上皮型钙粘蛋白基因稳定转染小鼠胚胎干细胞及对分化细胞黏附能力的影响(英文)

Stable transfection of E-cadherin gene into mouse embryonic stem cells and its effects on adhesive capacity of differentiated cells

背景:在胚胎期,上皮型钙粘蛋白的表达对于肝组织的形成有决定作用。目的:将上皮型钙粘蛋白基因转染小鼠胚胎干细胞,观察其对分化细胞间黏附能力变化的影响。设计、时间及地点:细胞学体外观察,于2007-12/2008-12在中山大学附属第一医院外科实验室完成。材料:清洁级BALB/c系孕13d小鼠由中山大学实验动物中心提供。BALB/c系小鼠胚胎干细胞由中山大学眼科中心黄冰教授建系和保存。含CMV启动子的真核表达质粒pEGFP-N1由中山大学医学院吕志跃博士惠赠。方法:取BALB/c小鼠新鲜肝脏组织,提取总RNA,反转录合成cDNA。以合成的cDNA为模板,进行PCR扩增目的片段,将其和pEGFP-N1双酶切后连接构建pEGFP-上皮型钙粘蛋白,转染小鼠胚胎干细胞并进行体外分化。主要观察指标:RT-PCR及免疫细胞化学检测上皮型钙粘蛋白在分化系统中的动态表达,同时观察分化细胞间黏附能力的变化。结果:基因转染的胚胎干细胞分化后1～17d稳定表达上皮型钙粘蛋白,而普通胚胎干细胞分化后上皮型钙粘蛋白的表达则逐渐降低。稳定表达上皮型钙粘蛋白的分化细胞间黏附能力明显增强,并在19d时仍能保持致密的细胞连接和多层生长状态,普通胚胎干细胞则由拟胚体结构逐渐分化为松散的细胞群落。结论:稳定表达上皮型钙粘蛋白的胚胎干细胞分化后,细胞间黏附能力明显增强,并保持多层细胞生长状态。

BACKGROUND：During the embryonic stage,E-cadherin expression plays a critical role in the formation of hepatic tissue.OBJECTIVE：E-cadherin gene was transfected into mouse embryonic stem cells （ESCs） to observe its effects on adhesive capacity of differentiated cells.DESIGN,TIME AND SETTING：A cytological in vitro observation was performed at the Laboratory of Surgery,First Hospital Affiliated to Sun Yat-sen University between December 2007 and December 2008.MATERIALS：BALB/c mice at gestational 13 days,of clean grade,were provided by Laboratory Animal Center,Sun Yat-sen University.BALB/c mouse ESCs were preserved by professor Huang Bing from the Department of Ophthalmology,Sun Yat-sen University.CMV promoter-containing eukaryotic expression plasmid pEGFP-N1 was gifted by doctor Lü Zhi-yue from Medical College,Sun Yat-sen University.METHODS：Total RNA was extracted from BALB/c mouse fresh hepatic tissue and synthesized into cDNA by reverse transcription （RT）.The synthesized cDNA was used as a template to perform a polymerase chain reaction （PCR） that amplifies a targeted fragment.Following double enzyme digestion,pEGFP-E-cadherin plasmids were reconstructed and transfected into mouse ESCs.In vitro differentiation of transfected mouse ESCs was performed.MAIN OUTCOME MEASURES：Detection of E-cadherin expression in the differentiation system using RT-PCR and immunocytochemistry and observation of adhesive capacity of differentiated cells.RESULTS：E-cadherin gene-transfected ESCs could stably express E-cadherin during differentiational 1-17 days,while non-transfected ESCs expressed a decreasing amount of E-cadherin.The adhesive capacity of differentiated cells that stably expressed E-cadherin was markedly enhanced.Compact cell connection and multi-layer growth state remained at 19 days.While non-transfected ESCs gradually changed from embryoid bodies into noncohesive cell populations.CONCLUSION：Differentiating E-cadherin ESCs exhibit markedly enhanced adhesive capacity and maintain multi-layer growth state.