睾丸间质细胞——研究自体吞噬的一种正常细胞模型

细胞在生理状态下自体吞噬出现的频率很低,很难用正常细胞来研究自体吞噬活动,一般都通过诱导自体吞噬来获得有关自体吞噬活动的资料。本实验观察了肝、肾、睾丸等组织的32种细胞,发现睾丸间质细胞中自体吞噬出现频率远远高于其他细胞,平均每100个细胞切面中可以看到25个自噬小体,从而为研究自体吞噬的过程和机理提供了一个正常细胞模型。本实验还观察到睾丸间质细胞的自体吞噬活动可分为前自噬小体、早期自噬小体和晚期自噬小体三个阶段,是一个连续的过程。前自噬小体和早期自噬小体不含溶酶体酶,只有在自噬小体与溶酶体接触后,才从后者获取溶酶体酶并将其内容物消化分解,成为晚期自噬小体。由自体吞噬所产生的残余体并不在睾丸间质细胞内积聚,而是通过胞吐作用排出细胞外。     

卵形鲳鲹血细胞发生的观察

对卵形鲳鲹(Trachinotus ovatus)肝脏、脾脏、头肾和体肾4种组织器官内各种血细胞的发生和外周血液的血细胞分类组成进行了观察。结果表明:卵形鲳鲹的红细胞主要在脾脏、体肾和头肾中发生;淋巴细胞主要在体肾、头肾和脾脏中发生;粒细胞主要在头肾和体肾中发生;单核细胞在肝脏、头肾和脾脏中均有发生;而血栓细胞的发生在这4种组织中均未观察到。卵形鲳鲹在外周血液中的白细胞包括淋巴细胞、血栓细胞、嗜中性粒细胞和单核细胞。在这几种白细胞中,单核细胞数量最少,很难观察到,数量少于1%;嗜中性粒细胞稍多,有(9.25±5.64)%;血栓细胞占(38.23±21.86)%;淋巴细胞最多,达(52.52±21.65)%。     

鲤疱疹Ⅱ型病毒(CyHV-2)感染对异育银鲫(Carassius auratus gibelio)组织器官的损伤作用

为分析感染Cy HV-2异育银鲫(Carassius auratus gibelio)体内不同器官组织结构和功能的损伤,采集健康和患病异育银鲫头肾、脾脏、体肾、肠道、肝胰脏、鳃各组织样本,通过石蜡切片,HE染色对各器官组织进行组织病理学观察并对肠道和鳃进行扫描电镜观察。病鱼主要表现为,食欲减弱、离群独游、全身发黑、背鳍、尾鳍末端发白、眼球突出、肌肉充血,鳃呈鲜红色。在鳃丝末端由红色斑块,经解剖有红色腹水流出,组织器官广泛性损伤。组织病理学变化为,头肾和脾脏细胞溶解、免疫细胞侵润、免疫和造血系统严重破坏;体肾中肾小球萎缩、肾小管上皮细胞脱落、肾间质出现空泡、免疫细胞侵润;肠道绒毛增生、绒毛和微绒毛长度显著减少、粘膜下层显著增厚;肝胰脏中肝细胞出现溶解、中央静脉受损;鳃中毛细血管破坏、血管破裂或扩大、红细胞溢出、鳃小片基部细胞显著减少。结果表明,病毒对异育银鲫具有明显的致病性,可导致各器官组织发生病变,同时机体处于缺血、缺氧、营养不足、废物积累的内环境,这进一步加剧对异育银鲫主要的器官组织的损伤。     

雷公藤甲素诱导小鼠急性肝损伤的形态学研究

目的:观察雷公藤甲素诱导小鼠急性肝损伤的形态学特征,为进一步研究雷公藤甲素肝毒性的病理特点和毒理机制提供基础。方法:昆明种小鼠以雷公藤甲素LD50剂量(0.8 mg/kg)水溶液灌胃,分别于给药12 h及24 h后取肝组织,制备石蜡切片、冰冻切片,行常规HE染色、PCNA染色、TUNEL染色及光镜观察。部分肝组织经戊二醛固定、制备超薄切片,行透射电镜下观察。PCNA及TUNEL染色结果采用图像分析软件进行定量分析及统计学处理。结果:0.8 mg/kg雷公藤甲素灌胃后12 h即可诱导肝组织炎细胞浸润、结构破坏、肝细胞坏死及代偿性增生。透射电镜下可见肝细胞内细胞骨架结构异常、细胞器大量脱落、自噬体明显增多。PCNA及TUNEL染色结果表明,雷公藤甲素可诱导肝细胞出现显著的增殖及凋亡。结论:雷公藤甲素可诱导肝组织炎性反应发生,同时伴随肝细胞凋亡、坏死及代偿性增生。推测肝细胞自噬性凋亡是雷公藤甲素诱导急性肝损伤的关键病理环节。     

