线粒体功能障碍在尿酸损伤肾小管上皮细胞中的作用

目的:探讨线体功能在尿酸损伤肾小管上皮细胞中的作用。方法:体外培养大鼠肾小管上皮细胞,将其分为对照组(N)、尿酸0.1 mmol/L组(U1)、尿酸0.2 mmol/L组(U2)、尿酸0.4 mmol/L组(U3)、尿酸0.6 mmol/L组(U4)。观察细胞形态的改变,计算细胞凋亡率;检测细胞内活性氧(ROS)的生成情况;观察线粒体膜电位变化。结果:尿酸干预组肾小管上皮细胞形态较对照组发生明显变化。尿酸干预组肾小管上皮细胞凋亡率较对照组明显上升(P0.05)。尿酸干预组线粒体膜电位较对照组显著下降(P0.05),U2、U3、U4之间,随着尿酸浓度的增加线粒体膜电位逐渐下降(P0.05)。尿酸干预组活性氧的水平较对照组明显升高(P0.05),随尿酸浓度的增加活性氧的水平逐渐增加(P0.05)。相关性分析显示肾小管上皮细胞凋亡率与线粒体膜电位的降低和活性氧的生成显著正相关(r=0.964,r=0.932,P0.01)。结论:线粒体功能障碍参与了尿酸致肾小管上皮细胞损伤的过程,为尿酸性肾病的诊治提供新的靶点。     

精子线粒体膜电位评估男性生育力的作用研究进展

全球目前约有15％的适龄夫妇无法生育,其病因中男方因素占约20％,男方因素是导致适龄夫妇无法生育的重要原因之一[1,2].另外目前国内外最新流行病学显示男性精液质量呈逐年下降趋势[3].而不明原因的少、弱、畸形精子症,即特发性少、弱、畸形精子症,又占据男性不育中的较大比例,因此精液质量与男性生育力密切相关[45].目前...     

精子线粒体琥珀酸脱氢酶的检测及意义

目的 :采用一种简便、快速的检测精子线粒体琥珀酸脱氢酶 (SDH)的改良方法检测精子SDH ,并评价其与精子活动率及存活率的关系。　方法 :4 6例年龄为 2 5～ 36岁 (平均 31岁 )生育与不育男性精液标本 ,采用计算机辅助精液分析系统检测精子活动率 ,应用改良SDH检测法检测精子SDH ,通过死、活精子荧光分子探针染色检测精子存活率 ,分析精子活动率、存活率及精子SDH阳性率之间的关系。　结果 :4 6例生育与不育男性精子活动率为( 6 7.33± 7.37) % ,存活率为 ( 79.78± 7.6 5 ) % ,精子SDH阳性率为 ( 74 .74± 8.2 9) % ;精子活动率、存活率及精子SDH阳性率 3者之间均存在显著相关性 (P均 <0 .0 1)。　结论 :精子线粒体SDH检测对评价精子线粒体功能具有重要意义 ,还可作为精子存活率的辅助指标     

