丙烯硫脲对日本血吸虫酚酶抑制作用的组织化学研究

目的 通过组织化学方法研究丙烯硫脲对小鼠体内、体外日本血吸虫成虫酚酶(phenol oxidase，PO)活性的抑制作用。方法 将42d日本血吸虫活成虫置于含25mmol／L邻苯二酚的磷酸盐缓冲液(pH6．8)中，28℃下孵育30min，取出虫体置载玻片上，加2滴含0．1％戊巴比妥钠的PBS溶液，置Leica荧光显微镜下，分别在普通光照和紫外光激发下观察酚酶在虫体内的组织学定位。体外抑制实验时，加底物前先将成虫置于含5mmol／L丙烯硫脲的PBS溶液中，28℃下孵育10min；体内抑制实验时，在小鼠感染日本血吸虫尾蚴20d后，按300mg／kg隔日1次腹腔注射丙烯硫脲。结果 体外抑制实验组雌虫卵黄腺及子宫内虫卵卵壳和雄虫体壁表层的桔黄色荧光或赭褐色颜色反应均基本消失；体内抑制实验组雌虫子宫内未见虫卵或虫卵样物质，仅见大量泥沙样卵黄颗粒，其荧光或颜色反应也基本消失。结论 通过一种更灵敏的显示酚酶活性的组织学定位方法，观察到不仅雌虫含有酚酶，雄虫也有酚酶活性。证明丙烯硫脲对血吸虫成虫的酚酶活性有明显的抑制作用，从而可使小鼠体内血吸虫雌虫虫卵的形成被完全抑制。     

日本血吸虫酚氧化酶的电泳及酶活性的研究

目的：观察日本血吸虫成虫酚氧化酶（phenol oxidase,PO)的酶谱及其活性，方法：将42d活成虫置含0．05％戊巴比妥钠的RPMI1640培养基中23℃孵育8h后，分别收集雌，雄成虫，匀浆，超声粉碎，高速离心取上清（含PO），再进行聚丙烯酰胺凝胶电泳（PAGE）及酶染色后其酶谱；同时在紫外分光光度计下检测前后2个时点的A488(A1,A2），PO活性相对值＝（A2－A1）／(min.mg样品蛋白），其活性值用每分钟每毫克蛋白A488变化值表示。设立酚酶抑制剂（丙烯式硫脲）组，在该组成虫匀浆上清中预先加入丙烯基硫脲以观察其对酚氧化酶活性的抑制效果。结果：雌虫及雄虫均表现为迁移率相同的一条主带，但雄虫的酶带显示反应比雌虫弱。日本血吸虫雌虫及雄虫的酚氧化酶相对活性分别为0.165min^1.mg^-1和0．0805min^1.mg^-1；加入酚酶抑制剂后，酶活性被明显抑制，终浓度为5mmol的丙烯基硫脲对成虫酚酶活性的抑制率为85．03％，当丙烯基硫脲终浓度达25mmol时，酚酶活性被安全抑制，结论：不仅雌性成虫含有日本血吸虫酚氧化酶，而且雄性成虫也含有少量的酚氧化酶且两者酶分子相同，雄性成虫含有的少量酚氧化酶的生理意义有待于进一步研究。     

酚酶活性抑制对小鼠体内日本血吸虫产卵的影响

目的观察小鼠体内日本血吸虫雌虫酚酶活性被抑制后对其产卵的影响.方法小鼠感染日本血吸虫尾蚴20d后,按500mg/(kg*d)腹腔注射酚酶抑制剂-丙烯基硫脲,感染后第45d剖杀小鼠,观察感染小鼠体内日本血吸虫雌虫子宫及小鼠肝脏中虫卵的数量及形态结构.结果与感染对照组相比,实验组小鼠雌虫子宫内虫卵数及每克肝组织虫卵数均明显减少.虫卵形态表现异常,主要是缺失硬化的卵壳结构.结论日本血吸虫雌虫酚酶活性被完全抑制后,雌虫不能产卵;不完全抑制则只能产生无正常卵壳的异常虫卵,证明具有活性的酚酶是虫卵正常形成过程中必不可少的成分,提示血吸虫酚酶有可能作为抗血吸虫病的一个新靶位.     

