维甲酸对人神经母细胞瘤SK—N—SH细胞形态结构和分化标志物表达的影响

目的：观察维甲酸对人神经母细胞瘤SK-N-SH细胞形态与超微结构及其相关标志物表达的影响,以鉴定其对神经母细胞瘤细胞终末分化的诱导作用。方法：1μmol／L维甲酸处理SK—N—SH细胞,光镜、电镜和免疫细胞化学检测研究SK—N—SH细胞处理前后细胞形态、超微结构变化和神经元相关标志物的表达变化。结果：光镜与电镜观察结果显示,SK—N—SH细胞经1μmol／LRA处理后,细胞形态和超微结构产生了细胞呈极性状、伸出多个轴突树突状突起、细胞逐渐变小变圆并融合在一起形成类似神经节样结构、细胞表面微绒毛减少、核仁变少变小、常染色质增多、细胞器丰富发达等显著变化;免疫细胞化学检测显示经RA处理后SK-N-SH细胞NSE、MAP2、Synaptophysin的表达较对照组细胞明显加强。结论：维甲酸能改变SK—N—SH细胞形态和超微结构恶性表型特征,并促进与神经细胞相关的终末分化指标的表达,从而对人神经母细胞瘤细胞的终末分化具有显著的诱导作用。     

Terminal Differentiation of Normal Human Oral Keratinocytes Is Associated with Enhanced Cellular TGF-β and Phospholipase C-γ1 Levels and Apoptotic Cell Death

Subculture of primary normal human oral keratinocytes (NHOK) results in terminal differentiation, leading to cell death. To investigate whether the subculture-induced death of NHOK is due to apoptosis, we studied transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, DNA fragmentation, and expression of several apoptosis-associated genes from NHOK with different passage numbers. We also determined the effect of transforming growth factor β₁ (TGF-β₁) on the induction of apoptosis in NHOK. We were able to subculture primary NHOK up to the fifth passage, at which point cells showed morphological features of differentiation. Appearance of DNA fragmentation concurrently occurred with an increase in the number of TUNEL-positive cells with higher passage numbers. The level of cellular p53 proteins was gradually decreased by the continued passage of cells, whereas the levels of intracellular and secreted TGF-β and phospholipase C-γ1 (PLC-γ1) were significantly elevated by serial subculture. Exogenous TGF-β₁ also induced differentiation and apoptosis of proliferating NHOK. These data indicate that terminal differentiation of NHOK is associated with apoptosis, which is, in part, linked to elevated cellular levels of TGF-β and PLC-γ1.     

Apoptotic cleavage of NuMA at the C-terminal end is related to nuclear disruption and death amplification

NuMA is a nuclear matrix protein in interphase and distributes to the spindle poles during mitosis. While the essential function of NuMA for mitotic spindle assembly is well established, a structural role of NuMA in interphase nucleus has also been proposed. Several observations suggest that the apoptotic degradation of NuMA may relate to chromatin condensation and micronucleation. Here we demonstrate that four apoptotic cleavage sites are clustered at a junction between the globular tail and the central coiled-coil domains of NuMA. Cleavage of a caspase-6-sensitive site at D¹⁷⁰⁵ produced the R-form, a major tail-less product of NuMA during apoptosis. The other two cleavage sites were defined at D¹⁷²⁶ and D¹⁷⁴⁷ that were catalyzed, respectively, by caspase-3 and an unknown aspartase. A NuMA deletion mutant missing the entire cleavage region of residues 1701–1828 resisted degradation and protected cells from nuclear disruption upon apoptotic attack. Under such conditions, cytochrome c was released from mitochondria, but the subsequent apoptotic events such as caspase-3 activation, poly(ADP-ribose) polymerase degradation, and DNA fragmentation were attenuated. Conversely, the tail-less NuMA alone, a mutant mimicking the R-form, induced chromatin condensation and activated the death machinery. It supports that intact NuMA is a structural element in maintaining nuclear integrity.     

