晚期糖基化终末产物对人血管内皮细胞增殖及凋亡的影响

目的研究晚期糖基化终末产物(AGEs)对人血管内皮细胞增殖及凋亡的影响,探讨糖尿病难愈创面新生血管化障碍或延迟的机理.方法不同作用时间及浓度AGE修饰的人血清白蛋白(AGE-HSA)与人血管内皮细胞(ECV304)在体外共同培养,用四唑盐(MTT)比色试验和细胞计数检测AGE-HSA对血管内皮细胞的增殖抑制作用,并用台盼蓝排斥试验检测细胞活力.采用FITC Annexin-V及碘化丙碇(PI)染色,用流式细胞仪检测凋亡细胞百分率,同时应用荧光共聚焦显微镜观察凋亡或死亡细胞FITC Annexin-V和PI的荧光染色,并用透射电镜和光镜观察凋亡细胞特异性的形态学改变.结果内皮细胞在12.5、25和50μg/ml AGE-HSA培养条件下,第2天细胞增殖数量与对照组比较无明显差异性,第4天和第6天则显著低于对照组(P＜0.01);而内皮细胞经100或200μg/ml AGE-HSA干预6h,MTT法测其OD值与对照组比较有明显差异(P＜0.01),同时内皮细胞出现特异性的凋亡形态学变化以及FITC Annexin-V阳性和/或PI阳性的细胞逐渐增多,且凋亡或死亡细胞数量随AGE-HSA作用时间及浓度的增加而增多.结论AGE-HSA能抑制内皮细胞增殖,并可以诱导其凋亡,而且与作用时间及浓度相关.提示AGEs可能是引起糖尿病难愈创面中新生血管化障碍或者延迟的机制之一.     

五氯酚对大鼠睾丸支持细胞影响的研究

目的:检测五氯酚(PCP)对体外培养的大鼠睾丸支持细胞的毒性作用。方法:35日龄雄性SD大鼠4只,采用MTT法检测浓度为10-2、10-1、1、10μmol/LPCP对大鼠睾丸支持细胞活力的影响以及AnnexinV/PI流式细胞术检测PCP对大鼠支持细胞的毒性作用。结果:支持细胞的活力随PCP浓度的提高而明显降低并呈时间依赖效应;随浓度增高,细胞由胞质丰富、胞核饱满变为胞质稀疏、胞核皱缩、细胞数目减少,进而细胞界限不明显乃至消失;流式细胞仪检测结果表明,PCP导致坏死的支持细胞比例增加。结论:PCP可引起大鼠睾丸支持细胞坏死,细胞毒性作用明显。     

甲基乙二醛对人血管内皮细胞的毒性作用

目的 探讨甲基乙二醛（methylglyoxal,MGO)对人血管内皮细胞的毒性作用及其机制。方法 体外培养的人脐静脉血管内皮细胞与不同浓度MGO共同孵育后，通过形态学观察和原位末端标记（TUNEL）法检测细胞凋亡，并以荧光Annexin-V及碘化丙碇（PI）染色，经流式细胞仪定量检测凋亡细胞和死亡细胞，细胞内氧化水平以氧化敏感的荧光染料2，7-二氢二氯荧光素（DCFH）染色。用流式细胞仪测定。结果 MGO刺激后，内皮细胞形态和超微结构出现凋亡的特异性变化，并且TUNEL染色阳性，流式细胞仪测定显示凋亡细胞和死亡细胞数量与MGO呈时间和剂量依赖关系；同时，MGO刺激后细胞内氧化水平明显升高，其时效与量效关系与细胞凋亡和死亡的变化相似，抗氧化剂（N-乙酰半胱氨酸和丙丁酚）及羰基化合物清除剂（氨基胍）对MGO引起的细胞氧化应激和细胞毒性具有抑制作用。结论 MGO对人血管内皮细胞具有直接的毒性作用，其机制可能是诱导细胞氧化应激，这些结果提示，活性羰基化合物蓄积可能与慢性肾功能衰竭和糖尿病血管并发症的形成有关。     

