二氧化硅及柠檬酸铝对巨噬细胞膜K~+通透性影响的研究

通常认为矽肺发病的重要环节之一是肺泡巨噬细胞吞噬二氧化硅(SiO_2)粉尘颗粒后的崩解死亡。因此,凡能保护巨噬细胞的药物就可能具有防治矽肺的作用。柠檬酸铝(柠铝)对实验性动物矽肺以及临床矽肺病人的疗效可能正是与其体外保护巨噬细胞的作用有关。最近体外研究证明,SiO_     

人外周血单核巨噬细胞诱导、培养及鉴定

在重组人粒.巨噬细胞集落刺激因子(rhGM-CSF)作用下体外诱导培养人外周血单核巨噬细胞,并进行鉴定和功能检测.Ficoll密度梯度离心法分离人外周血单个核细胞(PBMc),含10%胎牛血清的RPMll640培养基和rhGM-CSF培养7d.光镜、扫描电镜及透射电镜观察细胞形态,鸡红细胞吞噬试验检测吞噬功能,流式细胞术检测单核巨噬细胞表面特征性标志CDl4等生物学技术鉴定贴壁细胞性质.获得的贴壁细胞具备巨噬细胞的形态学特征及免疫表型.有吞噬功能,纯度较高.人外周血单核细胞在rhGM-CSF诱导下向巨噬细胞分化,具有生物学活性,纯度较高.是一种简单易行的体外诱导培养巨噬细胞的方法.     

鸡胚巨噬细胞AcP酶变化及AcP酶与凋亡细胞的关系

本研究采用电镜及酶细胞化学的方法观察了鸡胚脾脏不同胚龄组巨噬细胞溶酶体酸性磷酸酶（AcP酶）的变化、凋亡实验组巨噬细胞及其AcP酶与凋亡细胞的关系。取10天、13天和17天鸡胚脾脏,按Gomori法显示AcP酶,各胚龄脾脏巨噬细胞AcP酶细胞化学反应阳性,按AcP酶染色阳性做溶酶体计数,结果显示随着胚龄的增加溶酶体数随之增加,尤以第17天组溶酶体数增加最为明显,所得数据经统计分析表明各胚龄组间溶酶体数的差异有统计学意义。凋亡实验组采用放线菌酮诱导15天鸡胚脾脏细胞凋亡,结果显示凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主。巨噬细胞未见凋亡,而是吞噬了大量的凋亡细胞和凋亡小体,AcP酶反应颗粒不仅出现在巨噬细胞的溶酶体、吞噬体,还见于高尔基复合体、内质网等。细胞AcP酶反应强度数字化结果表明：凋亡组酶活性显著高于对照组,差别有统计学意义,提示胚胎巨噬细胞在凋亡细胞出现时AcP酶活性增强,说明巨噬细胞吞噬和消化凋亡细胞或凋亡小体是通过AcP酶等活性物质来实现的。     

Toll样受体抗体抑制脂多糖激活巨噬细胞的吞噬活性

应用脂多糖(LPS)激活巨噬细胞后其吞噬能力大大增强, 以此为模型发现TLR2的多抗能部分抑制LPS激活巨噬细胞吞噬金葡萄球菌的能力, 也能部分阻断对U937细胞的吞噬活性. TRAIL或TNFα多克隆抗体同样能起到类似作用. 实验还发现低浓度血清培养亦在一定程度上抑制LPS激活巨噬细胞的吞噬作用. 结果证实TLR2能介导LPS的功能, 而某些血清因子参与了介导LPS的信号转导过程, 提示LPS激活巨噬细胞吞噬能力的提高与诱导表达TRAIL等细胞因子有关.     

Toll样受体抗体抑制脂多糖激活巨噬细胞的吞噬活行

应用脂多糖(LPS)激活巨噬细胞后其吞噬能力大大增强,以此为模型发现TLR2的多抗能部分抑制LPS激活巨噬细胞吞噬金葡萄球菌的能力,也能部分阻断对U937细胞的吞噬活性.TRAIL或TNFα多克隆抗体同样能起到类似作用.实验还发现低浓度血清培养亦在一定程度上抑制LPS激活巨噬细胞的吞噬作用.结果证实TLR2能介导LPS的功能,而某些血清因子参与了介导LPS的信号转导过程,提示LPS激活巨噬细胞吞噬能力的提高与诱导表达TRAIL等细胞因子有关.     

