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...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。展开更多
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 ...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.展开更多
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 ...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.展开更多
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, e...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.展开更多
文摘目的: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。
基金supported by the program of The Project Supported by Natural Science Basic Research Plan in Shaanxi Province of China(No.SJ08-ZD05)
文摘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.
文摘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.
基金partially supported by the CONACYT(Mexico)grant 0105961/10110/194/09.
文摘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.
