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Histochemical studies on the aging of various organ systems of animals and men.

关于动物和人类各种器官系统衰老的组织化学研究。

基本信息

批准号：
02454564
负责人：
NAGATA Tetsuji
金额：
$ 1.28万
依托单位：
Shinshu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454564/
关键词：
aging organ system histochemistry radioautography DNA synthesis RNA synthesis protein synthesis glucide synthesis

项目摘要

In order to elucidate the ultrastructural histochemical changes of tissues and cells in various organ systems in animals and men, tissue samples obtained from various aging stages starting from embryonic stage to newborn, suckling, weanling, juvenile and senescent stages of ddY mice bred in our laboratory, as well as biopsied samples of human tissues from juvenile, adult and senescent patients in clinical departments were collected and investigated by means of various histochemical procedures. The histochemical methods employed were fixations with routine chemical fixatives, such as buffered glutaraldehyde and osmium tetroxide followed by dehydration and plastic embedding for thick and thin sections, or rapidly frozen cryosections, or freezesubstituted or freeze-dried and embedded dry sections, which were routinely stained with toluidine blue for light microscopy or uranyl acetate and lead citrate for electron microscopy, or specific histochemical reactions such as enzyme histochemistr … More y, radioautography after administration of various kinds of precursors for macromolecular compounds such as ^3H-thymidine (for DNA), ^3H-uridine (RNA), ^<3H>-leucine (proteins), ^3H-glucosamine or ^<35>SO_4 (glucides), ^3H-glycerol (lipids) either in vivo or in vitro, immunohistochemical reactions for proteins, and lectin histochemistry with biotinyl-lectins in combination with ABC and DAB methods to demonstrate glycoconjugates.The results obtained are as follows.1. Cell and Tissue Culture. Established cell line, HeLa cells, primary cultures from rat liver cells, human normal and cancer thyroid tissues were cultured in vitro and ultrastructural morphology, incorporation of ^3H-labeled thymidine and uridine were demonstrated. The labeling index of ^3H-thymidine incorporation increased from the initiation of cell culture, showed a peak on days 3 and 4 in culture and decreased.2. Organs of Movement. The human lumber yellow ligaments from various kinds of diseased. patients in various ages were surgically taken out, chemically fixed or rapidly frozen, cryosectioned, freeze-dried, and both the morphology of elastic fibers and elementary analysis with an X-ray microanalyzer were performed. The results revealed that the fine structure of elastic fibers changed according to aging, classified into 4 types, and soluble calcium was found in the ligaments suggesting calcification and ossification.3. Circulatory Organs. Mouse spleen cells from various stages of aging mice were examined. Acid phosphatase activity was demonstrated in the lysosomes of reticulocytes, macrophages and littoral cells and the intensity of the activity, which was estimated by X-ray microanalysis on Ce end-products, increased after birth reaching the maximum at 2 weeks and decreased to 10 months. The incorporations of ^3H-thymidine, -uridine and -leucine increased after birth, reaching the maxima at 2 weeks and then decreased.4. Endocrine Organs. The thyroid tissues obtained surgically from human patients with goiters were examined with PCNA/cyclin immunohistochemical stainability equivalent to the incorporation of ^3H-thymidine. The results showed that the PCNA/cyclin positive cells were more than ^3H-thymidine. In primary cultured normal and tumor cells, intracellular localization of keratin and vimentin were demonstrated by immunohistochemical procedures and inhibitory effects of both TSH and anti-cancer drugs were observed. On the other hand, incorporation of ^3H-thymidine was observed in aging mouse