AnnexinⅤ法检测HepG2细胞凋亡

目的 应用Annexin V-PITC/PI技术检测化疗药物顺铂(CDDP)诱导肝癌细胞(HepG2)凋亡作用的特性.方法 将不同浓度的CDDP与肝癌细胞株HepG2共孵育4 h、15 h、24 h、36 h后,应用流式细胞术和Annexin V-PITC/PI双染免疫荧光法观察细胞凋亡情况.结果 CDDP可以诱导HepG2细胞发生凋亡,随着药物浓度的增加和作用时间的延长,发生凋亡和坏死细胞的比例增加.结论 化疗药物CDDP诱导肝肿瘤细胞凋亡呈时间和剂量依赖性.AnnexinⅤ-FITC /PI双标记流式细胞术可用于早期细胞凋亡的检测.     

龙葵碱诱导胰腺癌细胞Panc-1凋亡的实验研究

目的 探讨龙葵碱对胰腺癌细胞Panc-1的凋亡诱导作用.方法 采用经典方法培养胰腺癌细胞系Panc-1成功后,取对数生长期的细胞用于实验研究,用不同浓度(分别为20、30、40和50 μg/mL)的龙葵碱进行干预,采用倒置显微镜观察龙葵碱作用后Panc-1细胞的形态学改变;CCK-8比色法检测龙葵碱对Panc-1细胞增殖的抑制作用;用流式细胞术Annexin V-FITC/PI双染法检测细胞凋亡率.结果 龙葵碱显著抑制人胰腺癌细胞Panc-1生长,并具有浓度和时间依赖性.倒置显微镜观察发现,随着龙葵碱浓度增加和作用时间延长,Panc-1细胞表现出典型的凋亡形态改变;CCK-8比色法发现,随着龙葵碱浓度增加和作用时间延长,对Panc-1细胞的增殖抑制作用逐渐增强;流式细胞术检测发现,Panc-1细胞的凋亡率随着龙葵碱浓度增加而逐渐增加.结论 ①龙葵碱能够抑制胰腺癌细胞Panc-1的增殖,且此抑制作用具有时间和浓度依赖性.②龙葵碱具有诱导胰腺癌细胞Panc-1凋亡的作用,并具有浓度依赖性.     

直链烷基苯磺酸钠对雄性小鼠生精功能亚慢性毒性作用研究

目的 探讨直链烷基苯磺酸钠(LAS)对雄性小鼠生精功能的亚慢性毒性作用及毒性遗留作用. 方法 NIH种雄性小鼠给予LAS(630 mg/kg,315 mg/kg)灌胃,每日1次,连续经口染毒2个月,同时设对照组.在第4、8周及停止染毒后4周进行精子形态学观察,光镜观察睾丸组织形态学变化,透射电镜观察睾丸超微结构变化. 结果 实验组与对照组相比,染毒4周后精子畸形率上升,畸形主要表现在颈部,精子中部胞浆小滴增多.染毒8周精子畸形率升高更显著.组织学检查实验组小鼠染毒4周时精细小管内各级精母细胞和精子细胞数减少,生精细胞排列紊乱,随实验时间延长及染毒浓度的增加此异常改变更显著;超微结构观察显示睾丸生精上皮内支持细胞、间质细胞、精原细胞线粒体广泛异常改变,呈时间及浓度依赖性.上述异常改变在停止染毒后4周无明显恢复. 结论 LAS对雄性小鼠生精功能亚慢性毒性作用及毒性遗留作用表现为:使生精上皮内支持细胞、间质细胞及精原细胞出现广泛线粒体空泡样变,精子胞浆小滴出现率升高.     

