盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。     

盘基网柄菌细胞发育过程中尿囊酸酶的亚细胞定位

利用胶体金免疫电镜技术,观察了盘基网柄菌细胞分化与凋亡过程中胞内尿囊酸酶的位置变化。结果表明,在细胞聚集期细胞产生的尿囊酸酶主要分布于线粒体及周围细胞质内。到了细胞丘时期,尿囊酸酶只特异地存在于发生内自噬的线粒体内,且仅局限于线粒体因内自噬产生的空泡区域,这些发生线粒体内自噬的细胞将分化成前孢子细胞。随着前孢子细胞分化的进行,尿囊酸酶颗粒在细胞内分布逐渐减少,在靠近质膜处的空泡内还能观察到一些酶颗粒;而另一些细胞内,几乎所有的胞器内都能观察到酶颗粒,一直延续至柄细胞形成。从中可以看到尿囊酸酶在将发育成孢子细胞和柄细胞两种类型细胞内的分布位置明显不同,结果提示了尿囊酸酶蛋白与盘基网柄菌细胞分化和凋亡调控途径有密切关系。     

魏氏拟尾柱虫休眠包囊细胞器的电镜酶细胞化学研究

为研究纤毛虫在休眠状态下细胞生命活动的特征及细胞器的功能,应用电镜酶细胞化学方法显示,魏氏拟尾柱虫（Ｐａｒａｕｒｏｓｔｙｌａ ｗｅｉｓｓｅｉ）休眠包囊中,在自噬泡位置,酸性磷酸酶反应颗粒形成多种形态的泡状体,ＡＴＰ酶反应颗粒聚集形成泡状体或球状体,在分散分嘏的线粒体中于内膜位置形成少数琥珀酸脱氢酶反应颗粒。由所得结果推测,纤毛虫休眠包囊中的自噬泡消化现象是细胞在特殊生理条件下的重要生命活动,是休眠     

簇生岐柄菌的超微形态学研究

为揭示网柄细胞状黏菌的超微结构特征,对簇生岐柄菌Cavenderia fasciculata生活史过程中的孢子、黏变形体和柄细胞3种类型的细胞超微结构进行系统观察,并与目前仅知的3种网柄细胞状黏菌的超微结构进行比较。结果表明：簇生岐柄菌C. fasciculata的孢子、黏变形体和柄细胞内均有线粒体结构,随着细胞分化,线粒体内部结构逐渐破坏,并被自噬泡吞噬;孢子表面具有线状纹饰。     

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。     

蜜环菌侵染天麻皮层过程中酸性磷酸酶的细胞化学研究

被蜜环菌(Armillaria mellea)侵染的天麻(Gastrodia elata B1.)皮层中,由外至内形成三种类型的染菌细胞:菌丝结细胞、空腔细胞和消化细胞。外部两类细胞中的酸性磷酸酶定位显示,一些位于空腔细胞或衰老的菌丝结细胞中的菌丝内部逐渐产生大量酸性磷酸酶,随后菌丝发生自溶。这两类细胞中未发现明显的释放水解酶消化菌丝的现象。当菌丝进入消化细胞以后,情况与此不同,大量包含酸性磷酸酶的微小颗粒出现在菌丝周围,随后这些酶颗粒相互融合,形成包围菌丝的消化泡,菌丝被溶酶体水解酶所消化。最后消化泡变为包含代谢废物的残体。     

内溶酶体–自噬溶酶体对阿尔茨海默症病理进程的影响

自噬是在细胞受到胞内应激或饥饿条件下,依赖于溶酶体将胞内异常蛋白质以及受损细胞器降解的过程。内体是由细胞内吞形成的单层膜结构细胞器,它可以内吞进入细胞的异常蛋白质将其送入自噬体或通过内溶酶体–自噬溶酶体途径降解。由于自噬体与内体在形态与功能上相互联系又有相似之处,从而构成内溶酶体–自噬溶酶体系统。在阿尔茨海默症(Alzheimer’s disease,AD)患者的神经元中,两种异常蛋白质[β淀粉样物质(βamyloid,Aβ)和过度磷酸化的Tau蛋白]可以通过内溶酶体–自噬溶酶体系统清除;而当此系统功能受阻时,神经元中出现异常自噬体与内体形成的颗粒空泡变性体,导致AD病理改变加重。因此,内溶酶体–自噬溶酶体在阿尔茨海默病中扮演着重要角色。越来越多的研究结果提示,对内溶酶体–自噬溶酶体系统的调控可能为阿尔茨海默病的治疗提供新靶点和方向。     

