外源TGF-β1基因在小鼠ES细胞中的表达及其对细胞分化的影响  

EXPRESSION OF EXOGENOUS PORCINE TRANSFORMING GROWTH FACTOR BETA-1 GENE IN ES CELLS AND ITS EFFECT ON THEIR DIFFERENTIATION IN VITRO

作　　者：丛笑倩[1] 夏顺汉[1] 徐露霞 李秀兰[1] 施渭康[1] 姚(?) 

机构地区：[1]中国科学院上海细胞生物学研究所,上海200031

出　　处：《实验生物学报》1995年第2期173-189,共17页Acta Biologiae Experimentalis Sinica

基　　金：国家自然科学基金

摘　　要：本文利用逆转录病毒载体Dol,在其BamHⅠ酶切位点插入猪TGF-β1的1.7kbcDNA,构建成表达质粒,并用磷酸钙沉淀法将该质粒DNA转染到小鼠ES-5细胞,经G_(418)筛选获得抗G_(418)的ES-5细胞克隆(ES-T),经RNA点杂交,Northern印迹杂交证明有6个细胞克隆能表达外源猪TGF-β1的mRNA,其中两个杂交信号较强的克隆进一步用Southern印迹杂交,也证明猪TGF-β1基因已整合到ES-5细胞基因组。经SELISA和生物活性测定,证明ES-T细胞存在着过度表达的TGF-β1基因产物。ES-T细胞生长特性与其亲代ES-5细胞相比,未发现明显差异。经低浓度RA处理悬滴培养的ES-T6细胞,将形成的拟胚体移至用白明胶铺底的培养板中,则最终有95%左右的拟胚体分化出血管样结构,而ES-5细胞在相同培养和处理条件下,只有17.8%形成无规则的管状结构,这结果提示ES-T6细胞基因组中外源TGF-β_1的额外表达,在调节该细胞株分化形成血管样结构中起着重要作用。同时,我们的实验体系也提供了一个适合于研究内皮细胞发生和血管形成的模型。A TGF-β1 gene expression plasmid was constructed by inserting the porcine 1.7Kb TGF-β1 cDNA into BamHI site of retrovirus vector Dol. The plasmid DNA was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and tran-sfected ES-5 cells were then selected by stepwise increase in G418 concentration. Finally, we obtained 21 clones that could be stably grown in culture medium with G418 at 500 μg/ml and were designated as ES-T cells. Dot blot and Northern analysis of total RNA and polyA+ RNA extracted from those ES-T cells were shown in Fig. 2 and 3, demonstrating that 6 clones could express exogenous porcine TGF-β1 mRNA. The stronger hybridized signal in two clones (ES-T6 and ES-T16) of them were further proved by southern hybridization of genomic DNA from these ES-T cells with 1.7Kb TGF-β1 cDNA probe (Fig. 4). The product of TGF-β1 gene overexpression in ES-T 6 cells was shown in Fig. 5 and 6 by SE-LISA for TGF-β1 immunoreactivity to TGF-β1 antibodies and biological assay for CCL/64 cell growth inhibition, res-pectivly. With respect to some biological characteristics, ES-T 6 cells, like their parent ES-5 cells, retained their pluripo-tent properties and positive SSEA-1 antigen (Plate I, Fig. 1).ES-T6 cells were expanded and used for studies of in vitro differentiation. Both of ES-T 6 cells and control ES-5 cells could form a lot of simple aggregates and differentiate into embryoid bodies by hanging drop culture for 3 days in the presence of retinoic acid (RA) at 10-9mol/L. Then individual embryoid bodies were plated on gelatinized tissue culture wells. On the third day of further culture without RA, a large amounts of epithelial-like and round cells occurred around the embryoid bodies formed either from ES-T6 cells or ES-5 cells (Plate I, Fig. 4). However, with further culture of embryoid bodies, only the cells differentiated from ES-T6 embryoid bodies could arrange themselves and differentiate into a lot of radially arranged tubular structures (Plate Ⅱ Fig. 5).

关 键 词：胚胎干细胞 TGF-Β1基因 细胞分化 

分 类 号：Q28[生物学—细胞生物学]