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织的实验研究

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织,探索建立人-小鼠嵌合肝模型方法。15只SCID小鼠,以四氯化碳(CCL4)制备急性肝损伤模型,24h后行2/3肝切除,然后分为三个实验组细胞移植组(7只)、阴性对照组(3只)及空白对照组(5只);将人脐血单个核细胞悬液注入细胞移植组小鼠脾脏内,阴性对照组小鼠脾脏内注入等量磷酸盐缓冲液(PBS),空白对照组不注射细胞悬液和PBS。术后7d、14d及21d取小鼠肝组织观察病理变化、检测人白蛋白(ALB)及细胞角蛋白19(CK19),同时检测小鼠血清及肝组织匀浆中人ALB含量。全部小鼠表现出急性肝损伤组织学特征;细胞移植组小鼠术后7d、14d、21d肝组织内均见大量人ALB及CK19阳性表达细胞,血清及肝组织匀浆可检测出人ALB;阴性对照组小鼠肝组织未见人ALB及CK19阳性表达,血清及肝组织匀浆中未检测出人ALB。人脐血单个核细胞在部分肝切除的急性肝损伤小鼠肝组织内可大量分化为人肝细胞及胆管细胞,在建立模型方面已取得关键突破。     

全反式维甲酸调控自噬促进肝祖细胞成熟分化的作用研究

该研究探讨全反式维甲酸(all-trans retinoic acid,ATRA)体外诱导小鼠胚胎肝祖细胞HP14-19细胞成熟分化及ATRA对细胞自噬水平的调控作用。应用1μmol/L ATRA处理小鼠胚胎肝祖细胞HP14-19细胞,不同时间点进行荧光素酶报告基因检测ALB-Gluc活性,Real-time PCR检测肝细胞相关标志基因的表达,吲哚菁绿(indocyanine green,ICG)及过碘酸–希夫(periodicacid-schiff,PAS)染色检测细胞的成熟功能;透射电镜观察自噬体及细胞连接,ptf LC3质粒转染细胞,激光共聚焦显微镜观察自噬流,Western blot检测自噬相关标志蛋白质水平等综合分析自噬的变化情况。结果显示,与对照组相比,ATRA可显著增强HP14-19细胞ALB-Gluc活性,抑制肝前体细胞标志DLK和AFP的表达,促进成熟肝细胞标志ALB、CK18、TAT和Apo B的表达,ICG及PAS染色阳性细胞数显著增多(P0.05);透射电镜结果可见ATRA诱导组出现大量自噬体和自噬溶酶体,同时细胞间紧密连接增多,并且自噬相关标志蛋白质Beclin1、LC3-II、RAB7水平增高,P62水平无显著变化,LC3-II/LC3-I的比值明显增加(P0.05);激光共聚焦显微镜可见ATRA组细胞质内黄色斑点的自噬体及红色斑点的自噬溶酶体均较对照组明显增多,自噬抑制剂3-MA和Baflomycin可抑制ATRA诱导的HP14-19细胞的ICG摄取和糖原合成功能。综上所述,ATRA可能通过调节细胞自噬水平有效诱导小鼠胚胎肝祖细胞的分化成熟。     

趋化因子ENA-78与肾脏疾病

体外实验中,各种肾脏细胞都能在特定刺激下表达趋化因子及受体。动物模型和人类肾脏疾病的肾组织中,炎症细胞浸润同时出现趋化因子及受体表达增多。ENA-78是一种来源广泛、生物功能多样的趋化因子,通过与其受体相互作用,引起中性粒细胞等白细胞的趋化、活化,参与肾脏疾病的发生、发展。     

