Expression of Hsp70-2 in rhesus monkey testis during germ cell apoptosis induced by testosterone undecanoate

Hsp70-2 functions as a molecular chaperone that assists other proteins in their folding, transport and assembly into complexes, and is postulated to be linked to the mechanisms that inhibit apoptosis. Here we have determined the association between Hsp70-2 gene and germ cell apoptosis induced by a high dose of testosterone undecanoate (TU). In this study, in situ analysis of cell DNA fragmentation and expression of Hsp70-2 in TU-treated monkey testes were compared with the normal testes. The TUNEL analysis data showed that a large number of germ cell apoptosis occurred in the testes on Day 30 after TU injection. Therefore, we speculate that spermatogenesis failure in TU-treated monkey testis may be a result of the germ cell apoptosis induced by a high dose of TU. As compared with that of normal testes, however, the level of Hsp70-2 mRNA was only slightly decreased while that of Hsp70-2 protein was almost unchanged in the testes from Day 7 to day 30 at the early stage of the germ cell apoptosis after TU treatment, but the levels of both Hsp70-2 mRNA and protein dropped dramatically on Day 60 when a large number of germ cells had undergone apoptosis and were depleted. Therefore, it is suggested that the Hsp70-2 may be not a molecule to prevent germ cell apoptosis induced by injection of TU in the testes at the early stage.     

Bcl-2 and Bax are involved in experimental cryptorchidism-induced testicular germ cell apoptosis in rhesus monkey

Apoptosis occurs spontaneously during spermatogenesis. However, little is known about its regulation in primate. Using an experimental cryptorchidism model in rhesus monkey, we have investigated the relationship between apoptosis and the Bcl-2 family members, Bcl-2 and Bax. Apoptotic cells were identified by in situ end labeling of fragmented DNA. The expressions of Bcl-2 and Bax in the testes during the heat stress-induced testicular germ cell apoptosis were detected by immunohistochemistry and Western blot techniques. The results showed that the apoptotic signals increased after heat treatment and the most susceptible cell types were spermatocytes and spermatids. A redistribution of Bax from the cytoplasmic to nuclear localization in some germ cells was observed. However, its total expression levels in the cells remained unchanged in the cryptorchid testes as determined by Western blot analysis; on the other hand, Bcl-2 levels increased significantly in response to heat stress. The subcellular redistribution of Bax and the increase in Bcl-2 expression in the cryptorchid testis suggest an involvement of Bcl-2 family members in heat stress-induced germ-cell apoptosis.     

CISPLATIN-INDUCED GERM CELL APOPTOSIS IN MOUSE TESTES

The purpose of this study was to investigate whether exposure of male mice to cisplatin induces apoptosis in male germ cells and the possible role of apoptosis in cisplatin-induced testicular damage. Forty-eight male BALB/c mice were divided into cisplatin and control groups. The mice from the cisplatin group received a single intraperitoneal injection of cisplatin of either 1, 5, or 10 mg/kg. The control group received a single intraperitoneal injection of saline alone. The testes were removed on days 1, 3, and 7 after cisplatin administration, respectively. Following histological examination, apoptotic indices (AIs) were measured within seminiferous tubules of the mouse testes by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. A low incidence of spontaneous apoptosis was observed in controls, particularly in spermatogonia and spermatocytes of the mouse testes. After cisplatin administration, both increased AIs and decreased spermatozoa and spermatids were found in the seminiferous tubules of the mouse testes. Cisplatin-induced apoptosis was found in spermatogonia, spermatocytes, and spermatids of the mouse testes. In comparison to the control values, AIs increased 2.6- to 6.8-fold in cisplatin-treated mouse testes. AIs reached the highest level on day 1 following 1 mg/kg, on day 3 following 5 mg/kg, and on day 7 following treatment of 10 mg/kg cisplatin. The study showed that cisplatin-induced germ cell apoptosis in the mouse testes was related to both the dose response and the time course of response. It is suggested that cisplatin-induced germ cell apoptosis may result in decreased spermatogenesis, and the higher dose of cisplatin may delay the occurrence of apoptosis in the mouse testes.     