孕酮趋化诱导人精子线粒体功能与ROS产生的关系

目的:使用计算机辅助精液分析仪和流式细胞分析仪,比较经上游法处理前后、孕酮诱导上游法处理前后精子线粒体相对活性以及活性氧自由基(ROS)阳性精子比例的变化,研究受精精子的线粒体功能特点。方法:收集正常人类精子(达到《世界卫生组织人类精液检查与处理实验室手册》第5版标准),利用"上游法"获能培养1 h,获得"获能前组"和"获能后组"精子。获能后组的精子继续在37℃和5%二氧化碳培养箱中,使用"孕酮诱导"垂直上游法再处理1 h,获得孕酮诱导超激活运动精子,分别称为"孕酮诱导组"和"对照组"。分别使用线粒体膜电位特异性染色剂JC-1和ROS染色剂DCF,标记人类精子线粒体膜电位,并计算线粒体相对活性指数,标记ROS阳性细胞比例。结果:正常人类精子经获能处理及继续垂直上游后,线粒体相对活性(高活性线粒体/低活性线粒体比值)显著上升。获能前组、获能后组、对照组和孕酮诱导组的线粒体活性分别为1.42、6.23、14.36和12.33;获能前组、获能后组、对照组和孕酮诱导组含有均衡线粒体(含有等量高膜电位和低膜电位的线粒体)比例分别为21.64%、4.27%、5.03%和8.57%,说明孕酮诱导能够在激活线粒体活性的同时,维持低活性线粒体的比例。ROS同时在精子的头、颈、尾部出现,获能前组、获能后组、对照组和孕酮诱导组ROS阳性细胞比例分别为2.89%、0.7%,4.25%和1.90%。上游1 h后的精子ROS阳性比例显著低于上游前精子。再经过1 h垂直上游后,"对照组"精子的ROS阳性比例大大增加;孕酮诱导组精子的ROS阳性比例显著低于"对照组"。结论:孕酮体外诱导上游精子,能够显著提高精子运动速度,使其呈现超激活运动状态,同时抑制精子的线粒体相对活性持续增高,保持线粒体活性均衡,降低ROS产量,可能有利于体外受精率和胚胎质量的提高。     

人精子体外氧化损伤对线粒体tRNA~(LeuUUR)基因的影响

目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。     

番茄红素对人精子冷冻损伤的保护作用及机制研究

目的:通过在人精子冷冻保护液中添加不同浓度的番茄红素,研究番茄红素对精子冷冻损伤的可能保护作用及机制。方法:选择吉林省人类精子库捐精者的精液标本25份,每份精液一式4份,3∶1加入冷冻保护液后混匀,不含番茄红素者设为对照组(Ly0),而Ly2、Ly5、Ly10实验组混合液中分别含有2、5、10μmol/L的番茄红素,冷冻复苏精液进行常规分析,采用流式细胞术分析冻融后各组精子的凋亡率;采用硫代巴比妥酸(TBA)比色法检测精子中脂质过氧化产物丙二醛(MDA)浓度。利用JC-1标记法检测线粒体膜电位。结果:冻融后各组精子运动参数较新鲜精液参数均明显下降(P<0.01),Ly5组冻融后精子凋亡率[(25.68±4.36)%]较对照组[(33.26±4.78)%]显著下降(P<0.05);Ly5组线粒体膜电位水平[(66.18±14.23)%]显著高于对照组[(55.24±12.31)%],P<0.05;Ly2、Ly5、Ly10组的MDA水平与对照组比较,无统计学差异(P>0.05)。结论:在精液冷冻保护液中添加一定浓度的番茄红素可以减少线粒体氧化损伤,减轻活性氧对精子质膜的氧化应激损伤,从而提高精子的抗凋亡能力。     

黄精赞育胶囊对弱精子症患者精子线粒体膜电位的影响

目的:观察黄精赞育胶囊对弱精子症患者精子线粒体膜电位的影响及临床疗效。方法:弱精子症患者70例分药物治疗组(n=39)和空白对照组(n=31)做队列观察研究。药物治疗组予黄精赞育胶囊口服3个月,1次4粒,每日3次。服用时可备孕,期间若配偶怀孕则停止用药。空白对照组不予药物干预。观察初次就诊和3个月后精液参数变化,及JC-1染色后流式细胞仪检测精子线粒体膜电位(MMP)值的变化。结果:两组各失随访1例。药物治疗组总有效率为71.05%,服药过程中3例患者女方自然妊娠。药物治疗组35例患者完成观察期结束后检查,MMP%、精子总活力、前向运动精子百分率(PR%)、精子形态均有改善,其变化值较空白对照组30例患者的测定值有显著差异(P0.01),分别为△MMP%:(20.28±14.21)%vs(1.19±10.36)%;△PR%:(17.58±12.73)%vs(2.26±8.29)%;△总活力:(20.68±14.12)%vs(3.46±8.67)%;△正常形态精子百分率:(3.37±3.99)%vs(0.23±3.48)%。MMP%与精子总活力(r=0.69)、PR%(r=0.75)、正常形态精子百分率(r=0.26)具有显著相关性(P0.01)。结论:黄精赞育胶囊能提高精子MMP值,改善线粒体功能,从而有效增加精子活力及PR%并改善精子形态,对治疗弱精子症安全有效。     

