REGULATING EMBRYO CARDIOMYOCYTE CELL DIVISION--IMPLICATIONS FOR GENE THERAPY

两条生长因子途径在心脏功能调节中发挥重要作用 胚胎中的肌细胞增殖。这些是成纤维细胞的生长 成纤维细胞生长因子和转化生长因子-β因子及其同源受体 转化生长因子-BR)。我们的研究小组(Mima等人；1995)已经证明了成纤维细胞生长因子信号转导 是心肌细胞早期体内增殖所必需的 胚胎心脏(胚胎雏鸡发育的第1周)。按周-2 在发育过程中，心肌细胞的生长变得不依赖于成纤维细胞生长因子，而这 与心肌细胞周期动力学的延长相吻合。研究人员 Sporn和他的同事(Letterio et al；1994)已经证明了基因 小鼠接受转化生长因子-β的敲除导致显著的增厚 心肌壁，提示转化生长因子-β信号转导起负性作用 控制心肌细胞增殖。细胞培养研究也支持这一点。 结论。因此，对心肌细胞分裂的调节涉及到正性。 (成纤维细胞生长因子)和负性(转化生长因子-β)途径。我们建议今后尝试 控制成人心肌细胞的增殖需要了解 这两条信号通路以及它们是如何在 出生后。我们计划用鸡胚胎进行三组实验。(1) 探讨成纤维细胞生长因子信号在调控心肌细胞增殖中的作用 小管状胚胎雏鸡心脏中的肌细胞。这将涉及：(A) 心肌细胞FGFR1在日龄间的上调和下调 利用复制缺陷逆转录病毒进行胚胎发育的E3和E7 构建；以及(B)FGFR1和成纤维细胞生长因子配体在 使用双顺反子逆转录病毒载体的肌细胞。(2)审视负面影响 显性非显性转化生长因子-β调控心肌细胞分裂 受体的构建破坏了这一信号通路。(3)使用 复制缺陷型腺病毒载体研究转化生长因子-β和成纤维细胞生长因子的作用 在细胞周期中退出细胞周期的肌细胞中的信号 胚胎雏鸡发育的第二周。雏鸡胚胎被选中 作为模型系统是因为：a)雏鸡心脏的早期形态发生 胚胎与小鼠、大鼠或人的胚胎基本相同；b)细胞周期 鸡的调控与高等哺乳动物的调控没有什么不同；c) 雏鸡心脏发育过程中心肌细胞增殖的下降 与人类相似；d)它提供了许多实验优势 转基因插入和操控心脏前体细胞 显微外科手术。

Two growth factor pathways play major roles in the regulation of cardiac myocyte proliferation in the embryo. These are the fibroblast growth factors (FGF) and TGF-B factors and their cognate receptors (FGFR and TGF-BR). Our group (mima et al; 1995) has demonstrated that FGF signaling is essential for the in vivo proliferation of cardiac myocytes in early embryonic hearts (week-1 of the embryonic chick development). By week-2 of development myocyte growth becomes FGF-independent, and this coincides with a prolongation of myocyte cell cycle kinetics. Studies by Sporn and colleagues (letterio et al; 1994) have shown that genetic knockouts of TGF-B reception in the mouse cause a dramatic thickening of the myocardial wall, suggesting that TGF-B signaling plays a negative control on myocyte proliferation. Cell culture studies also support this conclusion. Thus, regulation of myocyte cell division involves positive (FGF) and negative (TGF-B) pathways. We suggest that future attempts to control myocyte proliferation in the adult will require an understanding of these two signaling pathways and how they are regulated pre- and postnatally. We plan three sets of experiments using chicken embryos. (1) explore the role of FGF signaling in regulating the proliferation of cardia myocytes in the tubular stage embryonic chick heart. This will involve; (a) the up- and down-regulation of FGFR1 in cardiac myocytes between days E3 and E7 of embryonic development using replication-defective retroviral constructs; and (b) the co-expression of FGFR1 plus the FGF ligand in myocytes using a dicistronic retroviral vector. (2) examine the negative control of myocyte cell division by TGF-B using dominant-negative TGF-B receptor constructs to disrupt this signaling pathway. (3) Using replication-defective adenoviral vectors, examine the role of TGF-B and FGF signaling in myocytes which are withdrawing from the cell cycle during the week-2 of embryonic chick development. The chick embryo was selected as a model system because: a) early morphogeneisis of the heart in chick embryos is essentially identical to that in mouse, rat or man; b) cell cycl regulation in the chicken is no differnet from that in higher mammals;c) the decline in myocyte proliferation during chick heart development mirrors that of man; d) it offers many experimental advantages for transgene insertion and manipilation of cardiac precursors by microsurgery.