Rhodamine 123(Rh-123)对癌细胞与正常细胞的作用比较及机制分析

Rh-123是一种活细胞线粒体的荧光探针。在黑暗的背景下可显示清晰的线粒体荧光图像。在一定的浓度下,它对正常细胞无明显毒性作用,所以是研究线粒体形态与功能的有利工具。近年来又发现Rh-123具有选择性地杀伤肿瘤细胞的作用。本实验选用人子宫颈癌HeLa细胞及正常人胚肾成纤维细胞为材料,用多种方法比较了Rh-123对它们的作用,并探讨其作用机制。

SELECTIVE TOXICITY OF RHODAMINE 123 ON CARCINOMA CELL IN VITRO

The selective toxicity of the mitochondria-specific cationic fluorescent dye rhodamine 123 (Rh-123) on He La cells in culture was studied. In this report, we demonstrate that with continuous exposure, Rh-123 markedly inhibited the growth of HeLa cells but had little effect on normal human kidney fibroblasts. With continuous exposure to Rh-123, the growth rate, colony forming ability, anl mitotic index of HeLa cells were decreased. The mechanism of toxicity of Rh-123 on HeLa cells was investigated by EM and enzyme cytochemistry stain. The mitochondria of carcinoma cells were the main targets for the inhibitory action of Rh-123, since they selectively accumulated the dye. At the dosage of Rh-123 which was toxic to HeLa cells, the structure and function of mitochondria were disrupted, as the mitochondria-related enzymes, i.e., ATPase, LDH and SDH were inhibited. The possible mechanism of the action of Rh-123 on HeLa cells is briefly discussed.

Rhodamine123介导的光动力学疗法对小鼠淋巴细胞增殖及骨髓干祖细胞集落形成的影响

目的 探讨Rhodamine12 3(Rh12 3)介导的光动力学疗法对小鼠淋巴细胞增殖及骨髓干祖细胞集落形成的影响。方法 以C5 7B/ 6小鼠为供鼠 ,BALB/c小鼠为受鼠 ,脾脏淋巴细胞混合培养 ,比较单纯Rh12 3、单纯激光照射、光动力学疗法对小鼠混合淋巴细胞增殖 (MTT法 )及骨髓粒单核巨噬细胞集落形成 (CFU -GM)的影响 (甲基纤维素培养基法 ) ;流式细胞仪检测混合淋巴细胞中CD3+ CD6 9+ 阳性率。结果 Rh12 3组及单纯激光组对细胞增殖无显著影响 ,光动力学组 2 0mW/cm2 以下剂量激光对细胞增殖无明显影响 ,30mW/cm2 以上明显抑制细胞增殖 ;30mW/cm2 以下剂量对CFU -GM集落无明显影响。光动力学疗法后细胞培养 2 4h ,CD3+ CD6 9+ 表达明显下降。结论 光动力学疗法在 30mW/cm2 激光照射时可抑制淋巴细胞增殖 ,降低早期活化T细胞表达 ,但不影响骨髓CFU

Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia

Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.

Effects of Taxotere on invasive potential and multidrug resistance phenotype in pancreatic carcinoma cell line SUIT-2

INTRODUCTIONDevelopment of drug-resistance to chemotherapyand subsequent metastasis of tumor are primarilyresponsible for treatment failure and the death fromcancer. There have been many previous studies onthe relationship between expression of multidrugresistance (MDR) phenotype P-glycoprotein (P-gp)and the malignant properties of tumors, but theresults are often conflicting[1-8]. The difference intumor types or MDR phenotype induced by specificagents might account for this discrepancy. Taxotere(TXT), a member of the family of taxanes, hasantitumor activity through its effect of promotingthe polymerization of tubulin[9,10].

Effects of Colchicine on Distribution of Mitochondria in 2-cell and Compacted 8-cell Mouse Embryos

The distribution of mitochondria during early development of mouse embryos was visualized bymitochondria-specific vital fluorescent dye, rhodamine 123(Rh 123). Mitochondrial clusters wasmarkedly conceotrated to perinuclear area in blastomere of normal 2-ccll embryos. In blastomere ofuncompacted 8-cell embryos, mitochondria were randomly distributed throughout the cytoplasm, butthey were reorganizcd to the cytocortices beneath the apposed surfaces of blastomere duringcompaction. As demonstrated in our study, colchicine (10 μg/ml) produced marked effect onmitochondrial distribution in blastomcre of 2-cell and compacted 8-cell embryos: mitochondriabecame scattered throughout the cytoplasm ofblastomere. It is suggested that the spatial distributionof mitochondria in early mouse embryo are maintained by microtubule.