线粒体靶向抗氧化剂Mitoquinone对人精子冻融氧化应激损伤的保护作用

目的:探讨线粒体靶向抗氧化剂Mitoquinone(Mito Q)对冻融人精子的保护作用。方法:选取60份健康生育男性精液标本,每份精液一式6份,不含Mito Q者设为对照组(G0),而G1、G2、G3、G4、G5实验组混合液中分别含有2 nmol/L、20 nmol/L、200 nmol/L、2μmol/L、20μmol/L Mito Q,37℃孵育1 h后检测活性氧(ROS)、丙二醛(MDA)和线粒体膜电位(MMP)变化。选取合适Mito Q浓度B1、B2组用于精子冷冻保存,B0组未添加Mito Q,B1、B2组在精子冷冻保护液中分别含有200 nmol/L和2μmol/L Mito Q,进行冷冻保存,检测冷冻复苏后的ROS水平、MDA含量和MMP改变。结果:新鲜精液添加Mito Q孵育后,G3组和G4组前向运动精子百分率[(30.8±10.2)%和(32.7±13.5)%]和总活动率[(70.6±9.0)%和(70.3±11.9)%]显著高于G0组[(17.6±5.0)%、(54.9±11.5)%](P0.05);随着Mito Q浓度的增加,ROS水平呈下降趋势,G3、G4、G5组(分别为86.5±31.6、93.6±42.0、45.1±15.0)显著低于G0组(160.8±39.7)(P0.05);MDA含量G3、G4组[分别为(0.9±0.5)、(0.9±0.5)μmol/mg]明显低于G0组[(1.9±1.1)μmol/mg](P0.05),而G5组[(1.7±0.7)μmol/mg]不但没有降低,反而显著高于G3、G4组(P0.05);与G0组MMP(1 701±251)相比,G5组(1 156±216)显著降低(P0.05),而G1、G2、G3、G4组(分别为1 810±298、1 995±437、1 950±334、1 582±314)无明显变化。冷冻复苏后各组前向运动精子百分率和总活动率均较新鲜精液明显下降(P0.01),B1组前向运动精子百分率[(3.2±2.3)%]较B0组[(0.8±0.6)%]明显改善(P0.05);B1组精子总活动率[(43.0±9.5)%]较B0组[(26.5±11.4)%]明显改善(P0.05);B1组ROS[(34.6±12.3)]和B2组ROS[(37.0±10.5)]均较B0组[(56.9±14.3)]显著下降(P0.05),B1组MDA[(1.4±0.5)μmol/mg]和B2组MDA[(1.4±0.6)μmol/mg]均较B0组[(2.6±1.0)μmol/mg]显著下降(P0.05),B1组MMP[(1 010.0±131.5)]和B2组MMP[(880.6±128.6)]均显著高于B0组[(721.1±24.8)](P0.05)。结论:在精液冻存液中添加200 nmol/L的Mito Q能有效提高人精子质量,可作为精液冷冻保护添加剂用于精液的冷冻保存。     

流式细胞术在精子功能检测中的应用

目的:研究流式细胞术在精子功能评价中的作用。方法:对104例男性不育患者和10例正常生育男性的精液标本进行精液量、精子密度、活动率、畸形率等精液常规分析(CASA法),对精子存活率、染色质结构和线粒体膜电位等进行流式细胞术分析,并将检测结果通过SAS软件进行统计学分析。结果:成组U检验结果表明:不育患者的精子与正常生育男性精子相比除了具有密度低(U=2.51,P=0.014 3)、活动率差(U=3.44,P=0.001)、畸形率高(U=-5.88,P<0.000 1)等特点外,经流式细胞术测定精子结果表明不育男性的精子质膜完整率(U=4.72,P<0.000 1)、线粒体膜电位正常率(U=-2.53,P=0.030 9)和染色质完整率(αt:U=-3.82,P=0.000 3;SDαt:U=-3.98,P=0.000 1;COMPαt:U=-3.57,P=0.000 5)均显著低于正常生育男性。多元逐步回归分析结果表明:精子质膜完整性与精子活动率(t=1.66,P=0.101 6)、精子的线粒体膜电位与精子活动率(t=3.33,P=0.001 4)、精子染色质的完整性与精子活动率(t=3.24,P=0.001 9)均呈正相关,而精子的线粒体膜电位与精子颈、尾部的畸形率呈负相关(t=-3.44,P=0.001)。结论:流式细胞仪测定对精子质量评定意义显著,对男性不育的诊断和治疗也具有较高的参考价值。     

