生育年龄妇女早期卵泡细胞凋亡和Bcl-2/BAX蛋白表达

目的:研究生育年龄妇女早期卵泡的细胞凋亡和Bcl-2/BAX蛋白表达。方法:12例卵巢组织标本来自进行妇科手术的生育年龄妇女(年龄23-38岁),并经过组织病理学检查证实无明显形态异常。利用末端标记法(TUNEL)和免疫组织化学方法研究早期卵泡(包括始基卵泡、间期和初级卵泡)细胞凋亡和Bcl-2/BAX蛋白表达。结果:早期卵泡内18.75%卵细胞表现TUNEL阳性,但是卵泡内未见TUNEL阳性颗粒细胞。BAX在早期卵泡的卵细胞内表达,阳性表达率76.07%;相反未见Bcl-2在卵细胞表达。另外,早期卵泡内颗粒细胞也没有表达Bcl-2和BAX。结论:生育年龄妇女早期卵泡的卵细胞发生凋亡,促凋亡蛋白BAX在卵细胞凋亡过程中发挥调节作用,由此提示BAX介导的卵细胞凋亡可能是生育年龄妇女早期卵泡闭锁的机制。     

PPARdelta and its activator PGI2 are reduced in diabetic embryopathy: involvement of PPARdelta activation in lipid metabolic and signalling pathways in rat embryo early organogenesis

Maternal diabetes significantly increases the risk of congenital malformations, and the mechanisms involved are not yet clarified. This study was designed to address peroxisome proliferator-activated receptor delta (PPARdelta) involvement in diabetic embryopathy. We investigated the concentrations of PPARdelta and its endogenous agonist prostaglandin (PG)I(2), as well as the effect of PPARdelta activation on lipid metabolism and PGE(2) concentrations in embryos from control and streptozotocin-induced diabetic rats during early organogenesis. Embryos from diabetic rats showed decreased concentrations of PPARdelta and its endogenous agonist PGI(2) when compared with controls. In embryos from control rats, the addition of the PPARdelta activators (cPGI(2) and PGA(1)) increased embryonic phospholipid levels and de novo phospholipid synthesis studied using (14)C-acetate as a tracer. PGE(2) formed from arachidonate released from phospholipid stores was also up-regulated by PPARdelta activators. In embryos from diabetic rats, reduced phospholipid synthesis and PGE(2) content were observed, and clearly up-regulated by cPGI(2) additions to values similar to those found in control embryos. These data suggest that PPARdelta may play an important role in lipid metabolic and signalling pathways during embryo organogenesis, developmental pathways that are altered in embryos from diabetic rats, possibly as a result of a reduction in levels of PPARdelta and its endogenous activator PGI(2).     

Apoptosis and the expression of Bax and Bcl-2 in hyperplasia and adenocarcinoma of the uterine endometrium

BACKGROUND: Apoptosis plays a crucial role in carcinogenesis in various tumours. This study was designed to investigate the occurrence of apoptosis and the expression of Bcl-2 and Bax proteins in endometrial tumours of corpus uteri. METHODS: Endometrial tissues were obtained from 20 patients with endometrioid adenocarcinoma, 16 patients with endometrial hyperplasia, and 4 patients with myoma uteri (which were used as controls). The occurrence of apoptosis was examined by using molecular biochemical techniques. The expression of Bcl-2 and Bax proteins was also investigated using immunohistochemical staining with appropriate antibodies. RESULTS: The labelling of DNA in situ indicated that apoptotic cells were sporadically seen in postmenopausal endometrium (5.2 +/- 2.1, n = 4) and endometrial hyperplasia without atypia (2.6 +/- 0.5, n = 9). In contrast, labelled cells were detected in atypical endometrial hyperplasia (15.9 +/- 2.2, n = 7), and their numbers increased intensely in adenocarcinoma (29.3 +/- 3.7, n = 20). Autoradiographic analysis revealed DNA laddering in many cases of carcinoma. Bcl-2 was highly immunopositive in hyperplasia without atypia (36.2 +/- 6.5%, n = 9), but was decreased in the atypical endometrial hyperplasia (16.3 +/- 4.8%, n = 7). Large fractions of the carcinoma (6.3 +/- 1.8%, n = 20) and normal endometrium (2.8 +/- 1.4%, n = 4) were immunonegative or slightly immunopositive to Bcl-2. In contrast, Bax immunoreactivity was more frequent and stronger in adenocarcinoma (43.6 +/- 4.1%, n = 20) than that in normal endometrium (17.6 +/- 6.7%, n = 4) and hyperplasia (7.2 +/- 2.2%, n = 16). CONCLUSIONS: These results suggest that cells in hyperplasia expressing Bcl-2 might have prolonged survival ability. Neoplastic cells in adenocarcinoma might show apoptosis in association with a decreased expression of Bcl-2 and an increased expression of Bax. Therefore, the frequency of apoptosis and the expression of Bcl-2 and Bax might be correlated with carcinogenesis in the uterine endometrium of humans.     