Fluorometric Viability Assessment of Capacitated and Acrosome-Reacted Boar Spermatozoa by Flow Cytometry

Sperm capacitation involves functional changes, such as the removal or appearance of specific molecules and changes in the plasma membrane;the acrosome reaction (AR) is an exocytotic event induced by calcium influx, enabling the spermatozoa to penetrate the zona pellucida. These processes can be achieved only if the spermatozoa have good viability;indeed, determination of sperm viability is used for the assessment of semen quality. Membrane integrity and mitochondrial activity are important viability parameters of spermatozoa and fluorescent techniques based on membrane permeability to dyes have been developed to determine these parameters. The aim of this work was to determine the viability of boar sperm (fresh, one hour of capacitation induction and 20 min of AR induction) by flow cytometry using propidium iodide (PI) (1.25 μg/mL) and rhodamine 123 (R123) (0.20 μg/mL). Aliquots of 5 × 105 sperm were incubated with each fluorochrome separately and simultaneously for 10 or 20 min, respectively, at 38℃. The proportion of labeled spermatozoa and their fluorescence intensities were measured using a flow cytometer. The fluorescence index (FI) with PI gradually increased during the incubation and we found significant differences between all the groups. With R123, the FI increased in the capacitated sperm but decreased in the acrosome-reacted sperm, with significant differences between the fresh and capacitated spermatozoa. Our results suggest that the increase in the R123 fluorescence intensity in capacitated spermatozoa is due to changes in the mitochondrial membrane activity because the spermatozoa experienced changes in membrane fluidity and flagellar activation during capacitation. The use of fluorochromes and flow cytometry is a good tool for monitoring many markers of sperm function. Although capacitation and AR processes have been well studied, there is still much information to be elucidated with regard to these complex processes.

α-三联噻吩对斜纹夜蛾SL细胞线粒体膜电位及细胞周期的影响

【目的】研究α-三联噻吩（α-T）对斜纹夜蛾（Spodoptera litura）SL细胞线粒体膜电位及细胞周期的影响。【方法】通过Rhodamine123染色和流式细胞术（flow cytometry,FCM）研究α-T处理SL细胞24 h和48 h后细胞线粒体膜电位的变化,并通过PI染色和FCM,测定α-T处理SL细胞24 h和48 h后细胞周期各时相百分率的变化。【结果】光活化后的α-T处理SL细胞24 h后,0.0625 μg·mL^-1浓度和0.1250 μg·mL^-1浓度处理组细胞线粒体膜电位的变化幅度不大,而0.5000 μg·mL-1浓度处理组细胞线粒体膜电位明显升高,48 h后高浓度处理组（1.0000 μg·mL^-1）导致线粒体膜电位明显去极化;处理24 h后,低浓度（0.0625 μg·mL^-1）处理组细胞被阻滞于S期,而高于此浓度的光照处理组细胞被阻滞于G2/M期,48 h后,浓度≤0.1250 μg·mL^-1的光照处理细胞被阻滞于G2/M期,而浓度＞0.1250 μg·mL^-1的光照处理组细胞被阻滞于S期。【结论】SL细胞经α-T处理后,细胞处于一系列复杂的动态变化中。当细胞本身不能扭转ROS造成的氧化损伤时,细胞线粒体膜电位和细胞周期时相即出现较大幅度的变化,这种变化决定着细胞的受损程度和细胞的死亡途径。

肺癌细胞和正常人气道上皮细胞线粒体膜电位的比较

目的:探讨正常气道上皮细胞和肺癌细胞线粒体膜电位的差别。方法:利用rhodam ine 123标记,采用流式细胞仪和激光共聚焦显微镜的方法分别检测正常气道上皮细胞HBE细胞和肺癌细胞SPC、A549、H446细胞的线粒体膜电位。结果:HBE细胞的线粒体膜电位明显低于SPC细胞和H446细胞,但和A549细胞无明显差别。结论:正常气道上皮细胞线粒体膜电位水平低于大部分肺癌细胞的线粒体膜电位,但和少数肺癌细胞并无明显差别。

Apoptosis susceptibility of tumor cells to arsenic trioxide and the inherent cellular level of reactive oxygen species

OBJECTIVE: To explore the association of inherent cellular reactive oxygen species (ROS) levels with susceptibility of the tumor cells to apoptosis induction by arsenic trioxide (As(2)O(3)). METHODS: Low concentration (2 micromol/L) of As(2)O(3) was administered to two cultured leukemic cell lines, NB4 and U937, and two esophageal carcinoma cell lines, EC1.71 (also named EC/CUHK1) and EC1867, to confirm the difference in apoptosis susceptibility of NB4 versus U937 and of EC1.71 versus EC1867. Dihydrogenrhodamine 123 (DHR123), used as a ROS capture agent, was incubated with cells in the absence of As(2)O(3). Fluorescence intensity of rhodamine 123, the product of cellular oxidation of DHR123, was detected by flow cytometry and ROS was measured. RESULTS: Low concentration of As(2)O(3) induced apoptosis was more likely to occur in NB4 and EC1.71 cells than in U937 and EC1867 cells, or NB4 was more sensitive than U937, and EC1.71 more sensitive than EC1867 to As(2)O(3). The inherent cellular ROS level is higher in NB4 than in U937, and also higher in EC1.71 than in EC1867. CONCLUSIONS: The difference in cellular ROS level is positively associated with cellular susceptibility to apoptosis induction by As(2)O(3). The inherent ROS level might be important in defining apoptotic susceptibility to As(2)O(3).