解偶联蛋白2在蜕膜巨噬细胞活性调控中的作用及与复发性流产的关系

目的探讨蜕膜巨噬细胞解偶联蛋白2(UCP2)表达、细胞内活性氧(ROS)、吞噬活性之间的关系及其在原因不明复发性流产发病中的作用。方法采用免疫磁珠法分离11例原因不明复发性流产患者和12例正常早孕妇女蜕膜巨噬细胞,流式细胞技术检测两组蜕膜巨噬细胞UCP2的表达、细胞内ROS水平;同时检测巨噬细胞吞噬活性。结果蜕膜巨噬细胞UCP2表达与细胞内ROS水平负相关(r=-0.572,P0.01),与巨噬细胞吞噬活性负相关(r=-0.462,P0.05)。与正常早孕妇女比较,原因不明复发性流产患者蜕膜巨噬细胞UCP2表达明显下降,细胞内ROS水平升高,其吞噬活性增强。结论UCP2通过控制巨噬细胞内ROS水平,调节细胞活性,在维持正常妊娠免疫耐受和原因不明复发性流产发病中起重要作用。     

抑制解偶联蛋白-2基因表达减轻急性脂肪肝细胞损伤

目的 探讨下调解偶联蛋白-2(UCP-2)基因的表达对脂肪肝细胞损伤的影响,为存在脂肪变性的供肝提供治疗靶点.方法 采用二步胶原酶灌注法分离C57BL/6 J-ob/ob转基因肥胖小鼠的原代脂肪肝细胞,用靶向小鼠UCP-2基因的RNA干扰慢病毒载体感染所获得的脂肪肝细胞,实现对UCP-2基}q的特异性敲减(实验组),另以空载体感染的脂肪肝细胞为对照.荧光显微镜镜检确定感染效率,实时聚合酶链反应鉴定敲减效果.以肿瘤坏死因子-α(TNF-α)作用于感染后的肝细胞,24 h后以碘化丙啶染色,流式细胞仪检测细胞凋亡情况;Western印迹法检测凋亡蛋白酶-3(Caspase-3)的活化.结果 UCP-2基因表达被有效抑制.UCP-2基因的敲减率约为75%.实验组的肝细胞凋亡率为(4.97±0.25)%.明显低于对照组的(21.13±1.28)%(P<0.05),其Caspase-3的活化程度也低于对照组.结论抑制UCP-2基因表达能减轻脂肪肝细胞的损伤.     

不同年龄妇女卵巢解偶联蛋白2的表达

解偶联蛋白2(uncouplingproteins2,UCP2)是UCPs家族的一个成员,在人体各种组织广泛表达[1],其生理作用还不甚清楚。动物研究表明,在控制活性氧(reactiveoxygenspecies,ROS)产生、调节组织巨噬细胞功能中起重要作用[2]。而且UCP2(-/-)小鼠产仔数量和频率下降[3]。根据衰老的自由基学说,ROS是引起细胞功能下降的主要原因。生殖功能下降是最早出现的年龄相关改变。Rousset等[3]在小鼠卵泡颗粒细胞检测到UCP2基因的表达,推测UCP2可能通过调节卵巢组织ROS水平参与卵巢的年龄相关改变。本研究观察UCP2在人卵巢组织的表达及其与年龄的…     

大鼠烫伤后不同时间切痂骨骼肌解偶联蛋白2、3 mRNA表达水平的改变

目的观察烫伤大鼠伤后不同时间切痂其骨骼肌解偶联蛋白(UCP)2、UCP3 mRNA表达水平的异同. 方法选用120只雄性Wistar大鼠,其中8只作为正常对照组;余下112只造成30%TBSAⅢ度烫伤后分成4组A组不切痂,分别于伤后8、24、96、120、168 h处死;B组伤后8 h切痂,于伤后24、96、120、168 h处死;C组伤后24 h切痂,伤后96、120、168 h处死;D组伤后96 h切痂,伤后120、168 h处死.测定各组大鼠各时相点的血清瘦素、肿瘤坏死因子(TNF)α含量以及腓肠肌UCP2、UCP3 mRNA表达水平. 结果 (1)血清瘦素水平A组大鼠伤后24～168 h均低于正常对照组(P<0.01),B、C、D组伤后120 h和(或)168 h均高于A组(P<0.01).(2)血清TNF-α水平A组伤后各时相点均高于正常对照组(P<0.01),B组伤后各时相点均低于A组(P<0.05或0.01).C组伤后168 h低于A组(P<0.05).(3)腓肠肌UCP2 mRNA的表达量A组大鼠在烫伤后8 h即已明显升高(P<0.01),24 h到达高峰,以后逐渐下降.B、C组伤后168 h时分别为0.32±0.20、0.35±0.15,明显低于同时相点A组 0.71±0.12(P<0.05).各组腓肠肌UCP3 mRNA表达的变化趋势与UCP2类似. 结论大鼠严重烫伤后UCP2、UCP3 mRNA表达上调可能是代谢率升高的重要因素之一,休克期切痂可降低这一表达,降低代谢率.     