MG-132对肝癌细胞BEL7402凋亡及超微结构影响的实验研究

目的:探讨泛素-蛋白酶体通路阻断剂MG-132对肝癌细胞BEL7402细胞凋亡和超微结构的影响。方法:以5和10μmol／L MG-132分别处理人肝癌BEL7402细胞24和48h,用流式细胞术检测细胞凋亡;DNA片段分析进一步证实凋亡存在,Western印迹检测Bax蛋白表达,比色法测Caspase-3活性变化,用透射电镜观察细胞超微结构的变化。结果:随着MG-132浓度的增加和处理时间延长,细胞凋亡率增加;细胞DNA抽提电泳发现特征性凋亡梯状条带;Bax蛋白表达增加;Caspase-3活化;电镜观察到典型凋亡细胞,线粒体等细胞器的形态变化与MG-132浓度和作用时间有关。结论:蛋白酶体抑制剂MG-132可诱导肝癌细胞BEL7402凋亡;线粒体损伤、Bax蛋白上调、Caspase-3激活可能在MG-132 诱导BEL7402细胞凋亡起重要作用。     

Ca2+和CaN凋亡信号通路参与皮质酮诱导的Leydig细胞凋亡中FasL的表达

目的观察在皮质酮诱导的大鼠Leydig细胞凋亡中,Ca2 和钙调神经磷酸酶(CaN)依赖的信号通路是否参与FasL表达的调控。方法利用钙定性探针Fluo-3/AM检测皮质酮作用下的Leydig细胞中Ca2 浓度变化。通过酶底物法测定CaN活性。以Westernblot检测FasL表达。用Annexin-Ⅴ-FITC和PI双标评价Leydig细胞凋亡率。结果经超生理剂量皮质酮处理的Leydig细胞中出现Ca2 浓度升高,CaN活性增加及FasL表达增加。环孢菌素A可抑制CaN活性,使FasL表达下调,细胞凋亡率下降。结论Ca2 和CaN依赖的信号通路参与了皮质酮诱导的大鼠Leydig细胞凋亡;CaN介导了由Ca2 引发的FasL表达,Ca2 和CaN在大鼠Leydig细胞凋亡过程中起重要作用。     

丹参酚酸B对过氧化氢引起的黑素细胞凋亡的保护作用研究

目的:研究丹参酚酸B对过氧化氢(H_2O_2)诱导的人表皮黑素细胞氧化应激及凋亡的保护作用。方法:采用MTT法检测H_2O_2对黑素细胞的活力抑制作用及丹参酚酸B的保护作用;Annexin-V/PI双染流式细胞术检测H_2O_2诱导黑素细胞凋亡的作用及丹参酚酸B的保护作用;DCFH-DA法流式细胞术检测H_2O_2促进黑素细胞活性氧簇(ROS)生成的作用及丹参酚酸B的抗氧化效果。结果:250μmol/LH_2O_2作用于黑素细胞24h可明显降低细胞活力、增加凋亡。0.6～10μmol/L丹参酚酸B预处理1h可明显保护黑素细胞,减少H_2O_2诱导的黑素细胞活力降低及凋亡。H_2O_2作用2h可显著诱导ROS生成,而丹参酚酸B可显著抑制H_2O_2诱导的ROS生成。结论:丹参酚酸B可能抑制活性氧生成,减少H_2O_2引起的黑素细胞凋亡,因而可能具有成为治疗白癜风新药的前景。     

VP-16诱导PC-3细胞凋亡和p53基因表达的研究

目的 研究ＶＰ－１６诱导雄激素非依赖型前列腺癌细胞ＰＣ－３凋亡和抑癌基因ｐ５３表达的关系。方法 以末端标记法（ＴＵＮＥＬ）和流式细胞术（ＦＣＭ）研究ＰＣ－３细胞凋亡及细胞周期分布;以免疫组化方法研究ｐ５３蛋白的表达。结果 末端标记法显示凋亡的ＰＣ－３细胞呈明显的形态学改变,流式细胞术和免疫组化检测结果表明,随ＶＰ－１６作用浓度的增加利用和作用时间的延长,ＰＣ－３细胞的凋亡率和其ｐ５３基因表达阳性的     