大鼠睾丸间质细胞的自体吞噬活动

本文结合超微结构和细胞化学观察,研究大鼠睾丸间质细胞(Leydig细胞)中溶酶体的结??构与功能。观察结果表明,大鼠睾丸间质细胞中高尔基体非常发达,在高尔基体的成熟面存在着CMP酶阳性反应的GERL系统,说明这种细胞有不断产生溶酶体的能力。细胞化学结果也证实在睾丸间质细胞有较多的初级和次级溶酶体。睾丸间质细胞不仅有较多的溶酶体,而且还有相当数量的自噬小体,存在着活跃的自体吞噬活动。自噬小体的界膜来源于特化的光面内质网或高尔基体膜囊,包围的内容物主要是光面内质网和少量线粒体。当自噬小体与溶酶体融合后即成为自体吞噬泡,由于酶的消化作用,自体吞噬泡内的细胞器有一系列形态变化。根据CMP酶细胞化学反应,可以区分自噬小体和自体吞噬泡,后者是一种次级溶酶体,呈CMP酶阳性反应。睾丸间质细胞是分泌雄性激素的内分泌细胞,其光面内质网和线粒体在类固醇激素分泌中起重要作用,自体吞噬活动的结果是去除部分内质网和线粒体,可能在细胞水平上起着对雄性激素分泌的调节作用。     

豚鼠Clara细胞GERL及其在低氧下变化的细胞化学观察

本文对正常和模拟海拔5000米高度24、48和72小时后豚鼠Clara细胞GERL进行了超微结构和ACP活性的电镜酶细胞化学观察。结果表明Clara细胞具发达的GERL,包括ACP阳性的致密膜囊、致密囊泡、有衣小泡和细胞衣被覆区,Clara细胞GERL参与了分泌颗粒的形成。在低氧下GERL肥大,ACP阳性分泌颗粒增多,提示低氧导致GERL功能亢进,形成分泌颗粒的作用增强。     

内分泌细胞中溶酶体对激素分泌调节的作用

本实验用外源性雄激素引起垂体促性腺激素细胞和睾丸间质细胞分泌抑制,对这两种细胞中的溶酶体及分泌吞噬和自体吞噬活动进行了超微结构形态观察和半定量分析。实验中应用了CMP酶细胞化学技术和免疫胶体金技术。研究结果显示,在分泌受抑制状态下,垂体促性腺激素细胞中溶酶体增多,分泌吞噬活动加强;与此同时,睾丸间质细胞也表现溶酶体增多、自体吞噬活动加强。这些结果不仅再次证明在分泌蛋白质激素细胞中溶酶体以分泌吞噬的方式参与了激素分泌调节,更重要的是初步证明在分泌类固醇激素细胞的分泌调节中,也有溶酶体的参与,其形式是自体吞噬作用。细胞通过自体吞噬作用得以在短时间内清除一部分合成激素的细胞器和其中的激素,这可能是分泌类固醇激素的细胞及时有效地调整激素分泌量的一项重要机制,与分泌蛋白质激素细胞的分泌吞噬有着相同的意义。     

胎肝细胞输注与全胚注射液治疗再生障碍性贫血的机理探讨

本文对胎肝细胞输注或全胚注射液治疗再生障碍性贫血的可能机理作了一些实验性探讨。研究结果表明: 1.胎肝细胞在培养或解体过程中释放某些刺激红系造血的因子,有利于已经损伤的造血功能的恢复。 2.对正常小鼠注射无细胞胎肝制剂或全胚注射液后,骨髓红系细胞的分裂指数明显升高,骨髓中粒/红比值趋于降低,反映了骨髓中红系细胞增生活跃的状态。 3.对正常小鼠注射无细胞胎肝制剂或全胚注射液后,外周血网织红细胞和腹腔巨噬细胞的吞噬指数趋于平行增高,其增高程度和持续时间随注射次数的增加而加强。 小鼠注射无细胞胎肝制剂或全胚注射液后,巨噬细胞吞噬指数的增加,反映了巨噬细胞激活,这种作用除了提高机体的非特异性免疫功能,增强机体的抵抗力外,还可能通过巨噬细胞的活化,直接或间接地调控机体红系细胞的增殖,因而,对巨噬细胞在造血调控中的作用以及它在再生障碍性贫血发病机理研究中的意义提出了讨论。     

