金纳米笼探针用于线粒体成像和光热损伤细胞

线粒体是许多细胞行为的关键调节细胞器,线粒体膜电位降低被认为是细胞凋亡所发生的最早事件之一,因此线粒体成像及其膜电位的检测分析,对疾病的检测与治疗有重要的科学意义.采用金纳米笼(Aunanocages,Au NCs)介导的光热损伤与温度敏感的药物释放相结合,开发了一种线粒体靶向的荧光纳米探针AuNCs/PLEL/JC/KLA.引入一种线粒体靶向肽(KLAKLAKKLAKLAK,KLA),作为纳米探针的“指向标”,指引着探针特异地靶向到细胞线粒体部位,随后在近红外光的照射下, AuNCs吸收光能转化为热量,实现光热介导的细胞损伤.同时,高温促使外层温敏水凝胶发生凝胶-溶胶转变,实现荧光染料(JC-10)的释放.所释放的JC-10荧光染料可根据线粒体的活力表现出两种荧光信号,用于监测线粒体膜电位的变化.总之,该荧光纳米探针不仅实现了线粒体靶向的荧光成像与损伤细胞,同时还可以监测线粒体膜电位的变化.     

多壁碳纳米管诱导A549细胞氧化应激与去极化线粒体膜电位

以人肺上皮细胞系A549为模型细胞, 探讨多壁碳纳米管的细胞毒性效应及其机制. A549细胞暴露于不同浓度(0~300 μg/mL)的多壁碳纳米管后, 用MTT比色法检测细胞活力和Hoechst 33342染色法观察细胞形态; 用活性氧(ROS)敏感探针2',7'-二氯荧光素二乙酸酯(DCFH-DA)结合流式细胞仪检测细胞内ROS水平; 用荧光探针JC-1结合激光共聚焦显微镜检测细胞线粒体膜电位ΔΨm的变化; 用免疫荧光和蛋白印迹法检测细胞氧化应激敏感蛋白血红素氧合酶-1(HO-1)的表达水平. 结果表明, 多壁碳纳米管可引起A549细胞活性降低、细胞内活性氧ROS过量产生以及谷胱甘肽GSH含量下降, 诱导细胞氧化应激效应; 抗氧化剂N-乙酰半胱氨酸(NAC)抑制多壁碳纳米管诱导的A549细胞内ROS的产生. 多壁碳纳米管处理A549细胞2 h后, 诱发细胞线粒体膜电位下降; 多壁碳纳米管诱导细胞氧化应激的同时伴有适应性应激蛋白HO-1的上调表达. 结果表明, 细胞氧化应激和线粒体膜电位去极化可能是多壁碳纳米管诱导A549细胞毒性效应的重要机制.     

3,4,5-三羟基苯甲酸通过线粒体途径诱导人肝癌细胞SMMC-7721的凋亡

采用四甲基偶氮唑盐(MTT)观察3,4,5-三羟基苯甲酸(TBA)对人肝癌SMMC-7721细胞的增殖抑制作用; 通过流式细胞仪检测细胞凋亡、细胞内活性氧(ROS)及线粒体膜电位的变化, 用比色法测定Caspase-9和Caspase-3蛋白活性, 探讨TBA 诱导SMMC-7721细胞凋亡的分子机制. 研究结果表明, TBA对SMMC-7721细胞生长具有显著的抑制作用并诱导其凋亡, 诱导SMMC-7721细胞凋亡作用可能通过线粒体信号传导通路实现.     

木香烃内酯诱导乳腺癌MCF-7细胞凋亡作用机制的研究

研究了木香烃内酯诱导人乳腺癌细胞MCF-7细胞凋亡的作用机制.采用流式细胞仪测定不同浓度木香烃内酯(0,2,4,8 μg/mL)作用于MCF-7细胞后细胞凋亡、活性氧(Reactive oxygen species,ROS)含量及线粒体跨膜电位(Mitochondrial transmembrane potential,MTP)的变化,气相色谱-质谱联用(GC-TOF/MS)技术分析加药组与未加药组的代谢差异物.结果表明,木香烃内酯能诱导MCF-7细胞凋亡,并具有浓度依赖性,能够促使ROS含量升高;MTP在2μg/mL木香烃内酯作用时升高,在4和8μg/mL时显著下降;基于GC-TOF/MS的细胞代谢组学研究,最终发现15种代谢差异物.基于上述结果,推测木香烃内酯通过引起ROS含量升高、MTP降低,扰乱线粒体的正常功能,进一步阻碍TCA循环,抑制ATP合成,扰乱了细胞内代谢物的平衡,并引起位于膜间隙的凋亡相关蛋白释放,最终导致MCF-7细胞的凋亡.     

