REGULATING EMBRYO CARDIOMYOCYTE CELL DIVISION--IMPLICATIONS FOR GENE THERAPY

两种生长因子途径在心脏的调节中起主要作用， 胚胎中的肌细胞增殖。这些是成纤维细胞的生长 因子（FGF）和TGF-β因子及其同源受体（FGFR和TGF-β受体） TGF-BR）。我们的研究小组（米马等; 1995）已经证明，FGF信号传导 是早期心肌细胞体内增殖所必需的 胚胎心脏（鸡胚发育的第1周）。第2周 的发展，肌细胞生长成为FGF的独立，这 与肌细胞细胞周期动力学的延长相一致。的研究 Sporn及其同事（Letterio et al; 1994）已经表明， 在小鼠中，TGF-β受体的敲除引起了细胞膜的急剧增厚， 心肌壁，表明TGF-β信号转导起负作用， 控制心肌细胞增殖。细胞培养研究也支持这一点 结论因此，肌细胞分裂的调节涉及到正性的 (FGF)和负性（TGF-β）途径。我们建议，今后的努力， 控制成人心肌细胞增殖需要了解 这两种信号通路以及它们在 出生后我们计划用鸡胚进行三组实验。（一） 探讨成纤维细胞生长因子信号通路在调控贲门增殖中的作用 鸡胚心脏管状期的肌细胞。这将涉及：（a） 心肌细胞中FGFR 1的上调和下调 使用复制缺陷型逆转录病毒的胚胎发育的E3和E7 构建体;和（B）FGFR 1加上FGF配体在 使用双顺反子逆转录病毒载体。(2)检查底片 使用显性负性TGF-β通过TGF-β控制肌细胞分裂 受体结构来破坏这种信号通路。(3)使用 复制缺陷型腺病毒载体，检查TGF-β和FGF的作用 从细胞周期中退出的肌细胞中的信号传导 鸡胚发育的第2周。选用鸡胚 作为模型系统，因为：a）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 鸡的调节与高等哺乳动物的调节没有区别;c） 鸡心脏发育过程中心肌细胞增殖的下降 反映了人的; d）它提供了许多实验优势， 转基因插入和操纵心脏前体细胞 显微手术

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.