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Effects of inhibition of gap junctional intercellular communication on neural differentiation of mouse embryonic stem cells in vitro

抑制间隙连接细胞间通讯对体外小鼠胚胎干细胞神经分化的影响

基本信息

批准号：
09670233
负责人：
OYAMADA Yumiko
金额：
$ 2.11万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

1. Establishment of an in vitro system for neural differentiation using mouse embryonic stem (ES) cells in vitroWe established an in vitro system for neural differentiation using mouse ES cells. Undifferentiated ES cells were cultured in suspension for 8 days and treated with all-trans retinoic acid during the latter half of the period. Thereafter, cell were transferred to adhesion culture. Neurites were observed in more than 90% of embryoid bodies. We dispersed embryoid bodies by treatment with trypsin and developed a dispersed cell culture system for early neuronal cells. In this system, cells positive for neuronal markers formed small colonies after one day of culture.2. Changes in gap junctional intercellular communication during neural differentiation of ES cells in vitroWe measured gap junctional intercellular communication by microinjection tracer coupling with neurobiotin. Microinjected neurobiotin was found to spread into all cells constituting colonies that expressed A2B5 at day 1 of adhesion culture, but the dye did not spread into flat cells when there was no A2B5 expression around the colony. After day 3 of adhesion culture, neurobiotin injected into a single cell in the colony was restricted to small portions of the colony. These results suggest that compartmentalization of gap junctional communication within neural cells decreases rapidly during the process of neural differentiation.3. Preparation of a dominant-negative Cx43 expression vectorWe prepared a dominant-negative Cx43-green fluorescent protein (GFP) expression vector and transfected into a cell line expressing wild-type Cx43. This vector showed dominant negative effects on wild-type Cx43.4. In vitro differentiation of ES cells deficient in Cx43We found that Cx43 -/- ES cells differentiated into spontaneously contracting cardiomyocytes in vitro similar to the wild-type cells although they showed a very small extent of Lucifer Yellow dye coupling at all stages of in vitro differentiation.

1.小鼠胚胎干细胞体外神经分化体系的建立我们建立了小鼠胚胎干细胞体外神经分化体系。将未分化的ES细胞悬浮培养8天，并在后半期用全反式维甲酸处理。此后，将细胞转移至粘附培养。在90%以上的胚状体中观察到神经突。我们用胰蛋白酶处理分散胚状体，并建立了早期神经元细胞的分散细胞培养系统。在该系统中，神经元标记物阳性的细胞在培养一天后形成小集落.胚胎干细胞体外神经分化过程中缝隙连接通讯的变化我们用微量注射示踪剂偶联神经生物素的方法测定了胚胎干细胞缝隙连接通讯。发现显微注射的神经生物素在粘附培养的第1天扩散到构成表达A2 B5的集落的所有细胞中，但是当在殖民地周围没有A2 B5表达时，染料没有扩散到扁平细胞中。在粘附培养第3天后，注射到殖民地中的单个细胞中的神经生物素被限制在殖民地的小部分。这些结果表明，在神经分化过程中，神经细胞内缝隙连接通讯的区室化程度迅速降低.制备显性阴性Cx43表达载体我们制备了显性阴性Cx43-绿色荧光蛋白（GFP）表达载体，并转染到表达野生型Cx43的细胞系中。该载体对野生型Cx43.4表现出显性负效应。Cx43缺陷的ES细胞的体外分化我们发现Cx43 -/- ES细胞在体外分化成与野生型细胞相似的自发收缩的心肌细胞，尽管它们在体外分化的所有阶段都显示出非常小程度的荧光黄染料偶联。