流式细胞术的最新进展

流式细胞术是一项集多种技术于一体的新型高科技细胞分析技术,是一种自动而迅速地进行细胞及免疫分析的标准方法。近年来在其基础上建立起来很多新型的流式细胞仪系统,具有体积小、功能全、价格低、使用方便、应用广泛等特点。本文综述了流式细胞术的最新进展及其应用。     

乙酸胁迫酵母细胞凋亡过程中单个线粒体损伤的拉曼光谱分析

应用激光光镊拉曼光谱技术(LTRS)作为一种非入侵分析线粒体的工具,结合氧电极法、紫外分光光度计法,研究乙酸胁迫酵母细胞后提取的线粒体,以及直接胁迫正常酵母细胞的离体线粒体的拉曼光谱.结果显示,乙酸胁迫酵母细胞后提取的线粒体的拉曼光谱,归属于核酸的谱峰(1081和1301 cm-1)、蛋白质的谱峰(872,1445,1604和1657 cm-1)、脂类的谱峰(1125,1301,1445和1657 cm-1)、Cyt c的特征峰(750和1125 cm-1)、线粒体呼吸特征峰(1604 cm-1),都随着诱导程度的加深而降低,与常规法测定的指标呼吸率,磷氧比,细胞色素C含量的降低,MDA含量的升高的变化趋势相似;乙酸直接胁迫线粒体的拉曼光谱,归属于核酸的谱峰(1081和1301 cm-1),蛋白质的谱峰(872,1445,1604和1657 cm-1),脂类的谱峰(1125,1301,1445和1657 cm-1),Cyt c的特征峰(750和1125 cm-1),线粒体呼吸特征峰(1604 cm-1),都随着诱导程度的加深而降低,与常规法测定的指标呼吸率、磷氧比、细胞色素C含量的降低,MDA含量的升高的变化趋势相似.结果表明,乙酸胁迫细胞过程中,乙酸进到细胞内可能直接作用于线粒体,引起线粒体内物质的释放,导致依赖于线粒体途径的细胞凋亡.     

用于检测生物体系中线粒体活性氧的荧光探针

In addition to being the energy powerhouse of the cell, mitochondria are an important source of reactive oxygen species (ROS) during the process of molecular oxygen metabolism. Mitochondrial ROS are closely associated with normal physiological functions as well as human diseases, and participate in cell signaling, nucleic acid and protein damage, and oxidative stress induction. However, the complicated interplay between mitochondrial ROS and the cellular pathological state has not been fully elucidated. It is expected that research on the mitochondrial ROS undertaking in the molecular pathogenesis of human diseases would benefit from development of efficient tools for the detection of these ROS. In recent years, an increasing number of fluorescent probes for mitochondrial ROS with high sensitivity and selectivity have been developed. Here, we present a review of the recent advances in small molecular fluorescent probes for selective detection of ROS inside the mitochondria. In this review, the design, synthesis, characteristics, and applications of the published fluorescent probes for mitochondrial ROS are discussed in detail.     

线粒体靶向的近红外HClO/ClO-荧光探针的研究进展

HClO/ClO-作为细胞质中一种重要的活性氧(ROS),源自线粒体,参与各种生理和病理过程,因此快速有效检测HClO/ClO-具有重要的生物学及生理学意义.荧光分析法因其灵敏度高、响应时间快、选择性高、成本低和操作简便等优点而备受关注.更重要的是,使用荧光探针可以在体外和体内可视化检测.近年来,为了研究HClO/Cl...     