京尼平抑制解偶联蛋白2对激素非依赖性前列腺癌细胞能量代谢的影响

目的:探讨京尼平对线粒体解偶联蛋白2(UCP-2)的抑制作用是否参与到激素非依赖性前列腺癌细胞(PC-3)的能量代谢中。方法:培养PC-3细胞并分别使用40、80、160μmol/L京尼平干预48 h,MTT法检测各组细胞的增殖情况;RT-PCR检测细胞内UCP-2 mRNA的表达水平;可见分光光度法测定细胞内丙酮酸含量和线粒体琥珀酸脱氢酶活性。结果:随着京尼平浓度的升高,PC-3细胞的增殖活性、细胞内UCP-2 mRNA的表达水平、细胞内丙酮酸含量和线粒体琥珀酸脱氢酶活性均逐渐下降,即随着京尼平对UCP-2抑制的增强,细胞增殖活性逐渐降低,细胞内丙酮酸含量和线粒体琥珀酸脱氢酶活性亦随之降低。结论:京尼平可能通过降低细胞内丙酮酸含量和线粒体琥珀酸脱氢酶活性的方式参与激素非依赖性前列腺癌细胞的能量代谢中,参与方式可能与抑制UCP-2有关。     

解偶联蛋白2调节胰岛β细胞胰岛素分泌的研究进展

糖稳态(glucose homeostasis)对机体的代谢平衡至关重要，主要通过胰腺β细胞对升高的血糖浓度增加相应的胰岛素分泌来实现。当进餐后血糖水平升高时胰岛素分泌随之升高，而空腹时胰岛素分泌受抑。胰岛素基因的转录在维持分泌颗粒中充足的胰岛素储备方面有着重要作用，但对于血糖实时波动的调节，分泌颗粒中胰岛素的释放却依赖一个与糖代谢有关的复杂的信号转导系统。     

人类精子表面附睾蛋白激酶抑制剂Eppin和精囊凝固蛋白Semenogelin的相互作用研究

目的:探讨人类精子表面一种附睾蛋白激酶抑制剂Epp in和精囊凝固蛋白Sem enogelin(Sg)的相互关系。方法:①用抗Epp in的抗体与精子和精浆中Epp in蛋白进行免疫沉淀反应;②免疫荧光法在精浆和精子表面共同显色定位;③重组Epp in(rEpp in)和重组精囊凝固蛋白Sg(rSg)共同孵育,用抗Epp in抗体或抗Sg抗体进行免疫共沉淀;④W estern印迹检测rEpp in和rSg相互作用;⑤放射性125I-rSg与rEpp in结合达饱和后,用未标记的rSg竞争性结合分析;⑥放射性125I-rSg直接与印迹膜上的rEpp in行放射性自显影。结果:人精子表面蛋白Epp in与精囊蛋白Sg发生特异性结合,Sg分子结构中的半胱氨酸残基Cys-239是唯一的结合位点,Cys-239的还原及羧甲基化将阻碍125I-rEpp in与rSg的结合。结论:Epp in与Sg的结合是靠分子结构中二硫化键的参与。在人类射精后的精子表面,精囊蛋白Sg结合在精子表面的Epp in上。     