超生理剂量11-酸睾酮对人精液中脱落生殖细胞的影响

为研究大剂量睾酮对生精细胞的影响,对53例志愿接受11-酸睾酮的成年男性在给药前后及恢复期的精液进行分析,用瑞-姬氏染色进行脱落生殖细胞观察。结果为给药后30,60天脱落生殖细胞计数随精子计数减少而减少,初级精母细胞、次级精母细胞比例增多,精子细胞减少。细胞增多了凋亡脱落生殖细胞的各种形态变化,包括凋亡的初级、次级精母细胞和精子细胞。恢复期随精子计数的恢复,生精细胞的计数和形态也相应恢复。提示11-酸睾酮主要干扰了初级、次级精母细胞和精子细胞的正常分化发育。     

Expression of P16(INK4a) in testis of rhesus monkey during heat stress and testosterone undecanoate induced azoospermia or oligozoospermia

Previous studies on azoospermia or oligozoospermia induced by heat stress or high doses of testosterone mainly focused on germ cell apoptosis; no data regarding their possible effect on spermatogonia mitosis are available. We have established unilateral cryptorchid and testosterone undecanoate (TU)-treated monkey models and examined expression of P16(INK4a) in the testis to look at its possible role in azoospermia or oligozoospermia induced by the heat stress or the TU treatment. The results showed that both heat stress and TU were capable of inducing expression of P16(INK4a) mainly in spermatogonia and other types of germ cells as well as Sertoli cells at the later stage of germ cell apoptosis, namely on Day 10 after operation or on Day 60 after TU injection. It is, therefore, suggested for the first time that P16(INK4a) protein may inhibit the spermatogonia mitosis in the testis at the later stage of the germ cell apoptosis, resulting in arrest of spermatogenesis.     

重度妊娠高血压综合征患者血清诱导的脐血管内皮细胞凋亡失衡与肿瘤坏死因子的关系

目的:探讨重度妊娠高血压综合征(简称妊高征)患者血清对体外培养的脐血管内皮细胞(HUVEC)凋亡和对抑／促凋亡基因Bcl-2／Bax表达的影响以及与肿瘤坏死因子α(TNFα)的关系。方法:体外培养HIJVEC,加入重度妊高征患者血清和TNFα作为处理因素,免疫细胞化学检测Bcl-2／Bax蛋白表达;流式细胞仪做凋亡细胞记数;扫描电镜观察细胞变化。结果:轻重度妊高征患者血清处理后的HUVEC Bcl-2／Bax表达失衡,凋亡细胞数增加,扫描电镜可见经TNFα处理后的内皮细胞变圆,微绒毛消失,密度增高,可见凋亡小体。结论:TNFα可诱导体外培养的脐血管内皮细胞凋亡,并可引起凋亡相关基因Bcl-2／Bax表达失衡,可能是引起妊高征血管内皮细胞损伤的原因之一。     

丙烯腈对雄性小鼠的生殖毒性作用

目的探讨丙烯腈的雄性生殖毒作用机制。方法以雄性小鼠为研究对象,腹腔注射染毒,流式细胞仪检测分析不同观察终点、不同染毒剂量对小鼠睾丸组织各倍体细胞、各时相生精上皮细胞的影响。结果丙烯腈影响次级精母细胞、精子细胞和精子的形成,主要作用于生精上皮细胞的有丝分裂期;丙烯腈诱导雄性小鼠生精细胞凋亡,随着染毒剂量的增加和时间的延长,凋亡发生率增高,第21天的各染毒组细胞凋亡最明显(P<0.05)。结论丙烯腈具有明显的雄性生殖毒性,干扰精子形成。     

维甲酸致神经管畸形中牡蛎拮抗神经细胞凋亡的实验研究

目的 探讨维甲酸(retinoic acid,RA)致神经管畸形(neural tube defects,NTDs)中牡蛎拮抗神经细胞凋亡的分子机制。方法 建立NTDs模型并给予牡蛎拮抗,观察神经细胞的凋亡情况及凋亡相关蛋白Bcl-2、Bax与Caspase-3表达的变化。结果 1)RA组神经管上皮细胞凋亡指数明显高于对照组,牡蛎组凋亡指数明显低于RA组(P＜0.05)。2)RA组神经上皮细胞Bcl-2的表达明显低于对照组,牡蛎组Bcl-2的表达明显高于RA组(P＜0.05)。3)RA组Bax与Caspase-3的表达均明显高于对照组,牡蛎组Bax与Caspase-3的表达明显低于RA组(P＜0.05)。 结论 牡蛎可以拮抗RA引起的神经上皮细胞过度凋亡,其机理可能是通过上调Bcl-2的表达和下调Bax与Caspase-3的表达实现的。     