自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax表达的研究

目的：通过比较正常妊娠模型小鼠及自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax蛋白的表达，从细胞及分子水平探讨自然流产的发病机制。方法：建立正常妊娠模型CBAXBALB／c和自然流产模型CBAXDBA／2。用免疫组化SABC法测定两组模型孕13天蜕膜细胞Bcl-2和Bax蛋白的表达，并通过MIAS-2000医用彩色病理图像免疫组化测量系统对其表达进行半定量分析，其结果用平均灰度值表示；同时应用DNA缺口原位末端标记技术（TUNEL）测定两组模型孕13天蜕膜细胞凋亡情况。结果：与正常妊娠模型相比，自然流产模型蜕膜细胞Bcl-2蛋白的表达降低（P〈0．01）；Bax蛋白的表达明显升高（P〈0．01）。蜕膜细胞凋亡指数，自然流产模型明显高于正常妊娠模型（P〈0．01）。结论：早孕期蜕膜组织细胞凋亡异常是自然流产的机制之一，Bcl-2／Bax途径可能是诱导早孕期蜕膜细胞凋亡的重要因素。     

Bcl-2 和Bax蛋白在慢性病毒性肝炎肝组织中的表达

目的研究细胞凋亡相关的Ｂｃｌ－２和Ｂａｘ蛋白在慢性病毒性肝炎（ＣＨ）肝组织中表达的情况与该病发生发展的关系。方法对３５例不同病变程度的ＣＨ肝组织用免疫组化法检测Ｂｃｌ－２和Ｂａｘ的表达及定位。结果Ｂｃ１－２和Ｂａｘ分布基本一致，轻、中度ＣＨ主要见于碎屑样坏死（ＰＮ）区中的单个核细胞（ＭＮ），邻近ＰＮ区边缘的肝细胞浆中也有强阳性表达。ＨＥ染色观察，这些Ｂｃｌ－２和Ｂａｘ阳性肝细胞多呈重度水样变性或嗜酸性变。在重度ＣＨ肝组织中Ｂｃｌ－２和Ｂａｘ阳性的ＭＮ主要分布于重度ＰＮ区和桥接坏死区，周围部分肝细胞Ｂａｘ阳性，而Ｂｃｌ－２为阴性。在慢性重型肝炎，Ｂｃｌ－２和Ｂａｘ除在亚大块肝细胞坏死区浸润的ＭＮ胞浆内表达外，残存肝细胞亦呈阳性表达。结论Ｂｃｌ－２和Ｂａｘ在肝组织中的表达可能不仅与ＣＨ的细胞凋亡有密切关系，而且与慢性重型肝炎的发生发展有关     

Apoptosis and occurrence of Bcl-2, Bak, Bax, Fas and FasL in the developing and adult rat endocrine pancreas