肾周脂肪解偶联蛋白1表达对肾透明细胞癌患者预后的影响

目的评估肾周脂肪解耦连蛋白1(UCP1)表达对肾透明细胞癌(ccRCC)患者预后的影响。方法选取2013年2月至10月及2015年3月至10月收治的行后腹腔镜根治性肾切除术的ccRCC患者共98例,通过术前CT图像评估肾周脂肪厚度及黏连度。术后RT-qPCR检测肿瘤周围肾周脂肪UCP1,依据肾周脂肪UCP1 mRNA值,将患者分成高UCP1表达组(42例)与低UCP1组表达组(56例)。比较两组患者的一般临床资料、肾周脂肪厚度及黏连度,进一步采用Kaplan-Meier曲线评估两组间无进展生存期(PFS)的差异。采用单变量和多变量Cox回归分析确定PFS的潜在独立预后因素。结果高UCP1表达组的肾周脂肪厚度、肾周脂肪黏连比例、Fuhrman分级中Ⅲ~Ⅳ级比例和T分期中>T2期比例高于低UCP1表达组[(13.84±2.41)vs(10.75±1.99),42.86%vs 16.07%,28.57%vs 8.93%,21.43%vs 5.36%;P值分别为0.000,0.003,0.011,0.037]。随访期间(中位时间62.0个月),15例患者(12例高UCP1表达组,3例低UCP1表达组)发生肿瘤进展。Kaplan-Meier曲线显示,高UCP1表达组较低UCP1表达组PFS更差(71.43%vs 94.64%,P=0.001)。Cox回归分析显示,高UCP1表达和高T分期与低PFS显著相关(β=1.334,RR=3.796,95%CI=1.009~14.280,P=0.048;β=2.886,RR=17.930,95%CI=5.538~58.047,P=0.000)。结论肾周脂肪UCP1表达增加可能为ccRCC患者肿瘤进展的独立危险因素。术后联合肾周脂肪棕色化评估可能有助于临床更好的判断ccRCC患者的预后。     

活性氧引起的人类精子DNA链断裂

为探讨活性氧对人类精子DNA的损伤作用,分别用0、0.062 5、0.125、0.25、0.5及1.0 mM的H2O2和0、0.1、1.0、2.0、5.0 mM的β-NADPH在体外条件下处理精子,用单细胞凝胶电泳试验检测外源性H2O2和内源性O2引起的精子细胞DNA链断裂.结果显示用1.0 mMH2O2或5.0 mM NADPH处理精子30 min至4 h,彗星细胞百分率显著增加,平均彗尾也显著增长,并有明显的作用时间-效应关系.精子细胞用0.062 5 mM～1.0 mMH2O2或0.1～5.0 mM NADPH处理1 h,彗星细胞百分率与对照组比较显著增加,平均彗尾显著增长,并有明显的剂量-效应关系.这些研究结果表明外源性H2O2和β-NADPH在体外条件下刺激精了细胞产生的内源性O2可引起精子DNA链断裂.     

弱精子症患者精子中CRISP2基因及蛋白的初步研究

目的:探讨弱精子症患者精子中富含半胱氨酸的分泌蛋白2(CRISP2)mRNA及蛋白的表达水平,探讨其与精子活力的关系及相关的分子机制。方法:收集成年男性弱精子症患者精液78例,活力正常精液70例作为对照组,50% Percoll离心分离纯化后,提取精子总RNA及总蛋白,采用RT-PCR,SYBR Green实时定量PCR和Western印迹技术检测CRISP2 mRNA及蛋白相对表达量。结果:CRISP2基因在弱精子症患者精子中的表达量明显低于其在正常对照组精子中的表达量,下降达4.3倍;Western印迹发现CRISP2蛋白在弱精子症组较正常对照组下降达1.71倍,两组之间的表达量有明显的统计学差异(P<0.05)。结论:CRISP2基因及蛋白在弱精子症患者精子中的表达下调可能与精子活力下降有密切联系,提示CRISP2基因及蛋白可能成为弱精子症发病机制研究的一个分子靶标。     

An in vitro promoting role for hydrogen peroxide in human sperm capacitation

A complex process of maturation called capacitation is an essential step for spermatozoa to fertilize oocytes. Recent studies have shown that reactive oxygen species (ROS) can enhance the capacitation of human spermatozoa and sperm-zona interaction. We have investigated whether hydrogen peroxide (H₂O₂) could trigger capacitation of human spermatozoa and the acrosome reaction. The addition of catalase, a specific H₂0₂ scavenger, at the beginning of the capacitation process decreased the levels of both hyperactivation and induced-acrosome reaction whereas catalase added 15 min before the induction of the acrosome reaction by the calcium ionophore had no effect. Supplementation of the medium with H₂O₂ resulted in increased levels of hyperactivation and the acrosome reaction, whereas H₂O₂ added 15 min before induction of the acrosome reaction did not have any stimulatory effect. These results suggest that H₂O₂ may be involved in the capacitation process of human spermatozoa but not in the acrosome reaction.     