参附注射液对新生大鼠缺氧缺血性脑损伤的干预作用

目的:研究参附注射液对新生大鼠缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)的干预作用。方法:建立新生大鼠HIBD模型,应用HE染色、Annexin V/PI染色法、免疫组化染色法观察正常对照组、假手术组、生理盐水组及参附干预组在缺氧缺血后2 h、12 h、24 h、3天、7天、14天的病理变化、神经细胞凋亡及Bax、Bcl-2蛋白表达。结果:①缺氧缺血后生理盐水组海马CA1区病理变化明显,参附干预组上述变化减轻。②正常对照组及假手术组海马CA1区Bcl-2表达较弱,手术后该区Bcl-2表达仍弱,Bax表达增强,出现凋亡细胞,以24 h最明显;参附干预组Bcl-2表达较生理盐水组增强,而Bax表达减弱(P<0.05),凋亡细胞减少。结论:HIBD后Bax蛋白表达增强,细胞凋亡增加。参附可上调Bcl-2表达,下调Bax表达,使凋亡细胞减少,这可能是参附注射液治疗新生儿缺氧缺血性脑损伤的机制之一。     

丙烯腈对小鼠生精细胞Bcl-2、Bax蛋白表达的影响

目的探讨丙烯腈(AN)暴露对小鼠睾丸生精细胞Bcl-2、Bax蛋白表达水平的影响。方法将250只成年健康SPF级昆明种雄性小鼠,按体重随机分为阴性对照组、3个AN染毒组和阳性对照组,阴性对照组用生理盐水,各染毒组分别以1.25、2.50、5.00mg/kg AN腹腔注射(注射剂量为0.01ml/g BW),每天1次,连续5天,阳性对照组注射环磷酰胺40mg/kg一次。分别于首日染毒后的第7、14、21、28和35天采用颈椎脱臼法处死小鼠,采用免疫组织化学法(SABC)检测生精细胞Bcl-2和Bax蛋白的表达。结果 5个观察时间点AN 2.50mg/kg组和21天AN 1.25mg/kg组Bcl-2平均光密度值均显著低于阴性对照组(P<0.05);除21天AN 1.25mg/kg组外,所有观察时间点AN各染毒组Bax平均光密度值均显著高于阴性对照组(P<0.05)。Bcl-2在中剂量组和阳性对照组各观察时间点降低幅度均较大;Bax在各剂量组的不同时间点均以14天时升高幅度最大。结论 AN可诱导生精细胞Bcl-2蛋白表达减弱,以AN 2.50mg/kg组最为显著;同时可诱导生精细胞Bax蛋白表达增强,以14天时变化幅度较明显。     

迷迭香酸拮抗H_2O_2诱导的人脐静脉内皮细胞凋亡及作用机制

目的研究迷迭香酸(Rosmarinic acid,RA)对H2O2诱导的人脐静脉内皮细胞株ECV304凋亡的抑制作用及其机制。方法以H2O2诱导ECV304细胞凋亡为模型,用MTT比色法检测RA对细胞活性的影响;流式细胞仪检测法和Tunel法检测RA对细胞凋亡率的影响;用Western-blotting检测凋亡相关基因Bcl-2、Bax蛋白的表达情况和第24hcaspase-3活性。结果用0.5mmol/LH2O2孵育细胞24h细胞凋亡明显。不同浓度的RA(1、3、10μmol/L)预处理细胞后,呈浓度依赖性的增加内皮细胞活性,降低细胞凋亡率,并且Bcl-2蛋白表达呈浓度依赖性的上调,而Bax蛋白的表达和caspase-3蛋白活性呈浓度依赖性下降。结论迷迭香酸可以拮抗H2O2诱导的内皮细胞凋亡,其抑制内皮细胞凋亡与激活Bcl-2活性有关。     

氢醌对人白血病细胞凋亡及相关蛋白Bcl-2、Bax及Caspase-3表达的影响

目的 研究氢醌对白血病细胞株U937细胞凋亡及相关蛋白Bcl-2、Bax及Caspase-3表达的影响。方法 用不同浓度的氢醌处理U937细胞24 h、48 h后,采用MTT法来检测细胞增殖抑制率;经瑞-吉染色后,观察细胞形态变化;采用流式细胞仪检测细胞凋亡;采用免疫印迹法检测Bcl-2、Bax及Caspase-3蛋白表达。结果 (1)U937细胞的增殖抑制率随氢醌作用时间及浓度的增加而增加,呈时间-剂量依赖性(P<0.05);(2)氢醌作用后,部分细胞体积增大,胞浆中有空泡,胞核发生畸形;(3)流式细胞术结果显示,细胞凋亡率随着氢醌作用时间及浓度的增加而增加,呈时间-剂量依赖性(P<0.05);(4)免疫印迹结果显示,与空白组相比较,染毒48h时,细胞Bax蛋白的表达量增加(P<0.05),而Bcl-2蛋白表达量减少(P<0.05),Bcl-2/Bax比值下降(P<0.05),染毒24 h时,细胞Caspase-3蛋白的表达量增加(P<0.05);与染毒24 h各组相比较,染毒48 h时细胞Caspase-3蛋白的表达量随时间延长呈增加趋势(P<0.05)。结论 氢醌可以诱导U937细胞凋亡,其机制可能与调控凋亡相关蛋白Bcl-2、Bax及Caspase-3的表达有关。     