正常大鼠肾脏细胞溶酶体膜的构成蛋白

溶酶体是细胞内对其吞噬之物质溶解及消化之主要场所,同时也是细胞自噬作用的主要细胞器。为了进一步了解此细胞器的功能与结构,我们采用免疫荧光标记法,通过5种针对大鼠肝细胞溶酶体膜蛋白的特异性单克隆抗体,对大鼠正常肾脏细胞溶酶体膜蛋白进行了标记,并通过NH_4Cl溶液对溶酶体作了膜膨胀处理,结果显示:(1)细胞内溶酶体膜蛋白是由多种蛋白所构成,其各种蛋白的含量是不同的;(2)所有溶酶体膜蛋白均表达于该细胞器之表面;(3)NH_4Cl溶液能有效地使溶酶体扩张,这将有和于进一步研究溶酶体的结构。     

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L ‐Leucyl‐L‐leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy‐dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.     

SCANNING ELECTRON MICROSCOPY AND ELECTRON MICROPROBE ANALYSIS OF SILICIFICATION PATTERNS IN INFLORESCENCE BRACTS OF AVENA SATIVA

Using scanning electron microscopy, we determined the kinds and distribution of epidermal cell types in Avena inflorescence bracts (glume, lemma, and palea). Electron microprobe analysis of silica deposition in these epidermal cells showed that silica cells constitute one of the important deposition sites. Probe ratio data indicate that the silica deposited is 74 % pure. Significant amounts of silica also become deposited in the trichomes and lesser amounts in the walls of long epidermal cells. None could be detected in the stomata. The possible functional significance of silica deposition in epidermal cells of these bracts is discussed.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.     

The interaction of the causative agent of melioidosis with the host's alveolar macrophages]

Studies were carried out on guinea pigs and albino rats, intranasally infected with P. pseudomallei C-141. The cells of bronchovesicular exudate were obtained from animals 1, 4 and 24 hours after infection. Electron microscopy was applied to study the process of interaction of the agent and alveolar macrophages. Bacteria were shown to form a capsule which permitted avoiding phagocytosis, when entering the host respiratory system. Microbes that failed to form a capsule were absorbed by macrophages and enclosed in a phagosome. Then some bacteria were destroyed by the lysosomal enzymes, the other synthesized a capsule, which protected them against the effect of phagolysosome content. There were also such microbes which escaped from a phagosome prior to fusion with lysosomes and parasitized in phagocytic cytoplasma forming a capsule there. By the end of the first 24 hours of observation the intact encapsulated microbe species were found to prevail in the host cells.     

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages

Summary A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0–45 min at 37° C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5–10 min of exposure and after 25–30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.     

Leishmania mexicana: acid phosphatase ultrastructural cytochemistry of infected mouse macrophage cultures treated with phenazine methosulfate

Bone marrow-derived mouse macrophage cultures infected with Leishmania mexicana amazonensis amastigotes were given a 2-hr pulse with 10 microM phenazine methosulfate (PMS), a cationic electron carrier which destroys the intracellular parasites. Cultures were fixed at different times after the PMS pulse and processed for the detection of acid phosphatase (AcP) activity at the electron microscopic level. Only a small proportion of nontreated, infected macrophages stained for AcP. In contrast, 2 to 6 hr after exposure to PMS, many infected cells displayed AcP-positive lysosomes and parasitophorous vacuoles. This increased AcP reactivity paralleled the reduction in the percentage of morphologically intact parasites. In addition, qualitative observations indicated that while nontreated infected cells contained only few recognizable lysosomes, the lysosomal complement noticeably increased a few hours after exposure to PMS. Most intact intracellular amastigotes were not stained, but damaged parasites were often positive for AcP. Twenty hours after the PMS pulse, the percentage of AcP-positive macrophages dropped to the levels initially present in noninfected cultures and all of the parasites were destroyed. Exposure of noninfected macrophages to PMS did not affect their AcP reactivity.     

