邻苯二甲酸二丁酯孕期暴露致仔鼠睾丸膜联蛋白A5的表达差异

背景：国内外关于邻苯二甲酸二丁酯导致雄性生殖系统畸形发生机制的探讨多集中于邻苯二甲酸二丁酯干扰胚胎期睾丸睾酮合成途径的研究，对其基因表达调控的因素及功能蛋白质之间相互干扰的网络效应却并不明了。蛋白质组学能够从整体水平反映蛋白质组分，并筛选功能相关蛋白。 目的：探讨邻苯二甲酸二丁酯胚胎期暴露对胎鼠睾丸蛋白表达谱的影响，分离并鉴定差异表达蛋白质。 设计、时间及地点：随机对照动物实验，实验于2006-08/2007-10在南京医科大学动物实验中心及南京医科大学生殖医学重点实验室完成。 材料：将孕鼠随机分为实验组和对照组，每组5只。妊娠14~18 d。 方法：实验组按800 mg/(kg?d)染毒孕鼠，对照组予大豆油5 mL/d。妊娠21d 取出胚胎大鼠睾丸，提取总蛋白，进行二维凝胶电泳分离和图像分析，筛选出的差异蛋白质点利用质谱技术进行鉴定，并选择关键蛋白进行验证。 主要观察指标：①两组蛋白的电泳差异比较。②酶切，MALDI-TOF分析，数据库检索，生物信息学检索结果。③两组蛋白Western blotting检测及免疫组织化学染色结果。 结果：共筛选出33个差异表达蛋白（t≥2.831，P < 0.05），其中14个通过质谱分析和SwissProt蛋白数据库检索得到鉴定，包括膜联蛋白A5、过氧化物酶6、泛素羧基末端水解酶L1等。运用Western blotting，验证了膜联蛋白A5在实验组表达量明显高于对照组，通过免疫组织化学方法，发现膜联蛋白A5主要定位在胎鼠睾丸Leydig细胞中。 结论：实验运用蛋白质组学方法，建立了邻苯二甲酸二丁酯孕期暴露雄性仔鼠睾丸与正常仔鼠睾丸蛋白质差异表达谱系，鉴定出14个蛋白点，确定了膜联蛋白A5在胎鼠睾丸的定位。     

X射线电离辐射对成年小鼠睾丸组织的损伤*

背景：X射线电离辐射对睾丸的损伤,最直观的是通过对组织切片进行染色,近而进行形态学观察,可以直观地观察到组织学的损伤。 目的：观察X射线电离辐射后成年小鼠睾丸组织损伤的形态学变化。 设计、时间及地点：随机对照动物实验,于2008-01/10在延安大学医学院实验室完成。 材料：BALB/c健康雄性的成年小鼠(6~8周龄)18只,均购自解放军第四军医大学,体质量(20.0±2.5) g,实验前2周饲养于室温(20±3) ℃,空气湿度50%以上,饲料垫料均干燥消毒,标准饲料饮水无限制。 方法：采用辐射剂量1.0 Gy和2.0 Gy的X射线对成年小鼠进行全身照射,于照射后2周取小鼠睾丸组织,称质量,制备标本,苏木精-伊红染色,示睾丸组织变化;对照组不进行照射。 主要观察指标：正常小鼠睾丸质量与照射后睾丸的质量之比;正常睾丸组织生精小管与照射后睾丸生精小管结构的变化的对比。 结果：正常小鼠体质量与辐射组体质量无明显差异,但睾丸质量/体质量之比随照射剂量不同出现明显差异。苏木精-伊红染色显示睾丸组织在正常对照组小鼠睾丸组织生精小管管腔形状规则,管壁基膜完整,管腔内生精细胞层数多,排列规则紧密,管腔内充满成熟的精子在照射后出现病理性变化：睾丸组织内各级生精细胞排列混乱,生精细胞剥离脱落,空洞形成,管腔内精子数量明显减少;随照射剂量增加,生精小管结构明显紊乱,管腔皱缩、塌陷,生精上皮明显变薄,排列松散,管腔内已无成熟精子。 结论：X射线辐射后小鼠睾丸质量明显减少,且随照射剂量的不同有明显差异,损伤程度随剂量的增加而加重。     