日本血吸虫各期虫体的酶联免疫电转移印迹分析及抗原定位研究

本文采用酶联免疫电转移印迹技术（EITB）分析、比较日本血吸虫不同发育时期虫体特定的组分蛋白分子、雌虫、雄虫和虫卵抗原分别与相应的雌、雄虫和虫卵免疫血清反应呈现17、20和8条蛋白带,这三种抗原与其它不同时期虫体免疫血清反应,三者间及彼此间均出现交叉反应,而雌、雄虫和虫卵抗原与其它寄生虫感染血清作用没发现有叉及反应。应用间接荧光抗体试验（IFA）和免疫酶染色试验（IES）对日本血吸虫抗原进行定位研究,其结果相似。血吸虫主要抗原物质来源于成虫表皮、肠上皮和卵内的毛蚴.     

日本血吸虫造卵物质组织化学动态观察

日本血吸虫造卵物质组织化学动态观察陈子宸，林英娇我国何毅勋［１，２］已阐明了日本血吸虫的发育和虫卵形成的生理，并以组织化学方法证实雌虫生殖细胞中的卵黄腺细胞中的卵黄颗粒球是制造卵壳物质的前身物，包含蛋白质、酚酶和酚类物质［１，２］。本研究以组织化学方...     

日本血吸虫表皮生长因子受体基因的鉴定和功能研究

目的鉴定日本血吸虫表皮生长因子受体基因(SjEGFR),并阐明其在日本血吸虫生长和生殖发育中的作用。方法通过5’-RACE和3’-RACE扩增、测序获取SjEGFR基因全长片段。采用荧光定量PCR(qPCR)技术检测该基因在日本血吸虫不同发育阶段(16、18、20、22、24、26 d)表达情况。使用整条虫体原位杂交法对感染24 d的日本血吸虫雌、雄虫SjEGFR基因转录本进行定位。通过体内持续RNA干扰(RNAi)技术特异性敲低SjEGFR基因,待虫体发育至30 d收集虫体。统计分析雌、雄虫回收数量以及虫体长度,通过明矾卡红染色观察虫体生长发育情况。结果首次鉴定了SjEGFR基因,其结构具有一个保守的酪氨酸激酶位点。整条虫体原位杂交定位显示该基因转录本在虫体各处均有表达,在雌、雄虫肠道部位呈现出明显着色的高表达。体内RNAi显示,SjEGFR基因表达被特异性敲低后,雌、雄虫生长受阻。RNAi组雌虫子宫内无虫卵,肠道内无色素沉积、卵黄腺发育迟滞、卵巢发育受阻;雄虫呈现睾丸个数减少、体积变小,出现了更多未成熟的精母细胞。结论特异性敲低SjEGFR基因表达可影响日本血吸虫生长和生殖发育,阻滞虫卵产生。     

吡喹酮和硝硫氰胺对日本血吸虫酚酶影响的研究

本文首次报道用日本血吸虫酚酶分子生物化学、酚酶组织化学和雌虫形态学三者相结合的方法,对日本血吸虫雌虫酚酶的变化过程进行动态观察.实验发现:经吡喹酮和硝硫氰胺口服1剂治疗后,兔体内日本血吸虫酚酶同功酶的含量逐渐减少,与组织化学定位观察酚酶反应的变化同步,雌虫子宫虫卵数也逐渐减少,出现变形虫卵或只见卵黄腺颗粒或无虫卵.实验充分表明,吡喹酮和硝硫氰胺治疗后,可进行性、不可逆地破坏雌虫酚酶,使雌虫不能形成正常虫卵或抑制雌虫产卵.说明上述药物的杀虫机理与破坏雌虫酚酶有密切关系.     

