放线菌酮诱导大鼠脾细胞凋亡的电镜观察

利用透射电镜观察了放线菌酮体内诱导大鼠脾细胞凋亡的形态学变化,结果显示,腹腔注射放线菌酮4小时后,大鼠脾细胞发生凋亡,凋亡脾细胞核和胞质发生一系列形态学变化.主要表现为胞核染色质浓缩、重排、呈不同形态;多数凋亡脾细胞的粗面内质网增殖,凋亡小体形成,其线粒体也大量增加,或单个分布或团聚,肿胀、空泡化.结果提示,凋亡脾细胞的主要细胞器主动参与凋亡过程.     

放线菌酮诱导大鼠脾细胞凋亡的电镜观察

利用透射电镜观察了放线菌酮体内诱导大鼠脾细胞凋亡的形态学变化，结果显示，腹腔注射放线菌酮４小时后，大鼠脾细胞发生凋亡，凋亡脾细胞核和胞质发生一系列形态学变化。主要表现为胞核染色质浓缩，重排，呈不同形态，多数凋亡脾细胞的粗面内质网增殖，凋亡小体形成，其线粒体也大量增加，或单个分布或团聚，肿胀，空泡化。结果提示，凋亡脾细胞的主要细胞器主动参与凋亡过程。     

实验性糖尿病对大鼠胸腺皮质上皮细胞的影响

目的　探讨糖尿病对大鼠胸腺皮质上皮细胞影响的形态学变化 ,了解其引起胸腺细胞凋亡及胸腺萎缩的可能机制。　方法　采用四氧嘧啶型糖尿病动物模型 ,电镜下观察了糖尿病大鼠胸腺皮质上皮细胞的超微结构变化。　结果　 2型上皮细胞发生 4种变化 :1 内分泌功能紊乱样变 ,以大量变性的空泡与囊泡为特征 ;2 退化样变 ,以大量的变性分泌泡、髓样小体和粗大张力细丝束为特征 ;3 吞噬细胞样变 ,以大量的溶酶体样致密颗粒及吞噬的凋亡细胞与小体为特征 ;4 细胞凋亡样变 ,以细胞凋亡的早期形态学变化为特征。 3型上皮细胞发生两种变化 :1 退化样变 ,伴有大量分泌肽类激素样的致密颗粒群。 2 增生样变 ,在其胞体周伴有大量的胶原原纤维。此外 ,在胸腺皮质中还发现大量的胸腺细胞凋亡及胸腺细胞数量减少。　结论　糖尿病可导致大鼠胸腺皮质上皮细胞发生多样性的超微结构变化 ,可能是影响胸腺细胞发育 ,并导致其凋亡及胸腺萎缩的主要机制之一。     

γ-射线诱导HL-60细胞凋亡的形态学研究

目的　观察γ－射线诱导ＨＬ－６０ 细胞凋亡的形态学变化。　方法　应用Ｈｏｅｃｈｓｔ３３２５８荧光显微镜及透射电子显微镜进行观察。　结果　γ－射线诱导ＨＬ－６０ 细胞死亡的主要方式是凋亡，凋亡细胞具有典型的形态特征，细胞核染色质凝集边聚；发现细胞核经多种形式形成核碎块；并可见具有不同内含物的凋亡小体；内质网增多，参与形成核碎块。　结论　同一因素诱导同一细胞凋亡，细胞核的变化可有多种形式。γ－射线诱导Ｋ５６２和ＣＥＭ 细胞凋亡的形态学变化与ＨＬ－６０ 细胞的不同，反映出同一因素诱导不同细胞凋亡的途径有一定的差异。     

放线菌酮诱导大鼠肠系膜淋巴结淋巴细胞凋亡

本实验利用放线菌酮建造细胞凋亡动物模型 ,用原位末端标记和电镜方法观察了大鼠肠系膜淋巴结淋巴细胞凋亡的形态学变化。结果显示放线菌酮处理 4h后 ,肠系膜淋巴结部分淋巴细胞处于凋亡状态 ,主要表现为细胞核凝聚 ,染色质边集 ,可见线粒体、内质网增殖 ,自噬体和凋亡小体形成。结果表明放线菌酮主要诱发肠系膜淋巴结生发中心的 B淋巴细胞凋亡 ,线粒体、内质网的增殖是细胞凋亡的一种主动行为。     

胸腺小体在胸腺发育中的形态发生及其功能探讨

用组织化学与免疫组织化学技术对８例人胎的胸腺进行研究，观察在其发育过程中胸腺小体的形态变化有增殖细胞核抗原，花生凝集素和细胞角蛋白等的表达特征。结果显示：胸腺小体是在胸腺发育阶段由胸腺上皮细胞呈同心圆状排列而成，且胎儿胸中单个和融合状的胸腺小体部较成体胸明显增多。围成胸腺小体的上皮细胞从周边中央，角蛋白明显减少，增殖能力逐渐减弱，表现为逐渐退化的过程。证实胸腺小体是处理退变的上皮细胞的场所。另外，     