精子凋亡率与精子密度、活率、活力及活性氧关系探讨

目的 探讨精子凋亡率与精子密度、活率、活力与精浆活性氧关系.方法 分析85例男性不育患者精液样本,采用计算机辅助精液分析进行精液参数检测,采用Annexin V/PI染色法检测精子凋亡率,采用TBA法测定精浆活性氧.结果 精子凋亡率与精子密度之间有显著负相关性(P＜0.01);与精子活率之间无显著相关性(P＞0.05):与精子活力之间有显著负相关性(P＜0.05);与精浆活性氧之间有显著正相关性(P＜0.05).结论 精子捌亡率与精子密度和活力有相关性,精子凋亡率增加可能是少、弱精子症患者不育的原因之一,精浆活性氧升高可能是精子凋亡率增加的原因之一.     

长时受精过程中活性氧及精子的DNA损伤对体外受精结局的影响

目的通过检测精液处理后不同时间的精子DNA损伤程度与活性氧的变化,分析对比长时受精与短时受精两种精卵孵育时间的实验室参数及临床结局,旨在探讨长时受精对体外受精-胚胎移植治疗中的不利影响.方法接受常规IVF受精的夫妇146例,共取卵146个周期,移植113个周期.在取卵当日采集精液,经处理后将精子浓度调整为10×106/ml,留取标本3份,各1.0ml.第一份标本定为加精后0h(即在CO2培养箱内培养至该患者卵子加精时检测),第二份标本定为加精后5h(即在CO2培养箱内培养至该患者卵子加精后5h后检测),第三份标本定为加精后20h(即在CO2培养箱内培养至该患者卵子加精后20h后检测),每份标本均用于检测H2O2、CTA和精子DNA损伤.精子DNA损伤采用精子吖啶橙染色方法测定,H2O2及CTA含量测定采用分光光度比色测定法.在胚胎移植日,除外取消移植的患者33名,其余的113名患者随机分成两组,A组57人选择短时受精的胚胎进行移植,B组56人选择长时受精的胚胎进行移植.结果精子DNA损伤、H2O2水平在加精后20h均明显高于授精时及授精后5h(P<0.05),CTA水平明显低于授精时及授精后5h(P<0.05).精子DNA损伤率在授精后三个时间均与H2O2浓度呈正相关(r值分别为0.688、0.532和0.491, P<0.05);精子DNA损伤率与授精后20h的CTA浓度呈负相关(r=-0.347, P<0.05).B组的多精受精率明显高于A组(P<0.05),A组的优胚率明显高于B组(P<0.05),两组间的受精率、卵裂率、植入率、临床妊娠率无统计学差异.结论长时受精时,精子可以产生过量的活性氧并且导致DNA损伤的精子增多,对卵子及胚胎有不利影响.短时受精在不影响受精率及卵裂率的前提下,可以降低多精受精率,提高优胚率.     

Seminal oxidative stress in patients with chronic prostatitis

Prostatitis has been associated with abnormal semen parameters and may be the cause of infertility in some patients. Sperm antibodies and impaired sperm motility have been observed in the semen of patients with prostatitis. More recently, seminal oxidative stress has been detected in patients with prostatitis or accessory gland infection. Oxidative stress is the result of elevated Reactive Oxygen Species (ROS) or depressed Total Anti-oxidant Capacity, or both. Granulocytes, in prostatic fluid may be responsible for the generation of ROS, which are known to impair sperm function. Abnormal levels of ROS, however, have also been detected in the absence of leucocytospermia. Several investigators have observed significant oxidative stress in the semen or prostatic fluid from patients with bacterial and abacterial forms of prostatitis, comparable with levels reported in men with recognized infertility. Whether prostatitis is a risk factor for infertility or not, remains controversial. Therefore, the role of seminal oxidative stress as either the result of prostatic inflammation or the cause of infertility is likewise debatable, but plausible. We summarize the results of our studies, as well as those of others who are dedicated to the research of prostatitis, and its potential effects on semen quality.     