Apoptotic cell death is thought to play a crucial role in the manifestation of insulin- and non-insulin dependent diabetes mellitus. Therefore, apoptosis and apoptotic markers were studied in the rat endocrine pancreas to get insight into the possible life cycle of Langerhans islets.The islets were investigated at 13 time points between day E19 and 18 months. At each time point, histologic sections were treated with the direct fluorescein-labelled TUNEL method and immunostained for pancreatic hormones (glucagon, insulin), apoptotic promoters (Bak, Bax, Fas, Fas Ligand) as well as for the anti-apoptotic peptide Bcl-2. All tissue sections were investigated using confocal laser scanning microscopy under identical settings for semiquantitative estimation of staining intensity. TUNEL-positive cells occurred in all pre- or postnatal stages.The findings indicated a biphasic apoptotic activity in the endocrine pancreas during the lifetime of rats. The first phase began at E19 and peaked at P5 accompanied by a considerable increase in Bak fluorescence staining intensity, while the second phase began at P30 and peaked at 18 months with increasing amounts of Fas and FasL staining intensities in the islet cells. The presented in situ data may be important for understanding the increased age-related vulnerability of islet cells and for studies of isolated and cultivated rat islets. Accepted: 9 May 2000     

抑郁模型大鼠杏仁核神经元凋亡及Bax/Bcl-2的变化

目的:探讨抑郁模型大鼠杏仁核神经元凋亡现象。方法:将成年健康雄性Wistar大鼠随机分为对照组(15只)和模型组(15只)。采用慢性强迫游泳(4周)制备慢性强迫游泳应激抑郁模型。采用TUNEL和流式细胞术检测杏仁核神经元凋亡和凋亡率,WesternBlot检测凋亡相关蛋白Bax/Bcl-2的表达变化。结果:模型组和对照组凋亡细胞阳性率分别为24.08±4.30和3.08±0.91,凋亡率分别为17.14±2.71和3.34±0.80,Bax/Bcl-2比值分别为1.73±0.15和0.92±0.07,差异均有统计学意义(P0.01)。结论:抑郁模型大鼠杏仁核存在明显的神经元凋亡,这可能是抑郁患者杏仁核体积异常的原因之一。     

Apoptosis in cultured rat islets of langerhans and occurrence of Bcl-2, Bak, Bax, Fas and Fas ligand

Isolated Langerhans islets are widely used for diabetic transplantation experiments and investigations of the mechanisms leading to the death or survival of insulin-producing cells in cultured islets. The present study was aimed at investigating programmed cell death and the role of apoptosis-associated peptides in insulin and glucagon cells of islets isolated from untreated rats and held in cultured suspension. Islets were removed from medium on days 0, 7, 14, 21 and 29, embedded in Epon, and semi-thin serial sections were prepared. At designated intervals, histologic sections were treated with the direct fluorescein-labelled TUNEL method and immunostained for pancreatic hormones (glucagon, insulin) and apoptotic peptides [Bak, Bax, Fas, Fas ligand (FasL)], as well as for the anti-apoptotic peptide Bcl-2. All tissue sections were investigated using confocal laser scanning microscopy under identical setting for semiquantitative estimation of staining intensity. The percentage of apoptotic cells was between 1.6 and 2.1% and most apoptotic cells were beta-cells. Corresponding cells often contained Bak and Bax. Fas and FasL were mostly detected in islet cells within the first week after preparing the cultured suspension. The insulin content was low (1.1 +/- 0.22 ng per islet) directly after isolation. It then increased progressively up to day 14, after which it began to decrease. Glucagon expression, on the other hand, remained high for the entire duration of the investigation. In conclusion, the islet beta-cells may recover after the isolation procedure, but after 4 weeks in culture, both the insulin content and Bcl-2 staining decrease. Moreover, apoptosis is mediated by different mechanisms after the isolation procedure and after culturing the islets for 1 month. The present data may be important for further studies on isolated, cultivated or transplanted islets.     