Protection of melatonin against damage of sperm mito-chondrial function induced by reactive oxygen species

Aim: To study the mitochondrial function damage of sperm in-duced by reactive oxygen species (ROS) and the protection of melatonin (MLT) against the damage. Methods: Normal function spermatozoa were selected from semen samples by Percoll gradi-ent centrifugation technique. The ROS generated by the hypoxan-thine xanthine oxidase system was incubated with the normal sper-matozoa in the presence or absence of MLT (6 retool/L) for 30 and 60 minutes.     

活性氧与勃起功能障碍

男性勃起功能障碍(ED)是男科常见疾病之一,当前全世界有15200万男性受到ED的困扰。近年来已经认识到活性氧(reactive oxygen species,ROS)在ED中起重要作用。本文总结近年来ROS在ED中作用的研究进展,并阐述ROS与ED中各种危险因子的关系以及抗氧化治疗进展,为开发治疗ED的药物提供新的方向。     

Analysis of the Changes of Movement Function and Viability in Human Spermatozoa Induced by Reactive Oxygen Species

Objective: To study the influence of reactive oxygen species (ROS) on the mobility and viability of human spermatozoa. Methods: Spermatozoa with normal function were selected from human semen samples by the Percoll gradient centrifugation technique. ROS were generated by the hypoxanthine-xanthine oxidase system and were incubated with the spermatozoa under aerobic environment. Movement parameters of spermatozoa were analyzed by the computer-assisted semen analysis (CASA) system. Results: Thirty min after incubation with ROS, the motility, curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) of the spermatozoa were significantly decreased (P<0.01), while the change in the amplitude of lateral head displacement (ALH) was insignificant (P>0.05). After 60 min incubation, the motility was almost lost with all the movement parameters close to zero. Conclusion: When normal spermatozoa were incubated with ROS, the mobility was decreased. It is believed that ROS may be one of t     

JC-1单标法检测精索静脉曲张患者精子线粒体膜电位的研究

目的:检测精索静脉曲张患者精子线粒体膜电位并探讨其临床意义。方法:将67例精索静脉曲张患者分为VC1组(精索静脉曲张1度,n=26)、VC2组(精索静脉曲张2度,n=21)和VC3组(精索静脉曲张3度,n=20),以正常生育男性为正常对照组(n=29)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后用荧光染料JC-1染色后上流式细胞仪分析,检测精子线粒体膜电位(JC-1+%)。结果:VC1、VC2、VC3组精子线粒体膜电位[(56.29±16.32)%,P<0.05;(45.04±13.21)%,P<0.01;(31.63±12.91)%,P<0.01]均显著低于正常对照组[(76.21±13.96)%]。96例标本中,JC-1+%与(a+b)级精子百分率呈显著正相关(r=0.693,P=0.000)。结论:精索静脉曲张可引起精子线粒体膜电位降低,可能是导致男性不育的重要原因之一。     

活性氧对河岸葡萄叶圆片抗氧化酶活性的影响

研究了离体处理下活性氧对河岸葡萄叶圆片抗氧化酶活性的影响.结果表明,外源H2O2和Fe2++H2O2处理4 h可引起河岸葡萄叶圆片细胞膜透性明显增加,引起APOD,GPOD和SOD活性明显下降,但低浓度活性氧可不同程度引起叶圆片CAT活性增加;细胞膜透性与活性氧浓度呈显著正相关.     