自噬在非酒精性脂肪肝合并糖尿病中的作用

在一系列应激状态下,自噬会作为一种适应性免疫应答反应出现,是细胞的自我保护机制之一。研究表明,在非酒精性脂肪肝病的早期阶段,自噬增加有助于肝内脂质降解,缓解病情;而在后期阶段,自噬增加却会加剧病情发展。胰岛素抵抗可能会促使非酒精性肝病发生糖尿病,β细胞功能缺陷和胰岛素抵抗是2型糖尿病发病的两种最主要病理生理机制。多项研究证实,自噬增加能提高胰岛素敏感性,对维持胰岛β细胞的结构、数量以及功能也有重要作用。旨在阐述自噬在非酒精性脂肪肝病以及非酒精性脂肪肝病合并糖尿病治疗中的作用。     

鲜切香菇贮藏过程中质构变化与自溶自噬分析

旨在明确鲜切香菇经过机械损伤后发生的应激生理以及自溶进程中细胞质构的变化。以香菇0912子实体新鲜切片为材料,研究低温和室温贮藏条件下香菇切片生理生化指标的变化趋势,利用显微技术研究老化组织中细胞结构的变化。结果显示低温贮藏条件有利于减缓鲜切香菇褐变、软化、蒸腾失重及抗氧化活性的下降。微观结构研究显示鲜切香菇经过机械损伤后激发了自溶自噬机制,细胞内出现胞壁断裂、胞膜消融、自噬体吞噬细胞器、大量DNA断裂、细胞核消失及细胞解体等现象。鲜切香菇切片在损伤后发生自溶自噬现象,微观组织结构的破坏,是菇体劣变的重要原因之一,在外界贮藏环境的共同作用下加速腐败的进程。     

鳗鲡爱德华氏菌病的组织病理学研究

鳗鲡爱德华氏菌病病变性质为化脓性炎症。症理分型主要有两种：化脓性肝炎型、混合型。较早发生病变部位主要在肝脏,其化脓性病杜按病程分为初、中、晚期。肾脏、脾脏、胃粘膜、体表出现散在的脓肿、脓性溃疡、炎性血栓及血斑。此外,肝脏、肾脏实质细胞均出现变性和坏列、血管充血、出血等。本文详细报告了肝脏、脾脏、肾脏的超微结构变化并初步讨论了细胞、组织损伤的过程及细胞病理学、超微病理学意义。     

鳜鱼病毒病原的检出及组织病理分析

经对患暴发性传染病的鳜组织及病变组织超微切片的电镜观察,在细胞质,核及间隙中都有病毒颗粒,对该病毒进行了分离提纯,这是一种直径约为２８０ｎｍ球形病毒,将粗提病毒液接种到培养细胞中,会引起细胞病变;病毒通过人工感染健康鳜,会引起鳜发病和死亡,对患病獗进行解剖和组织病理观察,表明病鳜的５种器官都发生了病变,研究结果表明分离的鳜病毒（ＳｉｎｉｐｅｒｃａｃｈｕａｔｓｉＶｉｒｕｓ,ＳＣＶ）是引起鳜流４行的病     

眼斑拟石首鱼虹彩病毒病的诊断

对广东省珠海市每年6—10月池塘养殖的眼斑拟石首鱼(Sciaenop socellatus)暴发不明原因疾病进行了研究。通过对该病的流行病学调查,结合症状观察、病原分离鉴定、病理学变化,结果显示:该病具有明显的传染性,解剖病鱼可见鳃丝贫血、脾脏和肾脏肿大、出血,通过镜检未检测到大量的寄生虫;从发病鱼的肝、脾、肾脏和肠中未分离到致病菌;在病鱼肝、脾和肾组织病理切片中观察到有大量肿大细胞;通过使用OIE 推荐的虹彩病毒特异性引物进行PCR扩增出570 bp左右的目的片段,MCP基因序列比对显示扩增获得的基因序列与真鲷虹彩病毒(RSIV)的基因序列同源性高达99.2%。综合以上结果表明导致眼斑拟石首鱼发病死亡的原因是感染了细胞肿大属虹彩病毒而引起的虹彩病毒病。     

黄鳝体表溃疡病病原菌的分离与鉴定

研究首次报道从患体表溃疡病黄鳝的肝脏、肾脏、脾脏等分离到1株细菌, 经动物回归试验鉴定其为病原菌。形态学观察、生理生化试验和16S rRNA基因鉴定, 结果证实该病原菌株为温和气单胞菌(Aeromonas sobria)。通过致病性试验证明其对鲤鱼和泥鳅均有致病力;药敏试验结果表明:分离菌株对头孢氨噻肟、头孢曲松钠、丙氟哌酸、洛美沙星、甲氧苄啶、氟哌酸、壮观霉素等高度敏感,而对氨苄青霉素、乙酰螺旋霉素等药物不敏感。       