丙酸睾酮对大鼠胸腺细胞凋亡及胸腺中Bcl-2、Bax表达的影响

目的探讨丙酸睾酮在大鼠胸腺退化过程中的作用及其对大鼠胸腺中Bcl-2和Bax表达的影响。方法雄性大鼠去势后给予丙酸睾酮皮下注射,分别于注射后第1、4、7天取材,用半薄切片甲苯胺蓝染色检测胸腺细胞凋亡情况,免疫组织化学法检测胸腺组织中Bcl-2和Bax的表达情况,采用t检验进行统计学处理。结果丙酸睾酮处理组大鼠胸腺组织中可发现较多的凋亡细胞和凋亡小体;去势组大鼠胸腺组织中Bcl-2表达量明显高于假手术组（P〈0.001）,丙酸睾酮处理后Bcl-2表达量明显减少（P〈0.001）;而Bax的表达正好相反,去势组大鼠胸腺组织中Bax表达量明显低于假手术组（P〈0.001）,给予丙酸睾酮后Bax表达量明显增加（P〈0.001）;结论丙酸睾酮可诱导大鼠胸腺细胞凋亡,并抑制大鼠胸腺组织中Bcl-2的表达,促进Bax的表达。     

钙中性蛋白酶2及Bax在肝纤维化大鼠肝组织中的表达变化及意义

 目的：探讨钙中性蛋白酶2（calpain 2）及Bcl-2相关X蛋白(Bax)在肝纤维化过程中的表达变化及其在肝纤维化过程中的可能作用。方法:  雄性Wistar大鼠40只,随机分为正常对照4周、8周组和肝纤维化模型4周、8周组,每组各10只。肝纤维化组按3 mL/kg体重的剂量皮下注射40% CCl4植物油溶液,每隔3 d注射1次,造模时间分别为4周和8周;对照组大鼠皮下注射等量植物油溶液。采用TUNEL法检测肝组织中肝细胞凋亡情况;real-time PCR检测肝组织中calpain 2及bax mRNA的表达变化。免疫组织化学法及Western blotting检测肝组织中calpain 2及Bax蛋白的表达情况。 结果:  Real-time PCR检测发现肝纤维化4周和8周组大鼠肝组织中calpain 2及bax mRNA表达较相应的正常对照组显著增加。免疫组化及Western blotting检测显示肝纤维化4周组大鼠肝组织中calpain 2蛋白的表达与正常4周组比较无显著差异;随着肝纤维化程度的加重,肝纤维化8周时大鼠肝组织中calpain 2的表达显著增加;而肝组织中Bax的表达从肝纤维化4周时就显著增加,肝纤维化8周时达到高峰。此外,通过TUNEL法检测发现肝纤维化4周和8周组大鼠肝组织中肝细胞凋亡的数目较正常组大鼠显著增加。结论:  Calpain 2与Bax可能参与了肝纤维化的发生发展过程。     

凋亡相关蛋白Bcl-2及Bax在糖尿病大鼠下颌下腺内的表达

目的:观察糖尿病大鼠下颌下腺内凋亡相关蛋白Bcl-2和Pax表达变化.方法:SD雄性大鼠用链脲佐菌素复制糖尿病动物模型,H-E染色观察下颌下腺形态学变化,免疫组织化学SABC技术检测Bcl-2及Bax蛋白表达变化.结果:糖尿病组大鼠下颌下腺组织萎缩,间质纤维增生.与对照组比较,糖尿病组大鼠下颌下腺导管上皮细胞Bcl-2表达下降,Bax表达显著增加.结论:Bcl-2及Bax表达在糖尿病病理变化过程中出现了一定的变化规律.     

姜黄素对大鼠胸主动脉瘤Bcl-2和Bax表达的影响

目的 探讨姜黄素对大鼠胸主动脉瘤形成过程中Bcl-2和Bax表达的影响。方法 将30只成年雄性Wistar大鼠随机分为3组,即对照组、动脉瘤组和姜黄素治疗组。采用氯化钙（CaCl2）诱导法制备胸主动脉瘤模型,术后4周取材。HE染色及地衣红染色观察胸主动脉组织学改变;免疫组织化学和Western blotting方法检测动脉瘤壁Bcl-2和Bax的表达。结果 姜黄素可明显抑制胸主动脉的扩张。与对照组比较,动脉瘤组Bax表达水平明显升高,Bcl-2表达水平降低,姜黄素能有效降低动脉瘤中Bax的表达,升高Bcl-2表达水平。 结论 姜黄素可以促进胸主动脉瘤Bc1-2蛋白的表达,抑制Bax蛋白的表达,降低Bax/Bcl-2的比值。     