活性氧致人精子运动功能和存活力变化的分析

目的 :对活性氧 (ROS)作用后精子的运动参数和存活率的变化进行分析 ,以证实ROS是否为引起精子运动功能障碍的病因之一。　方法 :采用Percoll梯度离心法选择具有正常生理功能的人精子作为正常精子模型 ,应用次黄嘌呤 黄嘌呤氧化酶体系产生ROS ,在有氧环境下与精子模型孵育后 ,运用计算机辅助精液分析 (CASA)系统 ,分析ROS攻击后精子运动参数的改变。　结果 :与对照组相比 ,正常精子模型与ROS孵育 30min后 ,精子活动率、曲线速度 (VCL)、直线速度 (VSL)、平均路径速度 (VAP)均明显下降 (P <0 .0 0 1) ,头侧摆幅度 (ALH)尚无明显改变 (P >0 .0 5 ) ,而与ROS孵育 6 0min后 ,精子运动功能近乎丧失 ,精子主要运动参数趋向于零。　结论 :ROS与正常精子作用后 ,可导致精子运动功能下降 ,从而证实ROS为精子运动功能障碍的病因之一。     

Reactive Oxygen Species Independent Cytotoxicity Induced by Radiocontrast Agents in Tubular Cells (LLC-PK1 and MDCK)

Purpose. Radiocontrast agents (RAs) cause renal tubular damage by hemodynamic imbalance, which could cause hypoxic stimulus and direct cytotoxicity. However, reactive oxygen species (ROS) could be an important factor in RAs' direct cytotoxicity. This study investigated the involvement of ROS in deleterious effects produced by RAs on normoxic and hypoxic renal tubular cells. Materials and Methods. LLC-PK1 and MDCK were exposed to diatrizoate and ioxaglate in normoxic and hypoxic conditions. Apoptotic and necrotic cell death were assessed by acridine orange/ethidium bromide and annexin V methods. Hydrogen peroxide, superoxide anion, and malondialdehyde levels were analyzed by, respectively, 2′,7′-dichlorofluorescein, luminal, and thiobarbituric acid. Antioxidant agents were used to prevent cellular RAs damage. Results. Diatrizoate and ioxaglate decreased cellular viability in both cells, and this effect was enhanced by hypoxic conditions. Diatrizoate induced more injury than ioxaglate to both cell lines. LLC-PK1 underwent necrosis, while MDCK cells underwent apoptosis when exposed to diatrizoate. These results could not be attributed to an increase in osmolality. RAs did not increase hydrogen peroxide, superoxide anion or malondialdehyde levels in both cells. Additionally, N-acetyl-L-cysteine (NAC), ascorbic acid, α-tocopherol, glutathione, β-carotene, allopurinol, cimetidine, and citric acid did not protect cells against RAs damage. Surprising, NAC increased the cellular damage induced by ioxaglate in the both cell lines. Conclusion. The present study shows that RAs induce damage in cultured tubular cells, especially in hypoxic conditions. ROS were not involved in the observed RAs' cytotoxicity, and NAC increased ioxaglate-induced tubular damage.     

Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa

This study was designed to evaluate the effects of semen processing on the generation of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in spermatozoa, and to develop reliable indexes for the evaluation of sperm quality during sperm preparation. Swim-up and density gradient centrifugation methods were used to separate semen in oligoasthenoteratozoospermia (OAT), leucocytospermia (LC) and normozoospermia groups. Levels of ROS and MMP were measured by flow cytometry. Before preparation, the patients with abnormal semen parameters had a lower MMP and higher ROS, and there was a negative correlation between MMP and ROS. The levels of MMP and ROS increased significantly, especially ROS produced by swim-up. A significant difference was found between the correlation of MMP and total normal motile sperm count after preparation in the OAT group. The level of ROS was associated with the amount of white blood cells in the LC group. The MMP can be used as an objective index to evaluate the sperm quality of OAT patients, and the combination of MMP and ROS can be used to assess the efficiency of sperm preparation in LC patients. These findings can guide selection of the ideal sperm separation technique for different sperm samples.