小鼠胚胎睾丸Leydig细胞培养、纯化及其功能研究

目的:探讨小鼠胚胎睾丸Leyd ig细胞体外培养、鉴定、纯化的方法,并进行形态学观察、分泌睾酮能力等生物学特性检测。方法:选择胎龄16 d的胎鼠睾丸,0.03%胶原酶Ⅰ消化,3β-羟基类固醇脱氢酶(HSD)染色鉴定及纯度测定,锥虫蓝染色检测细胞活率。放免法测定Leyd ig细胞不同培养时间及密度下分泌睾酮的水平。结果:Leyd ig细胞培养前和培养72 h后纯度分别为(45.10±1.66)%和(81.17±2.32)%;培养液中可检测到睾酮,睾酮水平与Leyd ig细胞数、培养时间相关,单个Leyd ig细胞睾酮分泌能力逐日下降。结论:该法分离的胚胎Leyd ig细胞纯度较高,生长性好,保持增殖和分泌睾酮的生物学特性,可应用于相关的研究。     

邻苯二甲酸二乙基己酯对新生小鼠睾丸及Leydig细胞影响的实验研究

目的:探讨邻苯二甲酸二乙基己酯(DEHP)对新生小鼠睾丸及Leyd ig细胞形态结构及功能的影响。方法:DEHP分别以低、中、高3组剂量[100、200、500 mg/(kg.d)]灌胃作用于怀孕12 d到产后3 d(GD12~PND3)的KM母鼠,观察DEHP对新生雄性仔鼠体重、睾丸重量、Leyd ig细胞形态结构和3β-羟基类固醇脱氢酶(3-βHSD)活性、酶反应面积的影响。结果:DEHP作用于母鼠后,其雄性子代幼鼠体重和睾丸重量减轻,睾丸Leyd ig细胞形态、超微结构发生改变;高剂量组Leyd ig细胞数量明显增多;低、中剂量组睾酮合成关键酶3β-HSD酶活性下降,酶反应面积减小,但高剂量组在仔鼠出生后15 d时酶活性降低[(吸光度值(0.154±0.011)vs空白对照组(0.222±0.013),P<0.01],而酶反应面积增大[(6 303.0±745.6)μm2vs空白对照组(5 091.4±214.4)μm2,P<0.01)]。结论:DEHP能影响新生雄性小鼠体重、睾丸重量、Leyd ig细胞的形态结构和3β-HSD活性,具有抗雄激素效应。     

Taurine ameliorates cytotoxic effects of Di(2-ethylhexyl) phthalate on Leydig cells

It has been revealed that di(2-ethylhexyl)phthalate (DEHP) has toxic impacts on the male reproductive system. Taurine (TAU) is an amino acid with antioxidant property and beneficial impacts on the male reproductive system. In this study, protective impacts of Taurine (TAU) on DEHP-induced Leydig TM3 cell toxicity were investigated. The cells exposed to DEHP (0.8 µmol) or TAU (100 mg/ml) for 24 hr. Cell viability (MTT assay), apoptosis, oxidative stress and testosterone level were examined. DEHP could significantly decrease the cell viability percentage, reduce testosterone level, increase apoptosis, elevate Bax/ Bcl-2 ratio and enhance caspase-3 and -9 activity in the TM3 cells. Additionally, DEHP significantly elevated malondialdehyde contents and reactive oxygen species levels. It also augmented superoxide dismutase and catalase activity in the Leydig cells. Co-treatment of DEHP with TAU increased viability and testosterone level, while oxidative stress and apoptosis significantly reduced. TAU could decrease Bax/Bcl-2 ratio and caspase-3 and -9 activity in the DEHP-intoxicated cells. Our results have clearly shown that TAU protects TM3 cells against oxidative stress and apoptosis induced by DEHP.     