雌激素对大鼠胸腺细胞凋亡及Bcl-2、Bax表达的影响

目的:探讨苯甲酸雌二醇对大鼠胸腺Bcl-2和Bax表达及细胞凋亡的影响及其机制.方法:雌性大鼠行卵巢切除术,给予苯甲酸雌二醇后,观察胸腺指数的变化,Hochest33342荧光染色及透射电镜标本观察胸腺细胞凋亡情况,免疫组织化学检测胸腺组织中Bcl-2和Bax的表达情况,原位杂交技术检测Bcl-2、Bax m RNA的表达情况.结果:双侧卵巢切除组大鼠胸腺指数较假手术组增加,双侧卵巢切除+雌激素组大鼠胸腺指数较双侧卵巢切除组减小;假手术组和双侧卵巢切除组大鼠胸腺组织中以正常胸腺细胞为主,偶见凋亡细胞或凋亡小体,双侧卵巢切除+雌激素组可见较多凋亡细胞和凋亡小体;双侧卵巢切除+雌激素组大鼠胸腺组织中Bcl-2表达较双侧卵巢切除组和假手术组增高明显降低,而Bax表达呈现相反趋势;Bcl-2 mRNA、Bax mRNA的表达与Bcl-2、Bax的表达呈一致性.结论:雌激素可以降低大鼠胸腺指数,抑制胸腺组织中Bcl-2的表达,促进Bax的表达,从而诱导大鼠胸腺细胞凋亡,促进雌性大鼠胸腺退化.     

心理应激对大鼠淋巴细胞凋亡及基因调控的影响

目的：探讨不同心理应激强度对大鼠外周血淋巴细胞凋亡及其调控基因Bcl-2、Bax基因表达的变化。方法：健康的雄性SD大鼠24只，随机分为对照组、中等强度心理应激组、高强度心理应激组，每组8只。分别对大鼠进行3周高、中等强度噪音诱发的心理应激，采用流式细胞仪测定各组淋巴细胞凋亡的情况以及用免疫组织化学法观察调控基因Bcl-2、Bax基因表达的变化。结果：①高强度应激组大鼠淋巴细胞群中存有典型的凋亡细胞，且凋亡细胞发生率明显高于对照组，而中等强度应激组大鼠淋巴细胞凋亡率不显著。②高强度心理应激组大鼠淋巴细胞的Bcl-2表达下调，而Bax的表达量明显增加。结论：高强度心理应激过程中存在着免疫细胞凋亡，而且其发生与相关调控基因的表达一致。     

镉影响大鼠胎盘发育及Bcl-2和Bax的表达

目的通过观察镉暴露后胎盘组织形态结构和凋亡相关蛋白表达量的改变，探讨镉对胎盘组织中细胞增殖与凋亡的影响。方法将孕6天（E6）的20只孕鼠随机分成染镉组和对照组，染镉组一次性腹腔注射氯化镉，剂量为2mgCd／kg体重。两组的孕鼠分别于E14和E18断椎处死，每只孕鼠随机取出6个胎盘，制备石蜡切片。行HE染色观察染镉后胎盘的形态改变，免疫组织化学技术显示镉对Bcl-2和Bax表达的影响。结果染镉组胎盘海绵带滋养层细胞、巨细胞和空泡化细胞增多，且迷路带及海绵带滋养细胞呈退行性改变。染镉组胎盘组织细胞中的Bcl-2蛋白表达量比相应的对照组表达量明显降低（E14：t=7．067，P〈0．01；E18：t=6．988，P〈0．01）；Bax蛋白表达量比相应的对照组表达量明显增多（E14：t=4．008，P〈0．01；E18：t=4．732，P〈0．01）；Bcl-2／Bax比值与相应对照组之间的差异有统计学意义（E14：t=9．517，P〈0．05；E18：t=23．380，P〈0．01）。结论镉可以通过影响Bcl-2和Bax的表达，改变Bcl-2／Bax比值，使胎盘组织细胞的增殖和凋亡平衡紊乱，导致胎盘发育异常。     