小鼠睾丸间质细胞体外原代培养方法的建立

背景：组织块培养睾丸生殖细胞对污染不容易控制,胰蛋白酶消化接种培养睾丸生殖细胞可能损伤细胞。 目的：建立一种切实可行的小鼠睾丸间质细胞体外原代培养方法。 方法：小鼠睾丸间质细胞的分离采用胶原酶消化法,纯化采用Percoll等密度梯度离心法,活率鉴定采用锥虫蓝拒染法,纯度鉴定采用3β-羟基固醇脱氢酶染色方法。 结果与结论：小鼠睾丸间质细胞纯度达可达90%以上;体外培养的细胞形态完整、增殖速度快、贴壁生长状态良好。说明实验成功建立了小鼠睾丸间质细胞的体外原代培养模型。     

增塑剂邻苯二甲酸二丁酯诱导类神经细胞PC12细胞凋亡的相关基因表达调控

目的探讨增塑剂邻苯二甲酸二丁酯对类神经细胞PC12细胞增殖能力的影响及其对PC12细胞凋亡相关基因表达的调控．为接触人群可能产生远期效应的生物学检测提供参考评价指标。方法体外培养类神经细胞株PC12细胞，培养基分别含有0，12．5，25，50mg／L邻苯二甲酸二丁酯，作用24，48，72和96h后，用细胞计数CCK-8法检测细胞的存活和增殖，台盼蓝染色绘制生长曲线，DNA凝胶电泳观察DNA—Ladder，RT—PCR检测p53，bcl-2，Bax等凋亡相关基因表达变化情况。结果邻苯二甲酸二丁酯在12．5mg／L时即表现出显著的抑制PC12细胞增殖的作用（P〈0．01），且随剂量增加抑制作用增强，同时出现DNA断裂阳性；凋亡相关基因均有表达，其中p53基因、Bax基因表达增加,bcl—2基因表达降低，且呈剂量相关关系（P〈0．01）。结论邻苯二甲酸二丁酯暴露可抑制PC12细胞增殖并诱导PC12细胞凋亡，同时通过调节凋亡相关基因p53、Bax、bcl-2基因的表达水平而在其凋亡发生中起重要作用。推测邻苯二甲酸二丁酯长期低剂量暴露的累积效应与阿尔茨海默病以及其他神经细胞凋亡相关性疾病的发生发展可能存在相关关系。     

猫正常脑组织X刀不同剂量照射后组织病理学改变

目的：观察Ｘ刀不同剂量照射猫正常脑组织后的病理学改变。方法：用磁共振成像（ＭＲＩ）Ｔ２加权扫描、光镜检查、电镜超微结构观察等方法,研究Ｘ刀不同剂量照射正常脑组织后,于不同时程所引起的放射反应。结果：照射后３月１５０Ｇｙ剂量,照射后６月１００Ｇｙ剂量和１５０Ｇｙ剂量,ＭＲＩＴ２加权扫描观察到受照射脑组织出现类圆形高信号灶;３０Ｇｙ剂量在１年观察时间内,光镜未见异常变化,但电镜下发现明确的病理改变。７０Ｇｙ剂量在照射后３月出现照射反应。随照射后时间延长,照射脑组织反应呈进行性改变。结论：Ｘ刀不同剂量对猫正常脑组织照射,在不同时程内可引起多种形态的病理改变。     

精原干细胞和睾丸组织的同种异体移植

背景：精原干细胞作为精子发生过程的基础和前提，其自我更新和分化途径目前仍不完全清楚。 目的：观察非免疫缺陷动物新生Wistar大鼠的精原干细胞和睾丸组织移植于去势成年Wistar大鼠后的成活及生长发育情况。 设计、时间及地点：组织细胞形态学水平的随机动物对照实验，于2007-04/08在广西医科大学实验动物中心外科实验室完成。 材料：选用健康新生7~9 d雄性Wistar大鼠为供体，受体为经过严格检疫合格的8~12周的成年雄性Wistar大鼠，体质量180~220 g。 方法：取新生雄性大鼠睾丸，采用两步法组合酶顺序消化制备大鼠精原干细胞悬液，以Percoll不连续密度梯度离心法初步纯化精原干细胞。取10只成年雄性大鼠，切除双侧睾丸形成去势大鼠，按随机数字表法分为2组，每组5只。精原干细胞悬液移植组将制取好的1 mL精原干细胞悬液在5 min内注射至受体背部皮下。睾丸组织块移植组将制备好的已剖开的睾丸组织植入受体背部皮下, 每个受体移植2个睾丸4块睾丸组织。 主要观察指标：移植物的生长发育情况，移植8周末移植物的组织学特点及C-kit免疫组化定性分析结果。 结果：精原干细胞悬液移植后，移植物未见成活生长。睾丸组织块移植后移植物部分成活，移植8周末组织学检查可见特征性的精曲小管和细胞结构，可见精子细胞，部分生精小管退化；睾丸间质中可见淋巴细胞浸润；免疫组化鉴定可见睾丸组织内C-kit阳性细胞表达。 结论：同种异体异位移植于非免疫缺陷鼠中，新生睾丸组织块可以成活并能形成精子细胞，而精原干细胞悬液移植后未见移植物生长。     