日本因吸虫各期虫体的酶联免疫电转移印迹分析及抗原定位研究

本文采用酶联免疫电转移印迹技术（ＥＩＴＢ）分析、比较日本血吸虫不同发育时期虫体特定的组分蛋白分子、雌虫、雄虫和虫卵抗原分别与相应的雌、雄虫和虫卵免疫血清反应呈现１７、２０和８条蛋白带，这三种抗原与其它不同时期虫体免疫血清反应，三者间及彼此间均出现交叉反应，而雌、雄虫和虫卵抗原与其它寄生虫感染血清作用没发现有叉及反应。应用间接荧光抗体试验（ＩＦＡ）和免疫酶染色试验（ＩＥＳ）对日本血吸虫抗的进行定位研     

湖北钉螺体内酚氧化酶及其组织学定位

目的 了解湖北钉螺体内是否存在酚氧化酶（P0），并观察其在钉螺体内分布情况。方法 酶组织化学染色法：湖北钉螺成螺（雄螺和雌螺）采自芜湖江滩，解剖后，将软体组织置含25mmol／L邻苯二酚的磷酸盐缓冲液（pH6．8）中，37℃孵育，体视显微镜下观察PO与底物反应后的产物在螺体内的分布情况。荧光组织化学染色法：解剖后的湖北钉螺成螺软体组织置含25mmol／L邻苯二酚的磷酸盐缓冲液（pH6．8）中，37℃孵育30min，吸去邻苯二酚，加入0．05％戊巴比妥钠溶液，5min后置荧光显微镜下观察PO催化产生的荧光物质在螺体内的分布情况。结果 酶组织化学染色法显示钉螺的肝脏初为淡浅灰色，5min时呈现浅灰色，15min时呈现灰色，30min时呈现黑色。荧光组织化学染色法显示钉螺的肝脏表面呈现荧光反应。结论 湖北钉螺体内存在PO，雌、雄螺体内的PO均定位于肝脏。     

枞酸钠对体外培养日本血吸虫成虫的杀虫效果

目的观察枞酸钠对体外培养的日本血吸虫雄性和雌性成虫的杀灭效果。方法小鼠感染日本血吸虫尾蚴5周后肝门静脉灌注法收集雌、雄成虫,体外培养于DMEM培养液中,加入不同浓度枞酸钠连续培养3d,观察虫体死亡及活力降低情况,并设空白对照组。虫体盐酸卡红染色,观察其损伤情况。采用Bradford标准曲线法测定药物对虫体蛋白质含量的影响。结果在含不同浓度枞酸钠的DMEM培养液中,雄虫及雌虫死亡率与活力降低率显著高于对照组;雄性成虫对枞酸钠的敏感性高于雌性成虫（P均〈0.05）。染色结果显示,雄虫虫体肠管膨大,均出现黑色或褐色条带或斑点;雌虫肠管内容物分布不均,部分虫体卵巢形状不规则,着色不均匀。蛋白质含量测定结果显示雄性与雌性虫体的蛋白质含量均较对照组显著降低（P均〈0.05）。结论枞酸钠具有体外杀灭日本血吸虫成虫的作用,其可能会影响日本血吸虫成虫的蛋白质代谢。     

吡喹酮治疗后华支睾吸虫的组织化学变化

华支睾吸虫实验感染的大鼠经吡喹酮治疗后,虫体的组织化学变化为:糖原在治疗后1h开始减少,24h明显减少,48h大部消失。治疗后1h虫体实质组织内蛋白质开始增加,24h实质组织、生殖器官内的蛋白质均明显增加。RNA在治疗后1h开始减少,24h逐渐减少,48h部分消失。琥珀酸脱氢酶、苹果酸脱氨酶和钙-三磷酸腺苷酶在治疗后1h开始弥散增加,24h明显增加。葡萄糖-6-磷酸脱氢酶在治疗后48h出现弥散性增加。但DNA、脂肪、碱性磷酸酶、酸性磷酸酶、单胺氧化酶和酚酶均未见明显变化。     