长吻鮠血细胞发生的研究

用Wright-Giemsa和PAS染色对长吻鮠头肾、肾脏、脾脏、肝脏等器官组织的涂片、印片染色观察发现,头肾、肾脏和脾脏是其主要造血器官。红细胞、粒细胞和淋巴细胞主要在肾脏和头肾中发生,其次是脾脏。单核细胞则主要在肾脏和脾脏中发生,头肾中也有少量单核细胞产生。肝脏中无原始型血细胞,可能不是其造血器官。红细胞的发育经历四个阶段,其胞体体积经历了由大到小,由小到大再变小的"两大两小"发育过程;粒细胞的发育经历五个阶段,其胞体体积均由大变小,双叶或多叶核的粒细胞可能是衰老的粒细胞亦即核的分叶是粒细胞衰老的标志;淋巴细胞和单核细胞的发育各经历了三个阶段,两者发育成熟过程中胞体体积均由大变小。巨噬细胞由单核细胞发育而来。原血细胞和部分早期幼稚血细胞可以进行有丝分裂,部分成熟红细胞和血栓细胞可以进行直接分裂。红细胞在整个发育过程中,PAS反应均呈阴性,各类白细胞的发育过程中,PAS反应由阴性到阳性并逐渐增强,这显示随着白细胞的逐渐发育成熟,细胞内糖原物质含量逐渐增多。     

Non-specific cellular responses of rainbow trout to Vibrio anguillarum and its extracellular products (ECPs)

We have studied the early inflammatory response induced by Vibrio anguillarum and by its extracellular products (ECPs) in rainbow trout after intraperitoneal injection. The results showed a very similar inflammatory response which included leucopenia, mainly due to lymphopenia, neutrophilia and an increase in the number of circulating monocytes. Melanomacrophages as well as immature leucocytes were frequently observed circulating in the blood of injected rainbow trout. Monocytes often contain phagocytosed bacteria and other, altered cells including erythrocytes and leucocytes. However, neutrophils only occasionally phagocytosed bacteria. Many circulating leucocytes showed important structural alterations. Neutrophils of trout injected with bacteria and ECPs also showed stronger PAS-staining than those of control trout as well as Döhle bodies and swollen granules. A marked vasodilatation was observed in the kidney and spleen which was coincidental with a mobilization of eosinophilic granular cells and an hypertrophy of sinusoidal endothelial cells showing an increase in the number of cytoplasmic granules. An increase in the number of macrophages and melanomacrophages in the kidney and spleen as well as oedema and leucocyte infiltration in the liver and gills were also noted.     

Peripheral blood and head kidney leucocyte populations during out-of-season (0+) parr-smolt transformation and seawater transfer of Atlantic salmon (Salmo salar L.)

Using monoclonal antibodies (MAb) and flow cytometry, Atlantic salmon neutrophils and Ig+ cells in blood and head kidney were studied in under-yearling out-of-season (0+) smolts, and 2 and 4 weeks after transfer to seawater. The parr-smolt transformation was induced using a phase advanced simulated natural photoperiod regime, and sampling of four fish was performed at regular intervals, starting on the date of the photoperiod initiation. During the freshwater period the proportion of neutrophils in the head kidney leucocytes (HKL) remained quite stable and only gradual changes in Ig+ cells were observed. In the peripheral blood leucocytes (PBL), the proportion of neutrophils markedly increased during the last month prior to seawater transfer. The most notable changes in the proportions of MAb+ leucocytes were observed in PBL after seawater transfer, with a significant increase in Ig+ cells and a significant decrease in neutrophils after two weeks in seawater. In the freshwater samples, although there were fluctuations, a decrease in the numbers of total leucocytes per millilitre blood and per gram head kidney during parr-smolt transformation was observed. The number of MAb+ cells in blood appeared to be relatively stable, while the number in head kidney tended to decrease. Following seawater transfer, the numbers of total and MAb+ leucocytes in both blood and head kidney increased markedly. The results suggest that changes in both distribution and numbers of leucocytes in peripheral blood and head kidney take place during parr-smolt transformation, and that marked changes are associated with seawater transfer. Some mechanisms possibly involved are indicated.     

Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan

A herpesvirus has been isolated for the first time from a population of European eels Anguilla anguilla cultured in a recirculated system in Taiwan. Syncytia formation was detected in EP-1 (eel epidermis) cell cultures inoculated with cell-free homogenates prepared from both integument and visceral organs of moribund fish. Inoculation of homogenates onto EK (eel kidney) cell cultures induced giant cell formation. Subsequent passages produced a consistent and progressive cytopathic effect (CPE) in cell cultures. In this study, EP-1 cell cultures infected with EEHV (European eel herpesvirus) were examined using an electron microscope. Numerous nucleocapsids of about 100 nm in diameter were found within the nucleus of infected cells, whereas enveloped particles were observed within the cytoplasm. The mature viral particle, about 235 nm in diameter, had an electron-dense core with a hexagonal nucleocapsid surrounded by a coarse capsule. Histopathological examination of moribund fish showed epithelial hyperplasia with intracytoplasmic metabolic inclusions in the skin. Macrophage aggregates were found in liver, spleen, and kidney. A pair of primers designed from channel catfish virus and salmonid herpesvirus 1 was used in a polymerase chain reaction. A 402 bp fragment was amplified and cloned from genomic DNA of EEHV. The nucleotide homology was 99% (298 of 300) with DNA polymerase of eel herpesvirus (anguillid herpesvirus). EEHV nucleic acids were detected within melanomacrophages in the skin, liver, spleen and kidney by in situ hybridization (ISH).     

The effects of splenectomy and subsequent starvation on the storage of haemosiderin by the melano-macrophages of rainbow trout Salmo gairdneri Richardson

In clinically normal, cachectic and diseased teleost fish, haemosiderin is observed mostly in the melano-macrophage centres of the spleen and to a much lesser extent in the centres of the kidney and liver. When rainbow trout that had been splenectomised were starved, accumulation of haemosiderin was diverted to the kidney melano-macrophages.     

军曹鱼血液指标及血细胞发生的观察

测定军曹鱼的血液指标,红细胞密度为2．69±0．86×106个/mm3,白细胞密度为1．50±0．09×104个/mm3;血 红蛋白含量为7．42±0．22g/L,红细胞渗透脆性为0．43±0．07g％,红细胞沉降速率为1．18±0．46mm/h。观察军曹鱼 外周血液涂片,可区分出红细胞、血栓细胞、淋巴细胞、嗜中性粒细胞和单核细胞,但没有发现嗜酸性粒细胞和嗜碱 性粒细胞。在外周血液涂片观察中还发现了较多未成熟的红细胞和嗜中性粒细胞以及少量正在分裂的红细胞。 血栓细胞、淋巴细胞、嗜中性粒细胞和单核细胞在白细胞中所占比例分别为61．20±6．30％,16．60±3．28％,16．00± 3．61％和6．20±3．90％。对肝脏、脾脏、头肾和中肾等四种造血组织进行了涂片观察,军曹鱼的血细胞主要在头肾 和肾脏产生;脾脏是军曹鱼粒细胞发生的另一个场所,而肝脏也具有产生粒细胞和淋巴细胞的作用。       

Isolation of leucocytes from the anterior kidney and spleen of rainbow trout in a self-generating density gradient

Separation of leucocytes from tissues and enrichment of specific types of leucocytes are essential first steps in studies of leucocyte function. We describe a simple and rapid method for separating and enriching leucocytes from the anterior kidney and spleen of rainbow trout. Leucocytes were separated on self-generating density gradients of Sepracell-MN, a colloidal silica-based medium. Recovery of leucocytes from the gradients was near 100% and was not affected by procedural variables such as cell suspension: Sepracell-MN ratio (separation ratio), initial temperature, centrifugation time, or number of cells per gradient. Two bands of leucocytes formed at separation ratios of 1 : 1, 1:1.5, 1:2 and 1 : 3. Recovery of selected leucocyte types could be maximized, and contamination by other cell types reduced, by selection of the appropriate separation ratio and cell band. Recovered leucocytes were responsive in assays of functional capability (adherence, phagocytosis, superoxide anion production). Leucocyte populations that adhered to glass were enriched for macrophages, but some neutrophils and lymphocytes (paricularly spleen lymphocytes) were also adherent. The most abundant leucocytes in the anterior kidney and spleen of the experimental fish were lymphocytes (respectively, 47 and 88%, including thrombocytes), neutrophils (35 and 7%), and macrophages (11 and 3%).