delta)-1), where a and b are constants. The parameters b and c(= ab) represent the rate of modulus change and initial modulus, respectively. The slope of F-delta curves a and the parameter c were higher in the medial wall than in the other sites, which is attributable to abundant stress fibres in endothelial cells in the medial wall. There were no differences in the parameter b among the three locations. These results indicate that endothelial cells are stiffer in the medial wall of aortic bifurcation than in the other regions.     

Atomic force microscopic measurement of the mechanical properties of intact endothelial cells in fresh arteries

Mechanical properties of living endothelial cells in the abdominal aortas and in the medial and lateral wall of aortic bifurcations obtained from rabbits were determined by means of an atomic force microscope (AFM), focusing on the locational differences. Force (F)-indentation (δ) curves of the cells were expressed by an exponential function: F=a(exp(bδ)−1), where a and b are constants. The parameters b and c(=ab) represent the rate of modulus change and initial modulus, respectively. The slope of F-δ curves a and the parameter c were higher in the medial wall than in the other sites, which is attributable to abundant stress fibres in endothelial cells in the medial wall. There were no differences in the parameter b among the three locations. These results indicate that endothelial cells are stiffer in the medial wall of aortic bifurcation than in the other regions.     

Imaging the lamellipodium of migrating epithelial cells in vivo by atomic force microscopy

Cell locomotion originates at a specific region of the cell surface, the leading edge of a migrating cell. Various factors have been proposed to contribute to the propulsion of a cell over the substratum. Rapid turnover processes of cytoskeletal elements inside the cell and insertion of new plasma membrane at the leading edge of the cell permit the extension of a cell in a given direction. Our goal was to image in vivo plasma membrane turnover by means of atomic force microscopy (AFM) and to resolve dynamic processes at the nanometer level. As an experimental model we used migrating kidney cells derived from the Madin-Darby canine kidney (MDCK) cell line that was transformed by alkaline stress. These so-called MDCK-F cells exhibit spontaneous calcium-dependent oscillatory activity of plasma membrane potential associated with cell locomotion. We imaged cells during migration and observed dynamic invagination processes in the cell surface close to the leading edge, indicating internalization of plasma membrane. Invaginations were prevented by removal of calcium from the perfusate. During calcium reduction plasma membrane uncoupled from the underlying cytoskeleton and lipidic pores with diameters of about 30 nm could be disclosed and imaged. This study demonstrates that the AFM can readily trace dynamic physiological processes in vivo, emphasizing the potential role of calcium in maintaining plasma membrane integrity and function.     

Aldosterone receptor sites on plasma membrane of human vascular endothelium detected by a mechanical nanosensor

The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37°C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 ± 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 ± 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.     

年龄与位置对牙本质力学性质的影响

目的研究人牙本质弹性模量和硬度随年龄和位置的变化情况。方法采集下颌无龋坏第3磨牙,按照年龄分为青年、中年、老年3组。采用纳米压痕对牙本质切片的多个位置进行力学测试。结果外层和中层牙本质的弹性模量和硬度大于内层牙本质的弹性模量和硬度;随着年龄的增长,各个区域的牙本质弹性模量和硬度都增大。结论牙本质具有梯度力学特性,外层和中层牙本质具有很高的刚度,其抵抗变形的能力要强于内层牙本质。同时,随着年龄增长牙本质弹性模量和硬度增大。