茶多酚对ox-LDL诱导人脐静脉内皮细胞凋亡的抑制作用

目的:探讨茶多酚(teapolyphenols,TP)对氧化型低密度脂蛋白(oxidizedlowdensitylipoprotein,ox-LDL)诱导人脐静脉内皮细胞(humanumbilicalveinendothelialcell,HUVEC)凋亡的抑制作用及其机制。方法:实验分为四组:TP(25μg/ml)组、ox-LDL(200μg/ml)+TP(25μg/ml)组、ox-LDL(200μg/ml)组、对照组(等体积溶剂)。采用四唑盐(MTT)比色法测定细胞活性、吖啶橙荧光染色观察细胞凋亡的形态学变化,Westernblotting分析Bcl-2、Bax和caspase-3蛋白的表达。结果:ox-LDL可明显抑制HUVEC细胞增殖,TP与ox-LDL共同加入后,细胞增殖率明显上升,与ox-LDL组相比差异显著(P<0.05)。ox-LDL可诱导细胞发生凋亡,而TP能减弱其作用。Westernblotting结果:ox-LDL下调HUVEC细胞Bcl-2表达、上调Bax和caspase-3表达,TP与ox-LDL共同加入后,Bcl-2表达升高,Bax和caspase-3表达下降。结论:茶多酚对ox-LDL诱导的HUVEC凋亡具有抑制作用,且与上调Bcl-2蛋白和下调Bax和caspase-3蛋白表达有关。     

Induction of Cell Cycle Arrest and Apoptosis in Human Osteosarcoma U-2 OS Cells by Solanum lyratum Extracts

This research focused on a Chinese herb medicine, Solanum lyratum Thunb (Solanaceae) by ethanol extracts (SLE) for investigating the molecular anticancer mechanism in vitro for exploring the means of cell death through the effects on mitochondrial function. We found that SLE induced cytotoxic effects in human osteosacroma U-2 OS cells, and these effects include cell morphological changes, a decrease of the percentage of viable cells and induction of apoptosis. The results suggest that cell death induced by SLE is closely related to apoptosis based on the observations of DAPI staining and sub-G1 phase in U-2 OS cells. Flow cytometric assays also showed that SLE promoted the production of reactive oxygen species and nitric oxide but decreased the levels of mitochondrial membrane potential and promoted the activations of caspase-8 and -9 in U-2 OS cells. SLE inhibited the level of Bcl-2 but promoted the Bax level, and both proteins led to the release of cytochrome c from mitochondria to cytosol and activation of caspase-9 and -3, resulting in the apoptotic death which is mediated through the mitochondrial pathway. Taken together, SLE was demonstrated to be effective in killing U-2 OS osteosacroma cells via the ROS-promoted and mitochondria- and caspase-dependent apoptotic pathways.     

沉默信息调节因子1小干扰RNA诱导前列腺癌细胞PC3细胞凋亡

摘要:目的　观察沉默信息调节因子1（SIRT1）小干扰RNA（siRNA）对前列腺癌细胞PC3细胞生长增殖、DNA合成、细胞凋亡和Bcl-2和Bax蛋白的表达变化,探讨SIRT1在前列腺癌发生中的可能机制。方法　体外培养PC3细胞,分空白对照组（mock组）,转染阴性对照组（scramble siRNA组）和SIRT1 siRNA转染组;Western blot检测PC3细胞中SIRT1的干涉效能;MTT法测定PC3细胞的增殖率;BrdU掺入法测定DNA合成;流式细胞术检测细胞凋亡;Western blot检测PC3细胞中细胞凋亡关键调控因子Bcl-2和Bax的蛋白表达。结果　与对照组比较,SIRT1 siRNA组SIRT1蛋白表达降低（P＜0.01）,PC3细胞的增殖和DNA合成明显受抑制（P＜0.01）,细胞凋亡比例增加（P＜0.01）,Bcl-2蛋白表达减少,Bax的表达增加。结论　下调SIRT1的表达抑制细胞增殖和DNA合成,诱导前列腺癌PC3细胞发生凋亡,其机制可能与改变细胞凋亡关键调控因子Bcl-2和Bax的蛋白表达相关。     