精子发生相关基因及凋亡基因Dazl、Pgk2、Prm2、bax在卵巢异性移植小鼠睾丸、附睾组织中的表达***☆

背景：Dazl、Pgk2、Prm2和细胞凋亡相关基因bax均参与了精子发生的调控,这些基因的表达异常和缺失常常影响精子的发生。 目的：探讨卵巢移植对异性受体精子发生相关基因表达的影响。 方法：将1 d龄小鼠卵巢移植入成年雄性受体鼠肾囊下,分激素处理或不做激素处理两个实验组;于卵巢移植后第21天观察卵巢移植体,选择卵泡发育良好的受体鼠分别采集雄鼠睾丸、附睾提取总RNA,采用半定量RT-PCR分析目的基因表达情况。对照组为未进行卵巢移植的同龄正常雄鼠。 结果与结论：各组间目的基因的表达除Prm2的相对表达量在激素处理移植组显著高于对照组外(P < 0.05),其余各组间各基因的表达差异无显著性意义(P > 0.05)。初步证实在卵巢异性移植构建雌、雄性腺同体的生理环境中,卵巢移植体对受体雄鼠几种精子发生相关基因及凋亡基因的表达无明显影响。提示卵巢移植对雄性受体小鼠的精子发生在分子水平上无明显的损害作用。     

顽固性癫痫的神经病理学改变

目的探讨顽固性癫癎的神经病理学改变.方法对35例顽固性癫癎病人采用皮质脑电图描记下致癎灶切除,切除的组织送病理检查.结果术后病理报告肿瘤10例;血管畸形5例;颅内囊肿6例;炎性肉芽肿3例;外伤性软化灶3例.余8例为神经元变性,胶质细胞增生,含铁黄色素沉积,淋巴细胞侵润等.结论顽固性癫癎的致癎灶组织均存在着病理改变,且病理改变与临床效果密切相关.     

肌营养不良症模型鼠骨骼肌的组织病理学研究

目的　比较肌营养不良症模型 (mdx)鼠、C57鼠和Duchenne型肌营养不良症 (DMD)患者骨骼肌的组织病理学改变 ,以及dystrophin在肌细胞膜上的分布。方法　取mdx鼠、C57鼠和DMD患者骨骼肌作常规HE染色 ,比较其组织学改变 ;同时对mdx鼠、C57鼠骨骼肌作dystrophin的免疫组化染色 ,比较dystrophin在肌细胞膜上的分布。结果　mdx鼠骨骼肌肌纤维大小不等 ,轮廓变圆 ,肌间隙增宽 ,少量脂肪、结缔组织增生 ,细胞核中心移位增多 ,部分肌纤维变性坏死 ,而DMD患者骨骼肌的改变和mdx鼠基本一致。mdx鼠肌细胞膜缺乏完整环行棕色条带 ,而C57鼠则呈一完整环行棕色条带 ,提示mdx鼠dystrophin蛋白缺乏。结论　mdx鼠有类似于DMD患者的骨骼肌组织病理学改变     

海洛因海绵状白质脑病的病理学改变

目的 研究海洛因海绵状白质脑病(HSLE)的病理学改变.方法 应用常规染色和免疫组化染色(MBP、GFAP、NF、CD45、CD54、CD20)方法对3例HSLE死亡患者的脑组织进行病理检查.结果 HSLE的病理改变以脑白质海绵状变性为主,灰质很少受累.脑组织受累程度依次为小脑半球白质、内囊后肢、胼胝体压部、脑干、顶枕叶深部白质以及额颢叶深部白质,皮质以及皮质下弓状纤维不受累或仅轻微受累.HSLE白质损害以粗大的有髓神经纤维为主,髓鞘的损害重于轴突损害;脑组织无炎症细胞浸润,血管内亦无炎症介质沉积.结论 脑白质广泛海绵状白质变性是HSLE显著的病理学特点,不同部位脑白质损害程度与临床和影像学表现一致,轴突损害可能继发于髓鞘损害,HSLE脑组织中无血管炎性改变.     