adrenal gland by light microscopic radioautography which resulted in high labeling index in both adrenal cortex and medulla during the fetal stage, then decreasing after birth to senescence, showing different indices in superficial and deeper layers of the cortex and medulla.5. Digestive Organs. The mucigen granules in the goblet cells of the colons of aging mice were examined by ^3H-glucosamine radioautography, X-ray microanalysis on S in the glycoproteins and lectin histochemistry. The results showed that the incorporation of ^3H-glucosamine and S contents increased after birth, reached the maxima at 2 weeks and decreased. The staining pattern with several lectins changed from fetal to senescent stages demonstrating the change of glycoconjugates composition. On the other hand in the liver, labeling indices of various kinds of celis composing the liver with ^3H-thymidine, ^3H-uridine, and ^3H-leucine were examined by both light and electron radioautography The results revealed that the labeling index of ^3H-thymidine in hepatocytes, sinusoidal endothelial cells and other cell types decreased from fetal stage and after birth reaching almost 0 in 2 years, demonstrating different pattern of frequencies. To the contrary, the incorporations of ^3H-uridine and ^3H-leucine increased after birth, reaching the maxima at 2 to 4 weeks and finally decreased. The pancreatic cells of aging mice also demonstrated similar pattern of ^3H-thymidine, ^3H-uridine and ^3H-leucine incorporations. In the pancreas, incorporations of ^3H-glucosamine, ^3Hglycerol revealed the similar pattern to uridine and leucine, reaching maxima at 2-4 weeks after birth.6. Respiratory Organs. The tracheas of aging mice were examined after administration of ^<35>SO_4 by light microscopic radioautography, which demonstrated incorporation of glycoproteins into chondrocytes and cartilaginous matrix of the tracheal cartilages. The number of silver grains demonstrating glycoprotein synthesis increased after birth to 2 weeks, reaching a maximum and decreased. The incorporation of ^3Hthymidine is now being examined.7. Uro-genital Organs. In the kidneys of mice at various ages, incorporation of ^3Hthymidine was examined by light microscopic radioautography and it was found that the labeling index of glomerular epithelial celli and uriniferous tubular cells increased during the fetal stage and decreased after birth showing different patterns in the superficial and deeper cortex. Lectin histochemistry also revealed changes of stainability to various kinds of lectins in respective ages, thus the stainability patters were categorized into 4 types. The testis, ovary, uterine tube and uterus of aging mice are now under investigation.8. Nervous and Sensory Organs. Nucleic acid synthesis in mouse ocular tissues was studied by ^3H-thymidine and ^3H-uridine radioautography. The incorporation of ^3H-thymidine was demonstrated in the retina and retinal pigment epithelium, The labeling indices of these cells were high in the fetal stage and decreased after birth to the senescence. However, the indices in respective sites of the retina, i. e. from anterior to equatorial and posterior, as well as from external to internal layers, at each stage of development and aging, differed variably. The RNA contents as expressed by the number of silver grains in each cell at different sites, layers, and stages, varied, increasing after birth and showing maxima at 1 week and decreased. Lectin histochemistry revealed that several kinds of lectins showed characteristic changes in staining paftems in the retina due to aging.From these results, histochemical changes in cells and tissues of many organs In various organ systems of experimental animals and men were demonstrated. It is expected that these results should be utilized in the fields of clinical medicine to elucidate the mechanism of aging and senescence in men in the future. Less

