Reis Ricardo A M, Cabral da Silva Maurício C, Loureiro dos Santos Nelson E, Bampton Edward, Taylor Jeremy S H, de Mello Fernando G, Linden Rafael
Laboratório de Neuroquímica, IBCCF(o), UFRJ, Rio de Janeiro 21949-900, Brazil.
J Neurobiol. 2002 Jan;50(1):13-23. doi: 10.1002/neu.10008.
Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.
脊椎动物外周神经系统中的神经元存活依赖于靶组织提供的神经营养因子。为了鉴定新的存活因子，我们研究了视网膜细胞分泌因子对鸡交感神经节神经元存活的影响。胚胎第10天的交感神经元在没有适当水平神经生长因子（NGF）的情况下，48小时后会经历程序性细胞死亡。来自胚胎第11天视网膜外植体并在体外培养4天的视网膜条件培养基（RCM），在48小时后能支持90%的胚胎第10天鸡交感神经元存活。来自纯化的鸡视网膜穆勒神经胶质细胞的条件培养基能支持近100%的胚胎第10天鸡交感神经元存活。抗NGF（1微克/毫升）阻断了NGF的存活效应，但未阻断RCM的营养效应。脑源性神经营养因子（BDNF）和神经营养因子4（NT4，0.1 - 50纳克/毫升）均不能支持胚胎第10天交感神经元的存活。嵌合免疫球蛋白受体TrkA、TrkB或TrkC的孵育对RCM诱导的交感神经元存活没有影响。抗胶质细胞源性神经营养因子（GDNF）、抗转化生长因子β（TGFβ）和抗睫状神经营养因子（CNTF）不能阻断存活效应，碱性成纤维细胞生长因子（FGFb，0.1 - 10纳摩尔）也不能模拟这种效应。50微摩尔的LY294002可阻断RCM诱导的交感神经元存活，而PD098059则不能。此外，与单独使用NGF相比（48小时后82%的存活率），RCM和NGF联合使用并未导致神经元存活率增加。RCM中的分泌因子保留在分子量高于100千道尔顿的亚组分中，与肝素结合，不受透析影响，但对热敏感。我们的结果表明存在一种高分子量的视网膜分泌因子，它能在培养中支持交感神经元存活。