药物对血吸虫雌虫酚酶组织化学和形态学影响的观察

本文报道用酚酶组织化学和雌虫形态学相结合的方法，分别对一次口服吡喹酮和硝硫氰胺６０ｍｇ／ｋｇ剂量治疗的实验兔，停药后不同时间解剖，取出雌虫和对照组的雌虫同时进行酚酶反应，测量虫体长度和计数雌虫虫卵数．实验发现；两药均随停药时间延长酚酶反应逐渐减弱以致呈可疑阳性，４８ｈ时几乎是阴性反应．随停药时间的延长虫体子宫虫卵数逐渐减少，出现变性虫卵或只见卵黄腺颗粒或雌虫子宫内无虫卵．虫体长度逐渐缩短。     

Enzyme histochemistry of experimental embryo-derived teratocarcinomas

Summary Teratocarcinomas were obtained by transplantation of 7 day old mouse egg-cylinders under the kidney capsule of adult isogeneic animals in order to visualize the histogenesis in the teratocarcinomas. The sequence of histochemical changes roughly corresponds to the events taking place in the developing organism. The origin of differentiated tissues could not be traced histochemically because of the changes which occur in the enzyme pattern during differentiation. No marker enzymes were found to enable us to link the differentiated tissues with the undifferentiated embryonal carcinoma cells. The latter cells displayed a low activity of oxydative enzymes and virtually no activity of acid hydrolases, but were rich in alkaline phosphatase. Histochemically they are similar to ecto-mesodermal cells of the egg-cylinder from which the teratocarcinomas were derived. In conjunction with previous ultrastructural findings these data indicate that embryonal carcinoma cells are cells which did not differentiate, but proliferate, retaining morphologic and probably some functional characteristics of undifferentiated ecto-mesodermal cells from the early postimplantation stages of development.

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Zusammenfassung Teratocarcinome wurden durch Transplantation von Eicylindern 7 Tage alter Mäuse unter die Nierenkapsel von erwachsenen isogenen Tieren erzeugt, um die Histogenese in den Teratocarcinomen zu verfolgen. Der Ablauf der histochemischen Veränderungen entspricht etwa dem des sich entwickelnden Organismus. Die Herkunft der differenzierten Gewebe konnte wegen der während der Differenzierung sich abspielenden Veränderung des Enzymmusters histochemisch nicht verfolgt werden. Marker-Enzyme, die es erlauben würden, die differenzierten Gewebe mit undifferenziertem embryonalen Gewebe in Verbindung zu bringen, konnten nicht gefunden werden. Die embryonalen Zellen zeigten eine geringe Aktivität der oxydativen Enzyme und praktisch keine Aktivität saurer Hydrolasen, waren aber reich an alkalischer Phosphatase. Histochemisch sind sie ähnlich den ecto-mesodermalen Zellen des Eicylinders, von dem das Teratocarcinom abstammte. Zusammen mit früheren elektronenmikroskopischen Feststellungen deuten diese Befunde darauf hin, daß embryonale Carcinomzellen Elemente darstellen, welche sich nicht differenzieren, sondern bei ihrem Wachstum morphologisch und wahrscheinlich auch manche funktionellen Charakteristika undifferenzierter ectodermaler Zellen aus den ersten Entwicklungsstadien beibehalten haben.

     

Comparison of the histological methods in the diagnostic of deer cysticercosis

Summary  Histochemical methods for the detection and diagnosis of the developmental stages of the canine tapeworm, from the genus Taenia found in the heart and lungs of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) hunted in Eastern Slovakia, is presented here. Detailed morphology of cysticerci (Cysticercus spp.), based on microscopic and histochemical analysis is described. For confirmation and demonstration of PAS-positive substances in the body of parasitic tissue (tegument and mesenchyme) the McManus — PAS method was used. The histochemical method according to Van Kossa was very effective for confirmation of calcareous corpuscles, which are one of the most important histological markers of cestode tissues (larva or adult).     