Apoptosis in the medaka embryo in the early developmental stage

Apoptosis is an important event of the development of various organs. In this study, we used in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to visualize the temporal and spatial distribution of apoptosis in the developing medaka embryo, which is a useful model for developmental biology and genetics. Most of the apoptotic cells were distributed in the central nervous system and tailbud. In the brain and retina, most of the apoptosis occurred in the restricted period. In situ hybridization against caspase 3A and caspase 3B showed that these were distributed in the tailbud and the head, respectively. These results suggested that two types of caspase 3 were involved in apoptosis in different areas.     

Bcl-2/Bax蛋白在生后大鼠卵巢不同发育时期的表达

目的 探讨Bcl-2/Bax与卵泡发育和凋亡的关系. 方法 用免疫组织化学技术和图像分析系统,对95只大鼠生后不同发育时期卵巢中凋亡相关因子Bcl-2/Bax的表达情况进行研究. 结果 0～360 d卵巢各级卵泡中的卵母细胞均呈Bcl-2/Bax阳性染色.正常卵细胞中Bcl-2反应强于Bax,退化卵细胞中Bax的反应强于Bcl-2.Bcl-2/Bax在卵泡细胞中的表达模式有所不同,21d前 Bcl-2为阳性,21～150d之间为Bcl-2阴性;180d以后正常卵泡为阴性,闭锁卵泡为阳性;Bax的表达,180d前全为阴性,180d后的表达同Bcl-2.膜细胞中Bcl-2/Bax的表达与卵泡细胞存在相似之处.Bcl-2/Bax在黄体中的表达只见于180d后. 结论 Bcl-2/Bax对卵细胞的生长、发育、存活和凋亡可能有重要的调节作用,其对卵泡细胞、膜细胞及黄体细胞的存活和凋亡的调节作用不明显.     

Bcl-2 expression in normal endometrium during the menstrual cycle.

Bcl-2 is a proto-oncogene initially described in the (14;18) translocation in follicular lymphoma. It has been shown to prolong cell survival by preventing apoptosis. Endometrium undergoes rapid proliferation and differentiation under hormone control and is thus an excellent model to study the hormone dependency of Bcl-2 expression. We studied Bcl-2 expression by an immunohistochemical method in 53 samples of normal endometrium randomly distributed throughout the menstrual cycle, as well as five samples of hyperplastic endometrium. Bcl-2 staining predominated in glandular cells and peaked at the end of the follicular phase. Bcl-2 expression disappeared at the onset of secretory activity. The stroma, surface lining epithelium and arterial vessels also displayed cyclic variations in Bcl-2 expression. These results strongly suggest hormone-dependent regulation of Bcl-2 expression, which could play an important role in tumorigenesis.     

A study of diclofenac-induced teratogenicity during organogenesis using a whole rat embryo culture model.

BACKGROUND: Diclofenac is a non-steroidal anti-inflammatory drug, commonly used by reproductive age women for the treatment of a variety of conditions. However, there is limited information regarding the teratogenic effects of this drug. METHODS: The effect of diclofenac on the developing embryo during the critical period of organogenesis was investigated by using a whole rat embryo culture model. Embryos were exposed to various concentrations of diclofenac and scored for growth and differentiation at the end of the culture period. RESULTS: Total developmental score and score for caudal neural tube, flexion and hindlimb were significantly lower in embryos exposed to high concentrations of diclofenac (7.5 and 15.0 microg/ml), but no difference in these parameters was observed when embryos were exposed to low concentration of diclofenac (1.5, 2.5 and 5.0 microg/ml). No significant differences in yolk sac diameter, crown-rump length and number of somites was found between embryos in the experimental and the control group. CONCLUSIONS: Our study has demonstrated that diclofenac exerts direct teratogenic effects on rat embryos. Until more is known about the effects of diclofenac (especially in moderate to high doses) in women of reproductive age, we suggest its use should be treated with caution.     

Role of Bcl-2 Family Members in Caspase-Independent Apoptosis during Chlamydia Infection

Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.