高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究

二氧化硫(SO₂)及其衍生物(亚硫酸盐、亚硫酸氢盐等)作为活性硫物种的重要组成部分,与生命体的生理过程有着密切的关系.此外,摄入过量的SO₂会对呼吸系统造成不可修复的损伤,例如呼吸系统损伤、心脑血管和神经疾病等.近红外发射荧光探针具有较强的组织穿透能力,可以有效地检测活体内的生物分子.因此,设计了一种基于迈克尔加成(Michael Addition)机理的线粒体靶向近红外SO₂荧光探针XA-SO₂.溶液光谱实验表明,该探针XA-SO₂对SO₂有着显著的选择性和灵敏度,并且具有快速检测SO₂的能力.值得注意的是,该荧光探针XA-SO₂具有较好的细胞膜渗透能力,成功检测到了细胞外/内源性SO₂.更重要的是,该探针XA-SO₂能够有效地在细胞的线粒体上富集,有望实现细胞线粒体内SO₂的动态检测.     

基于三苯基磷的线粒体靶向荧光探针研究进展

简述了线粒体内各种相关活性物质及实时变化是探索生命体奥秘的重要组成部分,而化学荧光探针则是研究该领域的重要工具之一。近年来,具有定位和检测双功能的线粒体荧光探针被相继报道,其中,基于三苯基磷的线粒体靶向荧光探针因具有良好检测性能而成为当前研究的热点。综述了近十年以三苯基磷为定位基团的线粒体靶向体荧光探针在检测活性氧和还原性物质等方面的研究进展,并归纳了文献报道中各探针的特点,以期为进一步探索构建新的线粒体靶向荧光探针提供借鉴与参考。     

核酸适配体功能化近红外量子点结合流式细胞术快速检测白血病细胞

利用作为肿瘤细胞识别分子的核酸适配体(Aptamer)的高特异性和高亲和力以及作为信号报告单元的近红外量子点(QDs)的高荧光发射强度和低生物背景干扰特性,构建了一种基于Aptamer功能化近红外QDs的新型纳米荧光探针,并进一步结合流式细胞术在单细胞荧光分析方面的高通量、简便和快速等优势,建立了一种检测白血病细胞的新方法.以基于Cell-SELEX(Cell-based systematic evolution of ligands by exponential enrichment)技术针对CCRF-CEM人急性白血病细胞筛选的特异性Aptamer Sgc8c为模型,构建了Sgc8c-QDs探针,其仅需与细胞样品培育30 min即可实现对缓冲液和血清中靶细胞的简单、快速和高特异性检测.与传统荧光染料标记技术相比,该方法不仅大大提高了分析灵敏度,还显示出对血清等复杂生物样品的高适用性和优良检测性能.鉴于Cell-SELEX技术在其它白血病细胞Aptamer筛选领域的应用潜力,该方法有望作为一种通用技术在白血病诊断及预后监测等方面发挥重要作用.     

水体微型颗粒的流式细胞术测定

应用流式细胞术（FCM）对水体中不同粒径级微型颗粒的数量、百分比及随时间变化进行了初步研究；仪器经流速校正，样品经过滤，外参法测定；结果表明FCM可快速、即时分析水体中的微型颗粒。     

流式细胞术散射光谱法测定痕量银的研究

以TritonX_100为乳化剂,用AgNO3与NaCl反应制备了AgCl微粒体系。应用流式细胞仪和荧光光度计分别研究了液相流式AgCl微粒体系的流式细胞术(FCM)光散射及动态散射光谱(DLS)特性,建立了流式细胞术散射光检测痕量Ag+的新方法。实验发现,Ag+质量浓度分别在1.50×10-4~5.00μg/mL、4.80×10-3~20.99μg/mL范围内,流式、动态AgCl微粒体系散射光强度与Ag+质量浓度都呈现线性关系(r=0.995 0和0.999 8),方法检出限分别为0.045、1.50 ng/mL。     

Detection of Intracellular Proteins and Biomarkers Using Modified Silica Nanoparticles and Flow Cytometry In vitro

In this paper, antibody-modified silica nanoparticles were successfully synthesized, and their average diameter was (109±9) nm. These particles were mixed with cell extracts to target a protein, then, an antibody labeled with fluorescein isothiocyanate(FITC) was added to form FITC-labeled nanoparticle complexes and the product was analyzed using flow cytometry. The results confirm that the intracellular proteins and biomarkers were precisely and sensitivity detected by the novel method in vitro. The FITC intensity of Akt antibody-conjugated particles was increased ≥ 10-fold compared with that of control samples in MCF-7 cells. Furthermore, it can also simultaneously measure several proteins when modified with different antibodies.     

