超高灵敏度荧光显微成像技术对光敏剂细胞内分布的初步研究

目的：探讨应用基于ICCD的超高灵敏度荧光显微成像系统研究光敏剂细胞内分布的可行性。方法：传代培养内皮细胞、食管癌细胞和肺癌细胞,将不同浓度血卟啉单甲醚(HMME)与细胞共同孵育不同时间。采用荧光显微镜及ICCD组成的荧光显微成像系统采集不同浓度及不同孵育时间条件下HMME的荧光图像,并采用计算机图像处理技术进行图像增强、滤波后计算其细胞浆与细胞核的平均荧光强度比值。同时应用激光共聚焦显微镜图像采集进行对比。结果：HMME浓度为5μg／ml时,荧光显微镜采集到HMME的荧光图像;HMME浓度升高到160μg／ml,激光共聚焦显微镜获得HMME的荧光图像。两组图像的特点都为胞浆中荧光强度较高,细胞核区荧光较弱;细胞浆与细胞核的比值约为2～3：1。结论：荧光显微镜和ICCD采集细胞内光敏剂的荧光图像灵敏度高,方法可靠、实用。HMME较多分布在细胞质中,细胞核吸收较少。     

三套荧光显微成像系统在光敏剂亚细胞定位研究中的应用比较

目的：对三套荧光显微成像系统在国产新型光敏剂HMME亚细胞定位研究中的应用特点及适用范围进行了比较与评价。方法：分别应用LSCM、CCD、ICCD荧光显微成像系统,选择特异性细胞器荧光探针Rhodamine-123、DIOC6(3)标记细胞内线粒体和内质网。采用细胞器-细胞荧光强度比值法,对HMME进行单细胞内分布的定性与定量研究。结果：LSCM和CCD成像系统能采集到浓度达到160μg／ml时的HMME的荧光图像,获得荧光探针图像信息显示所标记的细胞内线粒体和内质网平均荧光强度比值(J1/J2值)都明显高于细胞内J1/J2值。而ICCD成像系统只需HMME浓度为5μg/ml,荧光图像特点都呈胞浆中荧光强度较高且分布不均,细胞核区荧光较弱的中空现象。ICCD系统对细胞器探针荧光图像在空间分辨上不理想。结论：LSCM与CCD成像系统限于其探测灵敏度,对于弱荧光性光敏剂,适用于其高孵育浓度条件下的亚细胞定位研究。二者获得的结果相一致：孵育24h,HMME在鼠肺内皮细胞线粒体和内质网有分布而几乎不进入细胞核。ICCD成像系统可不受孵育浓度条件的限制,实现光敏剂极微弱荧光的有效探测,但空间分辨率较低。     

ZnPcS2P2在K562和HL-60细胞中的定位研究

目的:探讨ZnPcS2P2在K562细胞,HL-60细胞亚细胞结构中的精确定位,揭示光动力学疗法(photody-namic therapy,PDT)的作用机制。方法:将K562细胞,HL-60细胞与ZnPcS2P2共同孵育5 h。应用激光扫描共聚焦显微成像系统,选择特异性细胞器荧光探针(线粒体探针若丹明Rodanmine123、溶酶体探针LysoTrackerDND-26、内质网探针Dioc6(3)采用波形比较法对光敏剂进行亚细胞定位。结果:ZnPcS2P2在K562细胞,HL-60细胞中发出的荧光与负载的Rodanmine123、Lyso-TracKer DND-26、Dioc6(3)均有部分重叠,波形均有相似之处。ZnPc-S2P2在线粒体、溶酶体、内质网均有分布。结论:线粒体是ZnPcS2P2介导的PDT(ZnPcS2P2-PDT)光损伤的主要靶点,溶酶体、内质网也是ZnPcS2P2-PDT光损伤的靶点。     

基于HMME的PDT体外灭活HL60实验研究

目的:探讨血卟啉单甲醚(HMME)介导的光动力疗法(HMME-PDT)对HL60细胞的作用及PDT前后HL60细胞表面超微结构的变化。方法:CCK-8法检测光敏剂浓度和光照剂量对HL60细胞抑制率的影响,荧光分光光度计监测PDT过程中光敏剂荧光强度随时间的变化,Fluo 3-AM荧光探针检测不同浓度HMME作用后HL60细胞内Ca2+变化,原子力显微镜观测PDT作用前后不同扫描范围HL60细胞表面的超微结构图。结果:细胞灭活率呈光敏剂浓度-光剂量依赖关系,当HMME为50μg/mL,光照剂量为24 J/cm2时,灭活效率达到70%;随着光照时间的增加,光敏剂的荧光强度不断减弱,下降速率也逐渐变慢;随HMME作用浓度增加,钙离子浓度显著升高;HMME-PDT作用后HL60细胞表面结构出现明显变化。结论:HMME-PDT能有效灭活HL60细胞,光敏剂浓度和光剂量是影响PDT疗效的重要因素,PDT过程中伴随有光漂白现象的发生,细胞凋亡和钙离子浓度增加呈正相关,PDT作用前后细胞出现明显萎缩,细胞膜粗糙度增加。     

血卟啉单甲醚在A549肺癌细胞内亚细胞分布的动态变化

目的:探讨血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)在A549肺癌细胞内亚细胞分布的动态变化。方法:传代培养A549肺癌细胞,分别与光敏剂HMME孵育2 h和12 h。应用由荧光显微镜及电感耦合器材(Charge-coupled device,CCD)组成的高分辨率荧光显微成像系统,结合荧光探针标记技术,采用细胞器-细胞荧光强度比值法研究HMME在不同时间的亚细胞分布情况。结果:在2 h和12 h两个孵育时间点高尔基体的平均荧光强度比值(J1/J2)值都最高;随着孵育时间延长,A549细胞的四种细胞器J1/J2值都升高且溶酶体幅度最大。结论:孵育时间是影响HMME亚细胞分布的一个重要因素。随着孵育时间的延长,A549肺癌细胞各细胞器吸收HMME能力逐渐增强,尤以溶酶体显著。     