为了阐明动物和人各器官系统组织细胞的超微结构组织化学变化，本实验室饲养的ddY小鼠从胚胎期到新生儿、哺乳期、断奶期、幼年期和老年期各衰老阶段的组织样本，以及临床科室幼年期、成年期和老年期患者的人体组织活检样本，采用各种组织化学方法进行了研究。采用的组织化学方法是用常规化学固定剂固定，如缓冲戊二醛和四氧化锇，然后脱水和塑料包埋厚切片和薄切片，或快速冷冻冷冻切片，或冷冻替代或冷冻干燥和包埋干切片，常规用甲苯胺蓝染色，光镜染色，或醋酸铀酰和柠檬酸铅染色，电镜染色。更重要的是，给药后放射自显像的各种大分子化合物的前体，如^3H-胸腺嘧啶（DNA）， ^3H-尿苷（RNA）， ^<3H -亮氨酸（蛋白质），^3H-氨基葡萄糖或^<35>SO_4（葡萄糖），^3H-甘油（脂类）体内或体外，免疫组织化学反应的蛋白质，用生物素-凝集素结合ABC和DAB方法进行凝集素组织化学，以证明糖缀合物。得到的结果如下：1。细胞和组织培养。体外培养已建立的细胞系、HeLa细胞、大鼠肝细胞原代培养物、人正常甲状腺组织和癌甲状腺组织的超微结构形态、^ 3h标记的胸腺嘧啶和尿苷的结合。^ 3h -胸腺嘧啶掺入的标记指数从细胞培养开始逐渐升高，在培养第3、4天达到峰值，随后逐渐降低。运动器官。人体木材黄韧带病变多种多样。不同年龄的患者手术取出，化学固定或快速冷冻，冷冻切片，冷冻干燥，并进行弹性纤维形态和x射线微量分析仪的元素分析。结果表明，弹性纤维的精细结构随着年龄的增长而发生变化，可分为4种类型，韧带中存在可溶性钙，提示钙化和骨化。循环器官。对衰老小鼠不同阶段的脾脏细胞进行了检测。网状细胞、巨噬细胞和滨海细胞的溶酶体中均有酸性磷酸酶活性，通过对Ce终产物的x射线微量分析估计其活性强度在出生后增加，在2周时达到最大值，在10个月时下降。^ 3h -胸腺嘧啶、-尿嘧啶和-亮氨酸的掺入量在出生后增加，在2周时达到最大值，随后下降。内分泌器官。用PCNA/细胞周期蛋白免疫组化染色检测从人类甲状腺肿大患者手术获得的甲状腺组织，相当于加入^ 3h -胸腺嘧啶。结果显示PCNA/cyclin阳性细胞均大于^ 3h -胸腺嘧啶。在原代培养的正常细胞和肿瘤细胞中，通过免疫组织化学方法证实了角蛋白和波形蛋白在细胞内的定位，并观察了TSH和抗癌药物的抑制作用。另一方面，通过光镜放射自显像观察到^ 3h -胸苷嘧啶掺入衰老小鼠肾上腺，导致胎儿期肾上腺皮质和髓质的标记指数均较高，出生后逐渐下降至衰老，皮层和髓质的浅层和深层标记指数不同。消化器官。采用^ 3h -氨基葡萄糖放射显像、糖蛋白S的x射线微量分析和凝集素组织化学方法对衰老小鼠结肠杯状细胞中的黏原颗粒进行了检测。结果表明，^ 3h -氨基葡萄糖掺入量和S含量在出生后增加，在2周时达到最大值，随后下降。从胎儿到衰老阶段，几种凝集素的染色模式发生了变化，表明糖缀合物的组成发生了变化。另一方面，在肝脏中，用光波和电子射线自显像法检测了^ 3h -胸腺嘧啶、^ 3h -尿嘧啶和^ 3h -亮氨酸对组成肝脏的各种细胞的标记指数。结果表明，^ 3h -胸腺嘧啶在肝细胞、窦内皮细胞和其他类型细胞中的标记指数从胎儿期开始下降，出生后在2年内几乎达到0，呈现出不同的频率模式。相反，^ 3h -尿嘧啶和^ 3h -亮氨酸的掺入在出生后增加，在2 ~ 4周达到最大值，最后减少。衰老小鼠的胰腺细胞也表现出类似的^ 3h -胸腺嘧啶、^ 3h -尿嘧啶和^ 3h -亮氨酸结合的模式。在胰腺中，3h -氨基葡萄糖、3h甘油的掺入与尿苷和亮氨酸的模式相似，在出生后2-4周达到最大值。呼吸器官。用光镜放射自显影法对老龄小鼠的气管进行了观察，发现气管软骨的软骨细胞和软骨基质中存在糖蛋白。显示糖蛋白合成的银粒数在出生至2周后逐渐增加，达到最大值后逐渐减少。目前正在研究^3Hthymidine的掺入。Uro-genital器官。在不同年龄的小鼠肾脏中，用光镜放射自显像检查^3Hthymidine的掺入情况，发现肾小球上皮细胞和尿小管细胞的标记指数在胎儿期升高，出生后下降，在浅皮层和深层皮层表现出不同的模式。凝集素组织化学也揭示了不同年龄对各种凝集素的染色变化，可将其分为4种染色模式。老龄小鼠的睾丸、卵巢、输卵管和子宫正在研究中。神经和感觉器官。采用^ 3h -胸苷和^ 3h -尿苷放射自显影法研究了小鼠眼组织的核酸合成。^ 3h -胸腺嘧啶在视网膜和视网膜色素上皮中掺入，这些细胞的标记指数在胎儿期较高，出生后逐渐下降，直至衰老。然而，在视网膜的各个部位，即从前到赤道和后，以及从外层到内层，在每个发育和衰老阶段的指数是不同的。不同部位、不同层位和不同阶段的细胞中以银粒数表示的RNA含量有所不同，在出生后增加，在1周时达到最大值，然后下降。凝集素组织化学结果显示，几种凝集素在视网膜中因衰老而出现特征性的染色模式变化。从这些结果可以看出，在实验动物和人的各种器官系统中，许多器官的细胞和组织发生了组织化学变化。这些结果有望在临床医学领域得到应用，以阐明男性衰老和衰老的机制。少