COX-2及凋亡基因表达在二十二碳六烯酸抑制人胰腺癌细胞体外增殖中的变化

目的研究COX-2和凋亡相关基因Bcl-2、Bax在二十二碳六烯酸(DHA)抑制人胰腺癌细胞体外增殖中表达的变化,探讨DHA抑制肿瘤细胞的作用机制。方法采用四甲基偶氮唑蓝(MTT)实验、流式细胞技术对细胞增殖、细胞凋亡、细胞周期以及肿瘤相关蛋白COX-2和凋亡基因Bcl-2、Bax进行分析。结果DHA能以时间-剂量依赖关系抑制人胰腺癌细胞增殖,同时诱导细胞凋亡。DHA对正常人成纤维细胞的增殖无明显影响。流式细胞仪检测显示:G0-G1期细胞比例增高,S期细胞比例下降。DHA作用24h后,胰腺癌细胞的COX-2、Bcl-2、Bax蛋白表达下降。结论DHA下凋COX-2、Bcl-2、Bax的表达可能是其抑制胰腺癌细胞增殖的作用机制之一。     

新生鼠缺氧缺血性脑损伤凋亡基因的变化及神经节苷酯的治疗作用

【目的】探讨新生大鼠缺氧缺血性脑损伤(hypoxic-ischemia brain damage，HIBD)后Bcl-2、Bax基因表达与细胞凋亡的关系及神经节苷酯(gangliosides，GM1)对其的影响。【方法】建立新生鼠HIBD动物模型，用TUNEL法和免疫组化法观察缺氧缺血和GMI干预后不同时间点细胞凋亡情况及凋亡基因Bcl-2、Bax的变化。【结果】缺氧缺血组随时间延长脑组织中Bcl-2、Bax的表达增加，Bcl-2／Bax的比值下降，同时凋亡细胞数增加，表明Bcl-2、Bax两者比值的降低促进凋亡的发生。GM1干预组Bcl-2表达增加显著，降低了Bax的表达，凋亡细胞减少。【结论】GM1可能通过改变凋亡基因的表达而抑制神经细胞的凋亡过程。     

Genistein-induced apoptosis of p815 mastocytoma cells is mediated by Bax and augmented by a proteasome inhibitor,lactacystin

Although genistein has been demonstrated to induce apoptosis of various cells, there is no report of its effect on mast cell proliferation. Here we show that genistein reduced the viability of mast cell tumor cell lines, p815 and RBL-2H, but not of a human mast cell line, HMC-1. Further investigation on its growth-inhibitory mechanism was undertaken on p815 mastocytoma cells. Genistein induced G2/M arrest and subsequent apoptotic death. p815 cells undergoing apoptosis showed many apoptotic manifestations, such as reduction of mitochondrial membrane potential, release of cytochrome c to cytosol, translocation of apoptosis-inducing factor to nucleus, activation of caspase-3, nuclear condensation, and generation of DNA fragmentation. Genistein treatment resulted in the increase of Bax expression and its translocation into mitochondria, whereas expression levels of Bcl-2 remained unchanged. Proteasome activity decreased at the early time points after genistein treatment, but thereafter it fluctuated at increased levels. A proteasome inhibitor, lactacystin, potentiated the induction of apoptosis. Taken together, genistein-induced apoptosis of p815 mastocytoma cells is at least in part mediated by proteasome, Bax, apoptosis-inducing factor, and caspase and augmented by cotreatment with a proteasome inhibitor, lactacystin.     

罗格列酮对2型糖尿病大鼠心肌细胞凋亡及其基因表达的影响

目的　观察 2型糖尿病大鼠心肌细胞凋亡率、凋亡调控基因Bax、Bcl-2的表达变化及罗格列酮的干预作用。方法　8周龄SD大鼠高热量饮食一月后 ,腹腔注射小剂量链脲佐菌素 (STZ 3 0mg/kg) ,成模后分别于 12、2 4周宰杀动物 ,采用光镜、原位末端标记法、流式细胞术检测心肌细胞凋亡及心肌Bax、Bcl -2表达水平。结果　糖尿病大鼠心肌凋亡细胞数明显增多 ,Bax蛋白表达显著上调 ,Bax/Bcl-2比值增加 (P均 <0 0 1) ,Bcl -2蛋白表达有所下降 ,但无统计学意义。罗格列酮干预组凋亡率降低 (P <0 0 1) ,升高的Bax蛋白表达下调 ,降低的Bcl -2蛋白表达上调 ,但与糖尿病未治疗组均无统计学差异 (P >0 0 5 )。结论　心肌细胞凋亡在糖尿病大鼠心肌病变进程中起一定作用 ,罗格列酮可抑制心肌细胞凋亡 ,但与Bax、Bcl -2的调控关系不大。