原子力显微镜观测血卟啉单甲醚对细菌光动力杀伤作用

[目的]探讨血卟啉单甲醚(Hematoporyrin monomethyl Ether,HMME)对革兰氏阳性(G )、阴性(G-)菌的光动力杀伤作用.[方法]通过平板菌落计数法和原子力显微镜(AFM),观察细菌与HMME作用前后形貌的变化.[结论]当HMME浓度为50 μg/mL,可见光(光功密度为200 mW/cm2)光照30min时90%以上的金黄色葡萄球菌(Staphyrlococcus aureus)能被杀死,无光照时对S.aureus杀灭效果显著.同等条件下,无论光照还是无光照,HMME对大肠杆菌(E.coli)无明显的杀伤作用.AFM图像显示,S.aureus细菌表面破坏严重,完全碎裂成鱼鳞状的片状堆积.对HMME作用后的E.coli扫描可见,菌体原来光滑的表面变成网格状的裂纹排列.[讨论]HMME对G 有明显的光失活效应,而对G-效果不明显.AFM的超微图像显示HMME对细菌细胞的攻击位点主要在细胞膜上.AFM为我们研究光敏剂对细菌的光动力损伤作用机制的可视化提供了依据.     

光动力过程中线粒体膜电位和细胞存活关系

以1-anilionaphthalene-8-sulfonate(ANS)作荧光探针,通过其荧光光谱研究了苯硫基酞菁锌PcS)、苯硫基铝酞菁(AIPcS)和烷氧基铝酞菁(AIPc)这三种金属酞菁配合物作为光敏剂的光动力作用对癌细胞线粒体膜表面电位的影响．研究表明,光动力作用后线粒体膜表面电位降低,表面电荷数面密度增加．ZnPcS的影响最大,这与酶联免疫检测光动力作用后对癌细胞的杀伤效果相一致,提示细胞线粒体膜可能是金属酞菁配合物在光动力过程中的作用位点。通过比较细胞线粒体膜表面电位以及表面电荷数面密度与细胞存活之间的关系,阐述了光动力作用的物理学机制．同时,由于线粒体膜电位与细胞凋亡的密切关系,金属酞菁配合物对线粒体膜表面电位的影响提供了一个衡量药物疗效的判据。     

钙信号在光动力疗法中的产生及作用

光动力疗法是基于光敏剂选择性地积聚在肿瘤组织中,肿瘤接受光照后凋亡或坏死的一种细胞毒性治疗方法.光敏剂的亚细胞定位决定了细胞光敏损伤的初始位置,线粒体、内质网、细胞膜、溶酶体,细胞骨架等均可成为光敏损伤的靶点.细胞内Ca2 作为一个广泛意义上的信号分子,参与了多种信号转导途径,在光动力疗法诱导肿瘤细胞凋亡过程中起了重要作用.从光动力疗法造成的亚细胞损伤出发,探讨了光动力疗法中钙信号的产生机制,并简要介绍了钙信号在光动力疗法诱导肿瘤细胞凋亡中的作用机制.     

"激光成像式活检定位肿瘤的原理与技术研究"通过评审

2007年1月8日在福建省科技厅主持下,在福州对谢树森教授主持的福建省自然科学基金重大项目(2002F008)“激光成像式活检定位肿瘤的原理与技术研究”进行了评审,一致认为:1.该项目在理论研究方面取得了如下具有创新性的成果(1)首次利用三维荧光光谱比较研究了二磺基二邻苯二甲酰亚胺甲基酞菁锌、癌光啉和血啉甲醚等3种国内第二代新型光敏剂,以及第一代光敏剂血卟啉衍生物的光谱特性,得到了在不同激发波长下的发射波长强度分布曲线,以及最佳的激发和发射波长;(2)研究了用于鼻咽癌光活检的光敏剂血啉甲醚的超快时间分辨光谱特性,得到了其荧光寿…     

构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应

目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to delta pH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 microM) and ATPase activity of inverted inner membrane vesicles (Ki = 126 microM) and partially purified F1-ATPase (Ki = 177 microM). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 microM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. delta psi-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of delta psi in isolated mitochondria.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (K_i = 12 μM) and ATPase activity of inverted inner membrane vesicles (K_i = 126 μM) and partially purified F₁-ATPase (K_i = 177 μM). The smaller K_i for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.     

Modulations of food-derived substances on intestinal permeability in Caco-2 cell monolayers

The effects of more than 300 kinds of food extracts on intestinal permeability were investigated in Caco-2 cells with the use of model compounds: Lucifer Yellow (LY) for the paracellular pathway, Fluorescein (FC) for the monocarboxylic acid transporter-mediated pathway, and Rhodamine 123 (RH) for the p-glycoprotein-mediated efflux pathway. With several extracts of increasing or decreasing LY permeation, increasing FC or RH permeation was also observed, indicating modulation by dietary substances in several pathways for intestinal absorption.     

Synthesis and characterization of fluorescent Lucifer yellow-lipid conjugates

The syntheses of fluorescent lipid probes composed of Lucifer yellow dyes linked to either cholesterol or phospholipids are described. The spectral properties of these probes are characterized, and the probes are evaluated for use with model membranes and with live animal and plant cells. Of the probes synthesized, the cholesterol derivative is the easiest to prepare and appears to be the most useful because it readily labels the plasma membrane of live cells and maintains a high ratio of cell surface-to-cytoplasmic fluorescence.