食管癌病理学研究的新进展(二)

1998年国内作者对279例正常食管鳞状上皮及其病变进行了细胞凋亡的研究。典型凋亡细胞的形态学特征为：胞质红染，细胞核染色质明显浓缩，有时可见核裂解的碎片。典型凋亡小体呈圆形，胞质呈强嗜酸性，其中含核碎片，或仅为一强嗜酸性胞质小体，而不可见核碎片。从正常食管鳞状上皮到鳞癌，各个阶段内均可见散在的凋亡细胞和凋亡小     

阿霉素诱导人肝癌细胞株HCC—9204肝癌细胞凋亡的形态学 …

应用抗癌药阿霉素成功地诱导增减人肝癌细胞株ＨＣＣ－９２０４肝癌细胞发生凋亡，用２０μｍｏｌ．Ｌ＾－１阿霉素作用肝癌细胞３ｈ，换培养液继续培养１２ｈ后，肝癌细胞ＤＮＡ结构发生断裂，电泳呈梯状。肝癌细胞形态改变出现细胞膜小泡和凋讯小体形成等凋亡细胞特征，经透射电镜和扫描电镜观察显示，在体积缩小胞膜皱缩的肝癌细胞表面产生一种球形小体，有的肝癌细胞崩解为碎片状，形态学观察结构提示，肝癌细胞产生的球形小体可     

雌激素对大鼠胸腺细胞凋亡及Bcl-2、Bax表达的影响

目的:探讨苯甲酸雌二醇对大鼠胸腺Bcl-2和Bax表达及细胞凋亡的影响及其机制.方法:雌性大鼠行卵巢切除术,给予苯甲酸雌二醇后,观察胸腺指数的变化,Hochest33342荧光染色及透射电镜标本观察胸腺细胞凋亡情况,免疫组织化学检测胸腺组织中Bcl-2和Bax的表达情况,原位杂交技术检测Bcl-2、Bax m RNA的表达情况.结果:双侧卵巢切除组大鼠胸腺指数较假手术组增加,双侧卵巢切除+雌激素组大鼠胸腺指数较双侧卵巢切除组减小;假手术组和双侧卵巢切除组大鼠胸腺组织中以正常胸腺细胞为主,偶见凋亡细胞或凋亡小体,双侧卵巢切除+雌激素组可见较多凋亡细胞和凋亡小体;双侧卵巢切除+雌激素组大鼠胸腺组织中Bcl-2表达较双侧卵巢切除组和假手术组增高明显降低,而Bax表达呈现相反趋势;Bcl-2 mRNA、Bax mRNA的表达与Bcl-2、Bax的表达呈一致性.结论:雌激素可以降低大鼠胸腺指数,抑制胸腺组织中Bcl-2的表达,促进Bax的表达,从而诱导大鼠胸腺细胞凋亡,促进雌性大鼠胸腺退化.     

鸡胚脾脏凋亡细胞核及其环孔板的超微结构

目的：着重观察鸡胚脾脏凋亡细胞核及其环孔板的形态学变化。方法,用放线菌酮处理第１５天胚龄鸡胚,建立鸡胚脾脏细胞凋亡模型,用透射电镜进行观察。结果：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。凋亡细胞核在裂解分离为核碎块的过程中出现环孔板,凋亡细胞核完全裂解分离后,环孔板消失。结果：环孔板参与凋亡细胞核的分割以及核碎块的包囊,提示凋亡细胞核裂解成为核碎块形凋记录上体的过程是一个主     

CXCR4 tropic human immunodeficiency virus type 1 induces an apoptotic cascade in immature infected thymocytes that resembles thymocyte negative selection

HIV-1 often replicates in the thymus of infected individuals, causing thymocyte depletion and thymic dysfunction. Nevertheless, the mechanisms by which thymocyte depletion occurs are not clear. Here we report that HIV-1 infection induced apoptosis primarily in productively infected thymocytes; aldrithiol-2 or Efavirenz treatment largely abrogated HIV-1-induced apoptosis. Moreover, X4-HIV-1 induced apoptosis primarily in immature CD4+ CD8+ (DP) thymocytes whereas most mature CD4 or CD8 single-positive (SP) thymocytes were resistant to X4 HIV-1-induced apoptosis despite infection. Consistent with this, we observed significant induction of several genes involved in negative selection of DP thymocytes. Furthermore, treatment of thymocytes with cycloheximide abrogated HIV-1-induced apoptosis, implying a requirement for de novo protein synthesis. Our results suggest that HIV-1-induced apoptosis of thymocytes requires the activation of caspases and the participation of mitochondrial apoptosis effectors, which serve to amplify the apoptotic signal, a process similar to that elaborated during thymocyte negative selection.     

Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis

In glucocorticoid-treated rat thymocytes and the murine lymphoid cell lines L5178 and S49 the morphology of apoptosis is associated with chromatin cleavage. The cleavage is at internucleosomal sites, apparently through activation of an endogenous endonuclease. In variants of the cell lines selected for resistance to glucocorticoid, neither apoptosis nor chromatin cleavage were observed after steroid treatment, and steroid receptors were undetectable. In thymocytes, both the morphological changes of apoptosis and chromatin cleavage were inhibited by cycloheximide and actinomycin D. The calcium-magnesium ionophore A23187 induced apoptosis and chromatin cleavage in thymocytes, and these effects were also inhibited by cycloheximide. The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments. They also provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.     

Hormone-induced cell death. 2. Surface changes in thymocytes undergoing apoptosis

In vivo, apoptotic cells are swiftly recognized by phagocytes, presumably because of changes on their surface. This article describes surface changes in rat cortical thymocytes undergoing apoptosis induced by glucocorticoid treatment in vitro. Homogeneous populations of thymocytes early in apoptosis were prepared by isopyknic centrifugation. These cells were compared with purified nonapoptotic cells in terms of several surface characteristics, including binding to macrophages, surface ultrastructure, microelectrophoretic mobility (a measure of surface charge density), and ability to bind four lectins and four monoclonal antibodies to thymocyte antigens. Apoptotic cells bound to macrophages more avidly than did nonapoptotic cells by a process not dependent upon serum factors. Their surfaces lost microvilli and became " blistered ," apparently through fusion of vesicles of endoplasmic reticulum with the plasma membrane. The surface charge density of apoptotic cells was less than that of nonapoptotic cells. Surface antigens and lectin-binding sites were less abundant on apoptotic than on normal cells, in proportion to the general reduction in cell size observed in apoptosis. Differences between apoptotic and normal cells were not detected, however, in the relative quantities of exposed galactose, N-acetyl galactosamine, N-acetyl glucosamine, N-acetyl neuraminic acid, or of several surface antigens, including the major sialoglycoproteins of the thymocyte membrane. It appears that although several changes occur in the surface of apoptotic cells, many cell membrane structures remain intact. The changes responsible for the recognition of apoptotic cells by phagocytes are more subtle than those detectable by the binding of lectin and antibody probes, but preliminary data suggest that a lectin-sugar interaction is involved.     

Spontaneous apoptosis in human thymocytes.

Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain.     

Different Roles of a Rat Cortical Thymic Epithelial Cell Line In Vitro on Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis,Induction of Apoptosis,Nursing and Growth Promoting Activities

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.     

Different roles of a rat cortical thymic epithelial cell line in vitro on thymocytes and thymocyte hybridoma cells: phagocytosis,induction of apoptosis,nursing and growth promoting activities

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.     

Nonspecific esterase released from thymic macrophages accumulates in the apoptotic thymocytes: an indication for this enzyme participating in the clearance of apoptotic thymocytes

In the mouse thymus, a large number of developing thymocytes die through apoptosis each day. It has been proposed that thymic macrophages are responsible for clearance of the massive number of thymocytes that die through apoptosis. The detailed clearance mechanism by which macrophages remove the apoptotic cells is not clear. Our in vitro studies in this report show that nonspecific esterase (NSE), a cytochemical marker enzyme of macrophages, was secreted from thymic macrophages as a consequence of stimulation by interaction with thymocytes, and the esterase accumulated in these macrophage-binding thymocytes (MBT). TUNEL staining demonstrated that these MBT were undergoing apoptosis. The inability to exclude eosin Y and the presence of pores on the plasma membrane were further evidence for the disintegration of these MBT. In vivo, the release of NSE was evident by the presence of NSE activity in the extracellular space between the macrophages and apoptotic thymocytes under the transmission electron microscope after dexamethasone injection, which causes massive apoptosis of thymocytes. Inhibition study showed that the inhibition of NSE delayed the MBT progressing to the late apoptotic phase. These results suggest that the NSE released from macrophages is involved in the clearance of apoptotic thymocytes.     

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.     

Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ-irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non-thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti-CD2 and anti-LFA-1 antibodies. The immature CD3^−/+dull CD4⁺CD8⁺ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC-induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid-induced thymocyte apoptosis is p53-independent, glucocorticoids are conceivably not involved in TEC-induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.