Represa J, Bernd P
Department of Anatomy and Cell Biology, State University of New York, Brooklyn 11203.
Dev Biol. 1989 Jul;134(1):21-9. doi: 10.1016/0012-1606(89)90074-2.
The preceding paper (P. Bernd and J. Represa, 1989, Dev. Biol. 134) describes the characterization and localization of nerve growth factor (NGF) receptors in inner ear primordia, the otic vesicle (OV) and cochleovestibular ganglion (CVG), obtained from 72-hr (stage 19-20) quail embryos. The studies described in this paper investigated whether NGF serves as a mitogen, a survival factor, and/or a differentiation factor in this system. Explants of isolated OV and CVG were maintained for 24 hr in serum-free medium alone (M-199), M-199 containing serum, M-199 containing NGF, or M-199 containing both serum and NGF. [3H]Thymidine was also present for the entire culture period. Both OV and CVG incorporated greater amounts of [3H]thymidine in the presence of serum or NGF, and their combined effect was additive. NGF's effects were dose dependent, saturable, and specific (blocked by anti-NGF). NGF caused little or no morphological differentiation of OV and no increase in protein levels, in contrast to OV grown in the presence of serum. CVG had both cochlear and vestibular portions present in all cases, but the apparent size and protein content of CVG was increased in the presence of either serum or NGF. Effects of serum and NGF were completely, but reversibly, blocked by amiloride, suggesting that the Na+-H+ exchange system had been activated. In order to determine whether increases in [3H]thymidine incorporation were due to increased cell survival or perhaps to an increase in proliferation, explants were initially grown for a 24-hr period in serum-free medium, followed by reactivation for an additional 24 hr in medium containing serum and/or NGF. It is likely that cells requiring either serum or NGF for survival would die during a 24-hr period in their absence. Our results revealed that the level of [3H]thymidine incorporation in OV was the same after reactivation. In the case of CVG, only NGF treatment yielded similar results; [3H]thymidine incorporation was lower in CVG reactivated with serum. It appears, therefore, that serum has probable proliferative effects upon OV and CVG, as well as survival effects for CVG. NGF, however, does not appear to affect survival in either OV or CVG, so that increases in [3H]thymidine incorporation in response to NGF are most likely due to proliferative effects upon OV or CVG, at least at this embryonic stage.
前文（P. 伯恩德和J. 雷普雷萨，1989年，《发育生物学》第134卷）描述了从72小时（第19 - 20阶段）鹌鹑胚胎获取的内耳原基、耳泡（OV）和蜗神经节（CVG）中神经生长因子（NGF）受体的特征及定位。本文所述研究探讨了NGF在该系统中是否作为促有丝分裂因子、存活因子和/或分化因子。将分离的OV和CVG外植体分别在无血清培养基（M - 199）、含血清的M - 199、含NGF的M - 199或含血清和NGF的M - 199中培养24小时。整个培养期间均加入[³H]胸腺嘧啶核苷。在有血清或NGF存在时，OV和CVG均掺入了更多的[³H]胸腺嘧啶核苷，且二者的联合作用是相加的。NGF的作用具有剂量依赖性、可饱和性且具有特异性（可被抗NGF阻断）。与在有血清条件下生长的OV相比，NGF几乎未引起OV的形态分化，也未使蛋白质水平增加。在所有情况下，CVG均有耳蜗和前庭部分，但在有血清或NGF存在时，CVG的表观大小和蛋白质含量增加。血清和NGF的作用完全但可逆地被阿米洛利阻断，这表明Na⁺ - H⁺交换系统已被激活。为了确定[³H]胸腺嘧啶核苷掺入量的增加是由于细胞存活率提高还是增殖增加，外植体最初在无血清培养基中培养24小时，然后在含血清和/或NGF的培养基中再培养24小时以重新激活。在缺乏血清或NGF的情况下，那些需要血清或NGF才能存活的细胞很可能在24小时内死亡。我们的结果显示，重新激活后OV中[³H]胸腺嘧啶核苷的掺入水平相同。对于CVG，只有NGF处理产生了类似结果；用血清重新激活的CVG中[³H]胸腺嘧啶核苷的掺入量较低。因此，血清似乎对OV和CVG具有增殖作用，对CVG也有存活作用。然而，NGF似乎并未影响OV或CVG的存活，所以至少在这个胚胎阶段，对NGF反应时[³H]胸腺嘧啶核苷掺入量的增加很可能是由于对OV或CVG的增殖作用。