Spontaneous cytotoxicity of macrophages against pancreatic islet cells

Activated peritoneal macrophages were found to lyse syngeneic [3H]leucine-labeled pancreatic islet cells or rat insulinoma cells after 15 h of coculture at 37 degrees C. Lysis was verified by electron microscopic analysis. Islet cell lysis was dependent on the T:E ratio and was comparable with P815 and L929 tumor cells used as targets. The cytotoxic activity was localized in the adherent fraction of Corynebacterium parvum activated peritoneal cells and was destroyed by incubation of cells with macrophage-toxic silica particles. Syngeneic thyrocytes and hepatocytes were found to be resistant to the cytolytic action of activated macrophages. It has been shown previously that macrophages contribute to pancreatic islet inflammation. The present in vitro analysis demonstrates that macrophages can function as effector cells in islet destruction.     

A LIGHT AND ELECTRON MICROSCOPE STUDY OF INTERLAMELLAR POLYGLUCOSIDE BODIES IN OSCILLATORIA CHALYBIA

A light and electron microscopic study of interlamellar granules in Oscillatoria chalybia was made to determine their physiological nature. Oscillatoria chalybia was cultured under continuous light in media of high nitrogen content, moderate nitrogen content and low nitrogen content. Cultures growing vigorously in a medium of moderate nitrogen content were placed in darkness for an additional 96 hr. Periodic acid-Schiffs reagent tests were made on specimens from these 4 cultural conditions. Electron microscopic studies of interlamellar granules were correlated with the cytochemical tests. It is shown that diastase digestion will eliminate the PAS-positive substance and the interlamellar granules. Conclusions are that the interlamellar granules are polyglucoside in nature and that they vary in number and size with available nitrogen, light intensity and age of culture in such a way as to indicate that they are food reserves.     

ATP is stored in lysosomes of greater epithelial ridge supporting cells in newborn rat cochleae

Adenosine triphosphate (ATP), which plays a crucial role in both developing and mature cochleae, is released from greater epithelial ridge (GER) supporting cells of the rat cochlea, but the organelles in which ATP is stored have not yet been identified. Thus, we studied the organelles involved in ATP storage and suggest that lysosomes provide this function. GER supporting cells of newborn rats were isolated, purified, and cultured, and labeled vesicles within the supporting cells were identified via confocal microscopy and transmission electron microscopy (TEM). ATP release from GER supporting cells after glycyl-L -phenylalanine-β-naphthylamide (GPN) treatment was measured. The specifically labeled organelles observed by confocal microscopy and TEM were lysosomes, and GPN treatment enhanced ATP luminescence in the extracellular fluid of the supporting cells. The release of ATP from supporting cells was affected by changes in intra- and extracellular Ca²⁺ concentrations. In addition, changes in the intracellular Ca²⁺ caused by inhibiting the phospholipase signaling pathway affected the release of ATP from supporting cells. We demonstrated that ATP is stored in the lysosomes of GER supporting cells within newborn rat cochleae and that ATP release from GER supporting cells may be Ca²⁺-dependent.     

椭圆背角无齿蚌外套膜组织培养与未培养细胞分泌活动的扫描电镜观察

作者用扫描电镜及相差显微镜,对椭圆背角无齿蚌外套膜组织培养与未培养细胞的分泌活动进行了研究,观察到两者的分泌活动都是十分旺盛的。培养细胞有局部分泌和顶浆分泌。细胞分泌形态观察到三种:(1)分泌端形成由膜包裹的突起,突起逐渐伸长,基部变成细颈,最后脱离细胞成为分泌泡(局部分泌);(2)细胞端部伸出长足,将分泌物排到较远处分泌后,长足缩回恢复原状;(3)分泌端伸出很多细枝,分泌物随后如液流式涌出细胞(顶浆分泌)。取外套膜色线边组织为材料,培养后在组织块和细胞上有角质素(与贝壳最外层相似)类的茶褐色结晶和无定形分泌物形成;用去掉色线边的外表皮组织块培养,则有珍珠(与贝壳最内层相似)状的白色和淡黄色结晶生成。表明了细胞在适宜的条件下培养,所形成的分泌物的性质可能与活体相同。因此大批量培养细胞可能得到人们希望获得的细胞产物。