Dibutyl phthalate-induced neurotoxicity in the brain of immature and mature rat offspring

Purpose: We aim to investigate the neurotoxicity induced by perinatal exposure of dibutyl phthalate (DBP) on the immature and mature offspring animals using a rodent model. Methods: Pregnant rats were given intragastric administration of 500 mg/kg body weight DBP daily from gestational day 6 to postnatal day 21 while control animals received the same volume of edible corn oil. Serum estradiol and testosterone levels of the offspring were evaluated. Protein levels of AROM, ER-β, BDNF and p-CREB in the hippocampus were also measured. Results: Perinatal exposure of DBP significantly up-regulated the serum estradiol levels in both immature and mature offspring rats. DBP exposure also significantly down-regulated the testosterone levels in immature male and female rats and mature male rats but had no influence on the testosterone levels in mature female rats. DBP exposure up-regulated AROM, but down-regulated ER-β, BDNF and p-CREB expression in the hippocampus of immature rat offspring, while it had no influence on the levels of these proteins in the mature animals. Conclusions: Perinatal exposure of DBP could induce neurotoxicity in immature offspring rats through regulation of AROM, ER-β, BDNF and p-CREB expression, while it has no influence on mature offspring animals.     

Incubation periods and histopathological changes in mice injected stereotaxically in different brain areas with the 87V scrapie strain

Summary After stereotaxic injection into five different brain areas (cortex, caudate nucleus, substantia nigra, thalamus and cerebellum) of IM mice with the 87V scrapie strain, the cerebellum had the shortes incubation period. The vacuolation pattern was similar regardless of the area injected with extensive vacuolation in the thalamus, mesencephalon and hypothalamus. The pattern of amyloid plaques differed markedly depending on the area injected. In particular, no plaques were seen anywhere in the brain after injection into intact cerebellum, whereas injection into the four cerebral areas yielded plaques in the forebrain but not in the cerebellum. The incubation period after injection into bisected cerebella was much longer than after injection into intact cerebella. Mice injected on one side of bisected cerebellum had amyloid plaques in the forebrain but not in the cerebellum. There is a discussion of the finding that, although no plaques and virtually no vacuolation were seen in the cerebellum, the shortest incubation period occurred after injection into intact cerebellum.Supported in part by NIH grants NS 21349-06 and NS 25308-02     

Behavioural and histopathological alterations in mice with cerebral malaria

Different features of sensorimotor function and behaviour were studied in murine cerebral malaria (CM) and malaria without cerebral involvement (non-CM) applying the primary screen of the SHIRPA protocol. Histopathological analysis of distinct brain regions was performed and the relative size of haemorrhages and plugging of blood cells to brain vasculature was analysed. Animals suffering from CM develop a wide range of behavioural and functional alterations in the progressive course of the disease with a statistically significant impairment in all functional categories assessed 36 h prior to death when compared with control animals. Early functional indicators of cerebral phenotype are impairments in reflex and sensory system and in neuropsychiatric state. Deterioration in function is paralleled by the degree of histopathological changes with a statistically significant correlation between the SHIRPA score of CM animals and the mean size of brain haemorrhage. Furthermore, image analysis yielded that the relative area of the brain lesions was significantly larger in the forebrain and brainstem compared with the other regions of interest. Our results indicate that assessment of sensory and motor tasks by the SHIRPA primary screen is appropriate for the early in vivo discrimination of cerebral involvement in experimental murine malaria. Our findings also suggest a correlation between the degree of functional impairment and the size of the brain lesions as indicated by parenchymal haemorrhage. Applying the SHIRPA protocol in the functional characterization of animals suffering from CM might prove useful in the preclinical assessment of new antimalarial and potential neuroprotective therapies.     

Subclinical histopathological changes in the oculomotor nerve in diabetes mellitus.