In vitro effects of aqueous extract of fermented rooibos (Aspalathus linearis) on human sperm function

Aspalathus linearis (rooibos) is a herbal medicinal plant originally from South Africa's fynbos and well known for its medicinal effects in treating different medical conditions. Rooibos contains significant levels of antioxidants capable of inhibiting the production of reactive oxygen species, which may improve seminal parameters. This study focussed on investigating the direct effect of fermented rooibos on human sperm functions in vitro. Semen samples collected by masturbation from unproven fertile donors (n = 25) and infertile patients (n = 25) after 3–5 days’ abstinence were liquefied and centrifuged (300 × g; 10 min) in human tubular fluid medium containing 1% bovine serum albumin. Afterwards, semen samples (7.5 × 10⁶/ml) were incubated at 37°C for one hour with aqueous extract of fermented extract in sperm preparation medium (0, 0.10, 1.0, 10 and 100 μg/ml) and assessed. Our data showed that fermented rooibos did not affect functional sperm parameters (motility, vitality, intracellular reactive oxygen species and acrosome reaction, p > .05), in vitro except in the reduced percentage of intact mitochondrial membrane potential and DNA fragmentation (p < .05). The decrease in DNA fragmentation generates the possibility of using the extract in patients prior to assisted reproductive techniques.     

Effect of in vitro selenium supplementation on sperm quality in asthenoteratozoospermic men

Sperm DNA damage, excessive oxidative stress and decrease in motility ?may lead to low fertilisation or poor? assisted reproductive techniques outcomes in asthenoteratozoospermic ?men. Selenium was considered as essential element for male reproductive functions. Selenium has important role in enzymatic process for elimination of excessive reactive oxygen species and helps to maintain membrane integrity. The aim of this study was to determine the effect of selenium supplementation on sperm quality, DNA fragmentation, mitochondrial membrane potential and membrane lipid peroxidation during sperm sampling in vitro at different times. In this experimental study, semen samples were collected from 50 asthenoteratozoospermic men. Samples were divided into two groups as control group and test group (incubated with 2 μg/ml selenium at 37°C for 2, 4 and 6 hr). Motility and viability were assessed based on WHO 2010 criteria. Mitochondrial membrane potential, sperm DNA fragmentation and malondialdehyde levels were evaluated in each group. Results revealed that motility, viability and mitochondrial membrane potential were significantly higher in the test group (p < .05). Also malondialdehyde levels were significantly lower in the test group (p < .03). DNA fragmentation significantly decreased in the test group after 6 hr of incubation (p < .02). In conclusion, in vitro selenium supplementation may protect spermatozoa from maltreatment effect of reactive oxygen species (ROS) during sperm sampling via keeping enzymatic and antioxidant process in optimum condition.     

Relationship between acrosin activity of human spermatozoa and oxidative stress

Aim: To study the association between seminal oxidative stress and human sperm acrosin activity. Methods: It is a prospective study consisting of 30 infertile men and 12 fertile normozoospermic volunteers. A full history, clinical examination and scrotal ultrasound were done to exclude other related factors such as smoking and varicocele. Presence of white blood cells (WBCs) in semen samples was evaluated by peroxidase staining. Lipid peroxidation in spermatozoa was induced after incubating with ferrous sulphate (4 mmol/L) and sodium ascorbate (20 mmol/L). Induced peroxidation of spermatozoa was assessed by determining the production of thiobarbituric acid reactive substances (TBARS). Acrosin activity was measured using the gelatinolysis technique. The halo diameters around the sperm heads and the percentages of spermatozoa showing halo formation were evaluated. An acrosin activity index was calculated by multiplying the halo diameter by the halo formation rate. Results: A significant difference was obse     

Lipid peroxidation in human spermatozoa and maintenance of progressive sperm motility

Washed and deep frozen spermatozoa of 46 patients from an infertility clinic were separated into 3 different groups depending on their progressive motility (expressed as the sperm motile efficiency index according to Ishii et al ., 1977), determined 0 and 3  h after liquefaction, and were examined for their lipid peroxidation (LPO) potential by means of the thiobarbituric acid assay. Spontaneous and iron-catalysed generation (after 15, 30 and 60  min incubation) of thiobarbituric acid-reactive substances (TBARS) was measured spectrophotometrically. Spontaneous LPO revealed the highest generation of TBARS in the group of spermatozoa with initially normal progressive motility and decreased maintenance of progressive motility after 3  h of aerobic incubation. Iron-catalysed LPO generally revealed the highest amounts of TBARS after 60  min, especially in the aforementioned group with decreased motility maintenance. The differences between this group and the two other groups were highly significant. Consequently, spermatozoa with initially normal progressive motility but decreased maintenance of motility, generated higher amounts of stable LPO products than others, which suggests that loss of motility under aerobic incubation seems to be the consequence of enhanced LPO processes.     

Redox regulation of mammalian sperm capacitation

Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility.