Du Z W, Tsung H C, Yao Z
Shanghai Institute of Cell Biology, Academia Sinica, Shanghai 200031, China.
Shi Yan Sheng Wu Xue Bao. 1998 Jun;31(2):155-69.
We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells. ES-gamma cells retained undifferentiated morphology and positive alkaline phosphatase activity (Plate I, Fig. 1, 2), so it resembled ES-5 cells in terms of stem cell characteristics. When ES-gamma cells were subcutaneously inoculated into nude mouse and differentiated in vivo, tumorous nodules containing various tissue structures were obtained, demonstrating their pluripotent properties just like parent ES-5 cells. Contrasting with ES-5 cells, the histological features of tumors showed no cartilage tissues, but abundant muscle tissues and keratinized cyst like structures constituted by stratified squamous epithelia (Plate I, Fig. 3). Differentiating in vitro by hanging drop culture methods, ES-gamma cells differentiated mostly into fibroblast-like cells, (Plate II, Fig. 1-5). The above results indicated that overexpression of RAR gamma gene changed the cell type of ES cells differentiating in vivo and in vitro. During the differentiation of ES-5 cells induced by RA, a large number of cells rounded up, detached from the dish and tended to die. We suspected that this phenomenon may be apoptosis. The ultrastructure appearance of the dying cells displayed typical apoptotic changes including chromatin condensation and nuclear fragmentation (Plate I, Fig. 4, 5). Detection of DNA fragments using agarose gel electrophoresis showed characteristic laddered patterns of apoptotic DNA fragments (Fig. 5). The above results indicated that RA induced apoptosis of ES-5 cells in the course of differentiation. The percentage of apoptosis of ES-5 cells increased accordingly, with the increase of RA concentration (Fig. 6). With the same concentration of RA 10(-7) mol/L, the percentage of apoptotic of ES-gamma death was roughly one times more than that of ES-5 cells (Fig. 7), a fact indicating that RAR gamma may mediate the apoptotic signal transduction of ES cells by RA.
我们构建了含有小鼠视黄酸受体γ（RARγ）基因和新霉素基因的pSG5 - RARγ - neo质粒，并通过磷酸钙介导的转染将其导入胚胎干细胞ES - 5细胞。分离出一些对G418有抗性的克隆，通过对这些克隆进行RNA斑点印迹分析，建立了一个过表达RARγ基因的克隆，命名为ES - γ细胞系。对ES - γ细胞进行Northern印迹杂交和Southern印迹杂交分析（图3、4）表明，ES - γ细胞过表达外源RARγ mRNA，且外源RARγ cDNA整合到了ES细胞的基因组中。ES - γ细胞保持未分化的形态和碱性磷酸酶活性阳性（图版I，图1、2），因此在干细胞特性方面与ES - 5细胞相似。当将ES - γ细胞皮下接种到裸鼠体内并在体内分化时，获得了含有各种组织结构的肿瘤结节，表明它们与亲代ES - 5细胞一样具有多能性。与ES - 5细胞相比，肿瘤的组织学特征显示没有软骨组织，但有丰富的肌肉组织和由复层鳞状上皮构成的角化囊肿样结构（图版I，图3）。通过悬滴培养法在体外分化时，ES - γ细胞大多分化为成纤维细胞样细胞（图版II，图1 - 5）。上述结果表明，RARγ基因的过表达改变了ES细胞在体内和体外分化的细胞类型。在视黄酸（RA）诱导ES - 5细胞分化的过程中，大量细胞变圆，从培养皿中脱落并趋于死亡。我们怀疑这种现象可能是细胞凋亡。死亡细胞的超微结构表现出典型的凋亡变化，包括染色质浓缩和核碎片化（图版I，图4、5）。用琼脂糖凝胶电泳检测DNA片段显示出凋亡DNA片段的特征性梯状模式（图5）。上述结果表明，RA在ES - 5细胞分化过程中诱导了细胞凋亡。随着RA浓度的增加，ES - 5细胞的凋亡百分比相应增加（图6）。在相同浓度的RA （10⁻⁷mol/L）下，ES - γ细胞的凋亡百分比大约是ES - 5细胞的两倍（图7），这一事实表明RARγ可能介导了RA对ES细胞的凋亡信号转导。