Kallikrein gene expression in the rat anterior pituitary

The report of 'kallikrein-like' activity in the rat neuro-intermediate lobe (N-IL) and its possible involvement in pro-opiomelanocortin processing led us to explore the expression of the kallikrein gene(s) in the pituitary. Using 32P-labelled rat pancreatic kallikrein cDNA, we have shown positive hybridization for rat anterior pituitary poly(A)+ RNA, of identical size on Northern blots (approximately 1.0 kb) to rat kidney poly(A)+ RNA run in parallel. Prior adrenalectomy or ovariectomy decreased the level of kallikrein mRNA seen in the anterior pituitary; total RNA from rat N-IL showed no significant hybridization. On hybridization histochemistry the anterior pituitary was strongly positive, and the neural and intermediate lobes negative. The previously reported kallikrein-like activity in the N-IL is therefore probably due to a non-kallikrein kininogenase; in the anterior pituitary, kallikrein may have a physiological role in limited precursor proteolysis, but lack kininogen activity.     

印度雕蚀菌感染三带喙库蚊幼虫的组织化学研究

本文采用组织化学技术,对印度雕蚀菌感染的三带喙库蚊幼虫的糖原、蛋白质、核酸(DNA、RNA)进行显微摄影及组成定量的图像分析。结果表明感染的幼蚊组织中糖原、蛋白质、核酸的含量均明显地较正常的幼蚊为少,灰度定量测定也表明两组幼蚊之间的差别非常显著。提示该菌感染对幼蚊上述生化成分有明显的影响,可能是其对幼蚊致病的重要机制。     

Cytochemical detection of a class 3 aldehyde dehydrogenase in human hepatocellular carcinoma

The development of hepatocellular carcinoma in rodents treated with different chemical compounds is associated with the appearance in the cytosol of neoplastic liver cells of an unusual aldehyde dehydrogenase isozyme of class 3 (ALDH-3) which is very active with aromatic aldehydes. This tumor-associated isozyme is readily detected by enzyme cytochemistry using the substrate benzaldehyde with NADP as coenzyme. To determine whether human hepatocellular carcinomas express ALDH-3, the activity of this isozyme was examined in frozen sections from 68 echo-guided human liver biopsies. In 54 cases the guided biopsy was performed on one or more nodules suggestive for hepatocellular carcinoma found at ultrasonography within the liver parenchyma. The remaining 14 patients were affected by chronic active hepatitis or cirrhosis. An intense enzymatic activity was ascertained in 5 out of 36 hepatocellular carcinomas. In non-neoplastic liver, in macroregenerative nodules and in metastatic adenocarcinomas enzymatic activity was not detectable. ALDH-3-positive tumors were typical hepatocellular carcinomas (histological grade II and III). These results suggest that ALDH-3 is a phenotype associated with malignancy in human liver tumors.     

钉螺组织化学与酶组织化学的进一步研究

采用组织化学与酶组织化学方法,定位观察了钉螺软体内DNA、组蛋白分布及3种酶的活性部位.结果显示:DNA在睾丸含量最高,呈聚集状态;组蛋白主要分布在齿舌、阴茎实质部、卵巢等;细胞色素氧化酶在各系统分布广、活性均高;葡萄糖-6-磷酸脱氢酶在生殖系及中枢神经节活性最高;5’—核苷酸酶在卵巢和睾丸部活性较高.     

Effect of water irrigations on human colonic mucosa structure after sigmoidostomy

The colonic mucosa of patients with sigmoidostomies, who were operated on for rectal cancer, and treated thereafter for different periods of time with daily water irrigations to obtain complete evacuation, was investigated by histologic and histochemical methods. Microscopic examination of the test specimens showed that the epithelial continuity, the characteristic brush border, and the positive mucous reaction to PAS and Alcian blue-Alcian yellow methods were not significantly changed with respect to controls. In some test specimens a remarkable number of mitoses were evident in the crypts. Since this could result from water irrigation stimuli and/or might represent an early manifestation of a restoved carcinogenetic process, specific investigations were performed on the 0-acylated sialic acids of the colonic mucins, which have been reported to represent markers of early malignant changes in colorectal epithelial cells. The results did not reveal alterations of the sialomucins in the treated specimens with respect to controls. Read at the XXI Concegno Società Italiana di Istochimica, Capri, Italy, May 21 to 23, 1986.