To provide reference values for the oculomotor nerve, for example, fascicular area and myelinated fiber (MF) number and size distribution, in pathological states, and to determine whether oculomotor nerves from diabetic patients without a history of oculomotor palsy have subclinical structural alterations, the morphometric features of 15 control subjects and eight diabetic patients were evaluated at a proximal and a distal level. On average the control nerves had a fascicular area of 2.7 mm2, 22,311 MF, and a bimodal diameter distribution with peaks at approximately 5 to 6 and 10 to 11 microns and a range of 2 to 20 microns. In proximal nerve, glial bundles were found in one-half of the subjects and patients, with a peak of MF sizes that differed from fibers in nonglial areas. The nerves from diabetic patients departed from the nerves from control subjects in the following two respects: changed size distribution, suggesting atrophy of fibers, and microfasciculation of edge fibers in parts of the fascicles in one-half of the patients. We conclude that glial bundles are probably a normal variant. Microfasciculation occurred much more frequently in nerves from diabetic patients and may represent subclinical injury of unknown cause. The alteration in size distribution may reflect the diabetic state and is not necessarily a precursor to diabetic ophthalmoplegia.     

Histological and hormonal testicular changes in spinal cord patients

Hormonal dosages and testicular biopsy are discussed in paraplegic patients of several neurological levels. There are no absolute biological criteria but histology showed slowing of spermatogenesis.     

Immunization with cholinergic cell bodies induces histopathological changes in rat brains

We have previously shown that sera from patients with Alzheimer’s disease (AD) contain antibodies to the cell bodies (perikarya; PK) of purely cholinergicTorpedo neurons, and that repeated immunization of rats with this neuronal preparation for over a year induces learning and memory impairments. In the present study, we examined the brain morphology of cholinergic PK immunized rats relative to controls. Immunohistochemical studies of the brains of these rats revealed the accumulation of IgG in specific areas, such as, the hippocampus. Parallel histochemical studies demonstrated significant changes in the hippocampus, and in white matter areas. They included large vacuoles and necrotic nuclei in the granular layer of the dentate gyrus, tangle-like appearance in some pyramidal neurons of the hippocampus, and vacuolar degeneration accompanied by oligodendroglia hypertrophy in white matter tracts, such as, the corpus callosum and fimbria. In contrast, immunization withTorpedo cholinergic nerve terminals, that has no cognitive effects on the rat, also did not induce brain morphological changes. These findings suggest that the learning and memory deficits induced by immunizing rats with cholinergic PK are related to the observed brain morphological changes, and support the hypothesis that the antibodies to cholinergic neurons found in the sera of AD patients may play a role in neuronal degeneration in this disease.     

"Myopathic" changes in chorea-acanthocytosis. Clinical and histopathological studies

Four cases of chorea-acanthocytosis were studied with special reference to muscular changes. All the cases showed the clinical stigmata of oro-linguo-facial dyskinesia with tongue biting, mild neurogenic muscular involvement and acanthocytosis. Serum creatine kinase (CK) was persistently elevated, showing MM type isozyme predominance. Histopathological studies of the peroneus brevis muscle showed prominent small group atrophy, increase of small fibers on diameter analysis, frequent angulated fibers, and angulated fibers with increased acid phosphatase activity. These findings are compatible with chronic denervation. However, central nucleation (approximately 10%) and fiber splitting (2-8%) were also found in all cases. These are compatible with myopathic changes. No correlation of these "myopathic" changes and serum CK levels was found. The "myopathic" findings are probably secondary to chronic denervation.     

Muscle membrane-skeleton protein changes and histopathological characterization of muscle-eye-brain disease

Muscle-eye-brain disease belongs to congenital muscular dystrophies with central nervous system abnormalities. The etiology of MEB is still unknown, but abnormal immunoreactivity for laminin-2 has been reported. To evaluate disease progression in muscle tissue, 32 biopsy specimens from 17 muscle-eye-brain patients were analysed. The samples of four patients were studied by immunohistochemical techniques and by quantitative Western blotting. The samples showed a great variation in the muscle pathology. Regenerative fibers and mild fiber size variation were present in over 60%. At infancy, necrotic and regenerative fibers were common, while fat infiltration was the most prominent finding in the age group over five years. In quantitative studies, the amount of laminin alpha 2 chain was clearly reduced to 10-20% of normal. In contrast, laminin beta 2 chain was overexpressed in the Western blotting studies. These findings may reflect a yet unidentified primary disturbance in the basement membrane composition and function.