蕨类植物紫萁绒毡层细胞凋亡的细胞学特征

绒毡层凋亡过程是小孢子发生中的重要事件,以往的研究主要集中在被子植物,蕨类植物尚未见此方面的报道。该研究首次采用透射电镜和免疫荧光技术对蕨类植物紫萁(Osmunda japonica Thunb.)绒毡层细胞凋亡的细胞学过程进行了观察,以明确紫萁绒毡层细胞的发育类型和凋亡特征,为蕨类植物绒毡层细胞凋亡的深入研究以及孢子发育研究提供依据。结果显示：(1)紫萁的绒毡层属于复合型,即外层绒毡层为分泌型,该层细胞发育过程中液泡化,营养物质被吸收;内层绒毡层为原生质团型,经历了细胞凋亡的过程。(2)绒毡层内层细胞在凋亡过程中细胞壁和细胞膜降解,细胞质浓缩且空泡化;细胞核内陷、变形,染色质浓缩凝聚,形成多数小核仁,DAPI荧光由强变弱;线粒体、质体、内质网、高尔基体等细胞器逐渐退化,液泡中多包含纤维状物、絮状物、黑色嗜锇颗粒和小囊泡等;出现多泡体、多膜体和细胞质凋亡小体,上述特征与种子植物绒毡层凋亡特征基本一致。(3)与种子植物相比,紫萁绒毡层的细胞凋亡开始得早,在整个凋亡过程中没有核凋亡小体的产生;除了产生孢粉素外,绒毡层细胞内产生了大量的丝状物质、絮状物质和电子染色暗的颗粒物,这些物质可能用于...     

自噬与细胞代谢及疾病关系的研究进展

自噬是以细胞质空泡化为特征的依赖于溶酶体的一种降解途径,是真核细胞特有的普遍生命现象。自噬利用溶酶体降解自身损伤的细胞质和细胞器,降解产物可用于能量生成、新的蛋白质和质膜的合成,以供细胞代谢和老化损伤细胞成分的更新,维持细胞存活、分化、发育和内环境稳态。自噬广泛参与多种生理和病理过程。对自噬与细胞代谢及疾病发生的关系作一概述。     

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。     

Vital staining of autophagic vacuoles in differentiating cells of Dictyostelium discoideum

Abstract. Vital staining of differentiating slime mold cells of Dictyostelium discoideum was studied, with reference to autophagic vacuoles they contain. By microscopically comparing within individual cells neutral-red staining granules with acid phosphatase positive granules, we identified vitally stained granules as autophagic vacuoles. At the early stages of differentiation, although the majority of cells were strongly acid phosphatase positive and there was little difference in the number of autophagic vacuoles between prestalk and prespore cells, only the former (about 25% of the total) were strongly vitally stained. It was thus concluded that autophagic vacuoles of prestalk cells are intensely stained with neutral-red while those of prespore cells are only weakly stained. Strong vital staining of prestalk cells was bleached by lysosomotropic agents such as NH₄Cl, methylamine, and chloroquine which are known to increase intra-lysosomal pH. This suggests that autophagic vacuoles of prestalk cells are strongly stained because of their acidity.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Cyclic AMP is known to function as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the prespore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Abstract. Cyclic AMP is known to function as the chemo-tactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyosteliwn discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultra-microtechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the pre-spore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development

Abstract The novel gene dia2 (differentiation-associated gene 2) was originally isolated as a gene expressed specifically in response to initial differentiation of Dictyostelium discoideum Ax-2 cells. Using dia2^AS cells in which the dia2 expression was inactivated by the antisense RNA method, DIA2 protein was found to be required for cAMP signaling during cell aggregation. During late development, the DIA2 protein changed its location from the endoplasmic reticulum (ER) to prespore-specific vacuoles (PSVs) that are specifically present in prespore cells of the slug. In differentiating prestalk cells, however, DIA2 was found to be nearly lost from the cells. Importantly, exocytosis of PSVs from prespore cells and the subsequent spore differentiation were almost completely impaired in dia2^AS cells. In addition, spore induction by externally applied 8-bromo cAMP was significantly suppressed in dia2^AS cells. Taken together, these results strongly suggested that DIA2 might be closely involved in cAMP signaling and spore differentiation as well as in the initiation of differentiation during Dictyostelium development.     

The requirement for DIF for prestalk and stalk cell formation in Dictyostelium discoideum: a comparison of in vivo and in vitro differentiation conditions

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7-14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 Mr antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8-12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation (A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93-100), but do not become independent of DIF until the culmination stage when differentiating in vivo.     

Analysis of 5' nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.     

盘基网柄菌发育期间gp150蛋白与蛋白激酶A相关性的研究

盘基网柄菌进入多细胞发育阶段后,野生型KAx-3细胞的盘基网柄菌蛋白激酶A（DdPKA）活性分别在12,16,20h时显著升高,这一变化趋势与细胞形态学上的分化有关;而突变型AK127细胞（gp150蛋白缺失）的DdPKA活性则一直保持在较高水平,直至22h才缓慢下降。两种细胞类型中24h的DdPKA活性都再一次升高。总体而言,AK127细胞的DdPKA活性要比KAx-3细胞高。这表明AK127细胞可能因缺失了gp150蛋白而导致DdPKA活性调控失去控制。在KAx-3细胞的分化过程中,前柄细胞（prestalk cells,pst）DdPKA的活性在16～18h缓慢上升,但在20h时显著下降;前孢子细胞（prespore cells,psp）中DdPKA的活性在18h时显著下降,但在20h时又迅速恢复,并达到前柄细胞中DdPKA活性的两倍。激光共聚焦结果显示,在KAx-3发育的关键阶段,DdPKA两种亚基的胞内定位并不一致,DdPKA-R亚基在空间位置上更为靠近gp150蛋白,甚至互相重叠。以上结果表明,gp150蛋白可能通过影响DdPKA-R的活性来调控前柄细胞的凋亡和前孢子细胞的分化。