微流控芯片用于流式细胞术的基础研究

自行设计并加工了玻璃微流控芯片,并将其与流式细胞术相结合,采用羟丙基甲基纤维素(HPMC)的磷酸盐溶液为缓冲体系和鞘液,解决了微粒在微芯片中流动的若干问题,使其状态可以得到更有效的控制.采用自行组装的激光诱导荧光装置并结合动电聚焦技术,实现了对荧光微球的计数,并可通过荧光倒置显微镜实时观察到微通道内微球的实际流动情况.方法简单,操作方便,并且具有仪器体积小、试剂及样品用量少和分析速度快等优点.     

Cephalotaxine Inhibits the Survival of Leukemia Cells by Activating Mitochondrial Apoptosis Pathway and Inhibiting Autophagy Flow

Cephalotaxine (CET) is a natural alkaloid with potent antileukemia effects. However, its underlying molecular mechanism has not been well understood. In this study, we verified that CET significantly inhibited the viability of various leukemia cells, including HL-60, NB4, Jurkat, K562, Raji and MOLT-4. RNA-sequencing and bioinformatics analysis revealed that CET causes mitochondrial function change. Mechanism research indicated that CET activated the mitochondrial apoptosis pathway by reducing the mitochondrial membrane potential, downregulating anti-apoptotic Bcl-2 protein and upregulating pro-apoptotic Bak protein. In addition, the autophagy signaling pathway was highly enriched by RNA-seq analysis. Then, we found that CET blocked the fluorescence colocation of MitoTracker Green and LysoTracker Red and upregulated the level of LC3-II and p62, which indicated that autophagy flow was impaired. Further results demonstrated that CET could impair lysosomal acidification and block autophagy flow. Finally, inhibiting autophagy flow could aggravate apoptosis of HL-60 cells induced by CET. In summary, this study demonstrated that CET exerted antileukemia effects through activation of the mitochondria-dependent pathway and by impairing autophagy flow. Our research provides new insights into the molecular mechanisms of CET in the treatment of leukemia.     

Influence of Charging Current and Potential Drop on the Propagation of the Change in the Membrane Potential

Propagation of a change in a potential difference between two aqueous phases (W1 and W2) across a membrane was examined by using three membrane cells (A, B and C). At first, the cell A was electrically connected with the cell B by controlling the ionic compositions. By changing the connection with the cell A from the cell B to the cell C indicating the different membrane potential, the change of the membrane potential was propagated. The delay and decrement of the propagation was observed by setting capacitors or resistors in the electric circuit.     

A Novel Method for the Detection of Chlorpyrifos by Combining Quantum Dot-labeled Molecularly Imprinted Polymer with Flow Cytometry

Uniform-sized fluorescent molecularly imprinted polymers were prepared by one-step swelling and suspension polymerization, while chlorpyrifos, methacrylic acid, ethylene glycol dimethacrylate, and oil-soluble CdSe/ZnS quantum dots were used as the carrier, template molecule, functional monomer, cross-linker, and fluorophor, respectively. The morphology, adsorption dynamics, binding ability, and selectivity of quantum dot-labeled molecularly imprinted polymers were evaluated. The dosage of quantum dots for labeling the molecularly imprinted polymers was optimized. The results showed that the optimized dose of quantum dots was 200?µL using a concentration of 8.0?µM. The microsphere size was approximately 10?µm with a honeycombed surface. The quantum dot-labeled molecularly imprinted polymers had an even brightness and a high selectivity. In the presence of different concentrations of chlorpyrifos, a decrease in the fluorescence intensity of the quantum dot-labeled molecularly imprinted polymer was clearly identified by flow cytometry. The whole detection process was accomplished within 2?h including pretreatment. This method was used for the determination of chlorpyrifos in tap water samples.