Effects of 60 Hz electromagnetic field exposure on testicular germ cell apoptosis in mice

Aim: To evaluate the effects of 60 Hz extremely low frequency (ELF) elelctromagnetic field (EMF) exposure on germ cell apoptosis in the testis of mice. Methods: Adult male BALB/c mice (7 weeks of age) were exposed to a 60 Hz EMF of 0.1 mT or 0.5 mT for 24 h/day. A sham-exposed group served as the control. After 8 weeks of exposure, the mice were sacrificed. Germ cell apoptosis in the testis was assessed by histopathological examination, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and flow cytometric examination of isolated spermatogenic cells stained with 7 aminoactinomycin D (7-AAD). Results: EMF exposure did not significantly affect the body and testis weights, but significantly increased the incidence of germ cell death. The distinguishing morphological feature of EMF exposure was a decrement in the number of well organized seminiferous tubules. Quantitative analysis of TUNEL-positive germ cells showed a significantly higher apoptotic rate in the 0.5     

Tumor necrosis factor and interleukin-1 stimulate testosterone secretion in adult male rat Leydig cells in vitro

The actions of two cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), on testosterone production by dispersed adult testis cells and purified Leydig cells in culture were studied. In one set of experiments, testis cells from adult (90-day-old) rats were enzymatically dispersed. In another set of experiments, the dispersed testis cells were placed on a Percoll density gradient and were centrifuged to yield purified (greater than 85%) Leydig cells. Both whole testis cells and purified Leydig cells were cultured in the presence of varying doses of TNF or IL-1 with or without maximally stimulating doses of human chorionic gonadotropin (hCG). Both TNF and IL-1 stimulated basal secretion of testosterone in whole testis cells, as well as purified Leydig cells. Additionally, both TNF and IL-1 augmented maximally hCG stimulated testosterone secretion. Both cytokines stimulated testosterone secretion by dispersed testis cells as early as 4 hours, and the effect continued for up to 72 hours. The cytokines slightly, but significantly, stimulated testosterone production in purified Leydig cells after 24 hours, and continued for up to 72 hours. We have concluded from this data that TNF and IL-1 stimulate the testosterone secretion by adult rat Leydig cells. While this effect might be mediated through the action of the cytokines on testicular macrophages, there might also be a direct effect on the Leydig cell since augmentation of secretion occurred in purified Leydig cells, as well as whole testis cells. Therefore, TNF and IL-1 may serve as local regulators of Leydig cell function.     

邻苯二甲酸二-(2-乙基)己酯致小鼠隐睾睾丸和附睾的组织病理学改变

目的 :探讨邻苯二甲酸二 (2 乙基 )己酯 (DEHP)引起的小鼠隐睾睾丸和附睾的组织病理学改变。　方法 :妊娠KM小鼠 4 0只 ,随机分成 5组 ,分别为正常对照组 8只、玉米油对照组 8只、己烯雌酚 (DES)组 8只、DEHP低剂量组 [DEHP 10 0mg/ (kg·d) ]9只和DEHP高剂量组 [DEHP 5 0 0mg/ (kg·d) ]7只。自妊娠第 12d开始到分娩后 3d ,分别持续经口给予DEHP 10 0mg/ (kg·d)、5 0 0mg/ (kg·d)和DES 10 0 μg/ (kg·d)及玉米油 ,观察仔代雄小鼠的隐睾发生率及隐睾睾丸和附睾的组织病理学改变。　结果 :DEHP 5 0 0mg/ (kg·d)组染毒小鼠的隐睾发生率显著增高 ,睾丸和附睾的体积明显减小、重量减轻 ;睾丸生精上皮发育明显异常 ,精曲小管变薄、萎缩 ,间质细胞异常增生 ,电镜下其隐睾精曲小管上皮和间质细胞均出现明显的超微结构改变。同时附睾管腔中的精子数显著减少甚至缺乏。　结论 :高剂量 [5 0 0mg/ (kg·d) ]DEHP可能具有与DES类似的作用 ,是一种诱发隐睾的重要因子。小鼠在孕期及哺乳期接触DEHP后可引起雄性仔鼠性分化异常 ,诱导隐睾发生、睾丸生精上皮损害和生精过程障碍 ,从而对雄性仔鼠生育力产生不利影响。以上作用存在明确的量 效关系。     

Experimental cryptorchidism in the adult mouse. III. Qualitative and quantitative electron microscopic morphology of Leydig cells.