Centriolar Mechanisms of Differentiation and Replicative Aging of Higher Animal Cells

The centrosome (centriole) and the cytoskeleton produced by it are structures, which probably determine differentiation, morphogenesis, and switching on the mechanism of replicative aging in all somatic cells of multicellular animals. The mechanism of such programming of the events seems to include cytoskeleton influences and small RNAs related to the centrosome. 1) If these functions are really related with centrioles, the multicellular organism's cells which: a) initially lack centrioles (e.g., higher plant cells and also zygote and early blastomeres of some animals) or cytoskeleton (e.g., embryonic stem cells); or b) generate centrioles de novo (e.g., zygote and early blastomeres of some animals), will be totipotent and lack replicative aging. Consequently, the absence (constant or temporary) of the structure determining the counting of divisions also means the absence of counting of differentiation processes. 2) Although a particular damage to centrioles or cytoskeleton (e.g., in tumor cells) fails to make the cells totipotent (because the morphogenetic status of these cells, as differentiated from that of totipotent ones, is not zero), but such a transformation can suppress the initiation of the aging mechanism induced by these structures and, thus, make such cells replicatively "immortal".     

Ganglioside Expression During Differentiation of Chick Retinal Cells In Vitro

The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunoflourescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.     

南方红豆杉悬浮培养细胞凋亡的组织学定位及与细胞分化的关系初探

应用末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记(TUNEL)检测技术和显微图像定量分析方法研究了悬浮培养中南方红豆杉(Taxuschinensisvar．mairei)细胞团不同区域细胞凋亡的发生,并比较了不同区域的细胞形态。结果显示细胞团可以划分为三个不同的区域核心区的细胞结构已消失,可见一些分化良好的管状结构;中层区是由增殖能力旺盛的分生组织细胞所构成;外层区是由分化成熟的细胞所组成。细胞凋亡主要发生在细胞团分化成熟的外层区。以上结果说明细胞凋亡可能是分化终结的一种形式。     

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。     

胚胎干细胞的生物学特性及体外诱导分化

来源于早期胚胎的胚胎干细胞 (ES)和胎儿生殖脊干细胞 (EG)可在未分化状态下长期增殖培养 ,并保持其多向分化潜能 .体外培养ES细胞的条件已趋于稳定 ,国内外建立了来自人和多种动物早期胚胎的ES细胞系 .某些细胞因子和化学物质等可定向诱导ES细胞分化为各种不同类型的组织细胞 .ES细胞在胚胎发育、细胞分化、转基因动物、移植治疗、药物开发等领域具有广阔的应用前景 .     

胚胎干细胞分化为血管内皮细胞的分子调控机制

胚胎干细胞在不同的诱导条件下具有多向分化的潜能,多种胞内外信号途径参与其分化过程的调控。现就胚胎干细胞向血管内皮细胞分化的诱导条件及分子机制做一综述,并阐明不同阶段的内皮前体细胞所表达的不同分子标志,同时提出胚胎干细胞在再生医学中的应用前景。     

RCMV感染星形胶质细胞对神经干细胞分化的影响

探讨大鼠巨细胞病毒（rat cytomegalovirus,RCMV）感染大鼠星形胶质细胞后,对神经干细胞分化的影响。原代分离培养新生大鼠星形胶质细胞和胚胎海马神经干细胞,将星形胶质细胞感染RCMV后和神经干细胞在Transwell24孔共培养体系下进行共培养,同时设对照组;用免疫荧光染色等方法检测神经干细胞与感染RCMV的星形胶质细胞共培养后,其分化细胞中神经元微管相关蛋白（microtubule-associated protein 2,MAP2）和星形胶质细胞胶质纤维酸性蛋白（glial fibril—lary acidic protein,GFAP）的表达。结果发现,感染RCMV的星形胶质细胞与神经干细胞共培养时,神经干细胞分化减慢,分化成的神经元和星形胶质细胞比率低于对照组,提示星形胶质细胞感染RCMV后可抑制神经干细胞的分化,可能与RCMV影响星形胶质细胞合成和分泌各种营养因子,干扰了神经干细胞的分化进程有关。     

Hepatic Sinusoidal Endothelium Heterogeneity with Respect to the Recognition of Apoptotic Cells

The recognition and removal of human apoptotic peripheral lymphocytes in selected populations of periportal and perivenous endothelial cells was studied inin situandin vitroexperiments. Apoptotic peripheral blood lymphocytes once injected into the liver circulation were retained by the sinusoids showing a large heterogeneity of distribution: apoptotic cells are found in the periportal tract double the amount found in the perivenous region. Apoptotic PBL adhesion was lowered to a sixth of the control after preinjection with a sugar mixture (Mannose,N-acetylgalactosamine,N-acetylglucosamine,d-galactose), as suggested by the expression of modified surface glycoconjugates on the plasma membrane of apoptotic cells. A bimodal profile of the distribution of the hepatic sinusoidal cell population, regarding the number of galactose and mannose receptors and the porosity index, was found. Two endothelial cell subsets were present: low porosity cells (average index 14 ± 6%; periportal tract) with a high number of carbohydrate binding sites, and high porosity cells (average index 26 ± 7%; perivenous tract), with a low number of carbohydrate binding sites.     

The Strength of Mechanical Forces Determines the Differentiation of Alveolar Epithelial Cells

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山羊精原干细胞体外培养分化

探索山羊精原干细胞体外培养体系。收集2月龄关中奶山羊睾丸,一步酶法消化分离曲细精管细胞,台盼兰检测平均存活率82.7%,以1×106个/ml接种含15%胎牛血清DMEM/F12培养瓶,37℃、5%CO2和饱和湿度条件下培养,4周后FBS逐渐降至10%。原代培养以多突起和片状的睾丸体细胞铺壁生长为主,10天左右精原干细胞数量增加,可见二联体和四联体,3周左右有鸟巢状和山脉状集落形成,碱性磷酸酶染色阳性,培养30天集落数不断增加,散在分布有贴壁和漂浮精子,换液后精子丢失。挑取单集落重新接种铺壁的曲细精管体细胞饲养层后陆续有精子细胞及精子形成,主要分布于集落周围。     

小鼠肌母细胞的增殖与分化

通过对小鼠肌母细胞C2C12的培养,研究C2C12细胞的增殖与分化的关系以及胰岛素在细胞分化过程中的作用。在对照组中,C2C12细胞增殖占了明显的优势,细胞形态几乎没有发生变化;而在实验组中,C2C12细胞在换为分化培养基24小时后,就出现了部分细胞衰亡和死亡的现象,尤其是在48小时细胞的死亡率达到最高,存活细胞开始从增殖期进入分化期,72小时出现了少量肌管,在96小时细胞分化效果达到最好。而在添加了胰岛素的分化培养基中的细胞分化效果明显好于没有添加胰岛素的分化培养基中的细胞,结果表明,胰岛素促进C2C12细胞的分化。     

Heat Shock Stress Induces Cleavage and Activation of PAK2 in Apoptotic Cells

Heat shock induces a stress response in mammalian cells and can also lead to apoptotic cell death. Here we report that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be drastically activated in several cell types by heat shock. Immunoblot analysis revealed that this 36-kDa MBP kinase can be recognized by an antibody against the C-terminal region of a family of p21^Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as tools, we further demonstrated that heat shock can induce cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment in mouse Balb/c 3T3 and human Hep 3B cells. The kinetic profile of appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in these cells induced by heat shock. In addition, the heat shock-induced cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in heat shock-treated Hep 3B cells. Moreover, blockage of the activation of caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors of caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could substantially diminish the extent of heat shock-induced cleavage/activation of PAK2. Overall, our results point out that PAK2 is cleaved and activated during the heat shock-induced apoptotic cell death process and suggest that caspase-3 is involved in this process.