FUNCTIONAL ROLE OF EPH RPTK IN NEURONAL DIFFERENTIATION

本项目的目标是了解可能的作用， Eph受体酪氨酸激酶（RPTK）可能在神经元凋亡中起作用。 分化 RN 33 B细胞系在体内分化， 移植到新生和成年大鼠脑中。 该细胞系 显示了神经突生长的形成和典型的 内源性神经元移植到大鼠大脑皮层时， 海马体。 高水平的Eph RPTK配体表达， 中枢神经系统，沿着至少一种Eph的表达 RPTK配体在中枢神经系统中的表达，沿着 RN 33 B细胞中至少一种ephRTK的表达，使我们 假设这种蛋白质可以介导 在体内观察。 此外，已经有七种ph RPTK被 与轴突靶向有关，这表明RPTKs家族 参与了神经分化的过程 解决 问题，我们将筛选和测序来自RN##B的克隆 细胞使用RT-PCR与简并寡核苷酸为 eph RPTK的保守区域。 一旦Eph RPTK的类型 已经确定，在mRNA和蛋白质的发育研究 将使用北方印迹、西方印迹和 免疫组织化学技术对RN 33 B细胞进行体外研究。 的 将通过使用EphRPTK来测试其在分化中的可能作用。 显性阴性构建体，以使RN 33 B细胞的Eph RPTK，并分析其对移栽后形态建成的影响 注入新生大鼠的大脑 该分析将使一个重要的 对了解体外神经元分化的贡献 在体内，当神经细胞系用作模型系统时， 研究神经移植过程中培养物的分化。

The objective of this project is to understand the possible role that eph receptor tyrosine kinases (RPTK) may play during neuronal differentiation. The RN33B cell line differentiates in vivo after transplantation into the neonatal and adult rat brain. This cell line shows neurite outgrowth formation and a morphology typical of endogenous neurons when transplanted in the rat cerebral cortex or hippocampus. The high levels of eph RPTK ligand expression in the central nervous system, along with the expression of at least one eph RPTK ligand expression in the central nervous system, along with expression of at least one eph RTK in RN33B cells, has led us to hypothesize that this protein could mediate the morphogenesis observed in vivo. In addition, sever eph RPTKs have been implicated in axonal targeting, suggesting that this family of RPTKs are involved in the process of neural differentiation. To address the question, we will screen and sequence clones derived from RN##B cells using RT-PCR with degenerate oligonucleotides for the conserved regions of the eph RPTK. Once the type(s) of eph RPTK has been determined, developmental studies at the mRNA and protein level will be done with Northern blotting, Western blotting and immunohistochemistry techniques on RN33B cells in vitro. The possible role of eph RPTK in differentiation will be tested by the use of a dominant negative construct, to inactivate the RN33B cell eph RPTK, and analyze its effect on morphogenesis after transplanted into the neonatal rat brain. The analysis will make a significant contribution to the understanding of neuronal differentiation in vitro and in vivo, when a neural cell line is used as a model system to study differentiation in culture during neural grafting.