The morphology of Leydig cells of control and 28-day-old cryptorchid mice was studied by electron microscopy and stereologic techniques. Leydig cell profiles of control mice were larger in section when compared to cryptorchid mice, but no differences were observed in the distribution of organelles in Leydig cells in the two groups. Quantitatively, the absolute volumes of smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), mitochondria, lysosomes, multivesicular bodies, peroxisomes, cytoplasmic matrix, nucleus, lipid droplets, membrane whorls, ribosomal aggregates, and annulate lamellae per Leydig cell were reduced significantly after 28 days of cryptorchidism. However, the absolute volumes of these organelles per testis were not significantly different between control and cryptorchid mice, due to the increase in Leydig cell number per testis in the cryptorchid testis, compared to the controls, except that the absolute volume of Golgi per Leydig cell was not significantly different between control and cryptorchid rats, but the absolute volume of Leydig cell Golgi was significantly lower in control rats. Based on these results, we conclude that, morphologically, a 28-day cryptorchid mouse Leydig cell clearly approximates a "half unit" of a control Leydig cell.     

Changes in the expression of claudins and transepithelial electrical resistance of mouse Sertoli cells by Leydig cell coculture

In the testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of blood-testis barrier (BTB). To verify the role of paracrine interactions between the Sertoli and Leydig cells in the structure and function of BTB in testis, the expression of claudin-1 and -11, and transepithelial electrical resistance (TER) of the mouse Sertoli cells were examined under the Leydig cell coculture. TER of Sertoli cell monolayer was significantly larger under the Leydig cell coculture in comparison with the control culture. Meanwhile, the expression of claudin-1 slightly decreased and claudin-11 significantly increased in the Sertoli cells in the Leydig cell coculture compared with control. Testosterone significantly increased claudin-11 expression in cultured Sertoli cells. Taken together, it suggested that Leydig cell coculture changed the structure and functions of inter-Sertoli TJs in vitro. Interactions between Leydig and Sertoli cells might be involved in the development of functional blood testis barrier in mouse testis.     

The expression and function of androgen receptor coactivator p44 and protein arginine methyltransferase 5 in the developing testis and testicular tumors

PURPOSE: The role of androgen receptor coactivators in testicular development and cancer formation is unclear. p44/Mep50 was identified as an androgen receptor coactivator that functions in a complex with protein arginine methyltransferase 5. We studied the expression of p44 and protein arginine methyltransferase 5 in developing fetal testis and adult testicular tumors, including seminomas and Leydig cell tumors. MATERIALS AND METHODS: A total of 30 human fetal testes from abortuses at a gestational age of 10 to 40 weeks, 33 human seminomas and 11 human Leydig cell tumors were retrieved from the archives of the departments of pathology. Immunohistochemistry was performed with affinity purified p44 and IgG purified protein arginine methyltransferase 5 polyclonal antibodies. RESULTS: Protein arginine methyltransferase 5 and p44 were expressed predominantly as nuclear proteins in fetal Leydig cells and human adult nonneoplastic testes, including germ cells and Leydig cells, while they were expressed in the cytoplasm of germ cells of the fetal testis. Expression was strongest in the fetal testis during the second trimester. Compared to adult nonneoplastic testes, human seminoma and Leydig tumor cells showed a marked decrease in nuclear expression of p44 and protein arginine methyltransferase 5 with a concomitant marked increase in cytoplasmic expression of these proteins. Furthermore, average testicular size was increased by 29% in p44(+/-) heterzygotic mice. CONCLUSIONS: These results suggest distinct functions of the nuclear and the p44/protein arginine methyltransferase 5 complexes in the developing fetal testis and in the oncogenesis of testicular tumors. Further studies are needed to confirm the functional relevance of these findings.