Schwalb J M, Boulis N M, Gu M F, Winickoff J, Jackson P S, Irwin N, Benowitz L I
Department of Neurosurgery, Children's Hospital, Boston, Massachusetts 02115, USA.
J Neurosci. 1995 Aug;15(8):5514-25. doi: 10.1523/JNEUROSCI.15-08-05514.1995.
Unlike mammals, lower vertebrates can regenerate an injured optic nerve and other pathways of the CNS throughout life. We report here that in dissociated cell culture, goldfish retinal ganglion cells regenerate their axons in response to two factors derived from the sheath cells of the optic nerve. Axogenesis factor 1 (AF-1) is a small peptide (700-900 Da) that is inactivated by treatment with proteinase K but heat stable. A second factor, AF-2, is a polypeptide of ca 12 kDa. In the absence of these factors, dissociated retinal cells remained viable in serum-free, defined media for at least a week but showed little outgrowth, as visualized using the vital dye 5,6-carboxyfluorescein diacetate (5,6-CFDA). The addition of AF-1 induced up to 25% of cells in culture to extend processes > 75 microns in length by 6 d; AF-2 had a lesser but highly significant effect. To verify that neurite outgrowth was from retinal ganglion cells per se, we applied the lipophilic dye 4-Di-10-ASP to the optic tectum and allowed it to diffuse up the optic nerve for several days before culturing the retina. A far greater percentage of cells containing the dye showed axonal outgrowth than was observed from the overall cell population, indicating that ganglion cells are selective targets of the factors. The effects of AF-1 or AF-2 were not secondary to enhanced viability, since neither overall cell survival nor the number of retinal ganglion cells remaining in culture after 6 d was affected by the presence of the factors. The activity of AF-1 and AF-2 was not mimicked by several defined factors tested over a broad concentration range, for example, NGF, BDNF, NT-3, CNTF, taurine, retinoic acid, acidic or basic fibroblast growth factors. The concentration of AF-1 is considerably higher in CM than in optic nerve homogenates, suggesting that it is actively secreted; AF-2 has a similar concentration intra- and extracellularly. Insofar as AF-1 and AF-2 derive from cells of the optic nerve and act upon retinal ganglion cells, they are likely to be important in inducing optic nerve regeneration in vivo.
与哺乳动物不同，低等脊椎动物在其一生中都能够再生受损的视神经以及中枢神经系统的其他通路。我们在此报告，在解离细胞培养中，金鱼视网膜神经节细胞可响应源自视神经鞘细胞的两种因子而再生其轴突。轴突发生因子1（AF-1）是一种小肽（700 - 900道尔顿），经蛋白酶K处理后失活但耐热。第二种因子AF-2是一种约12 kDa的多肽。在没有这些因子的情况下，解离的视网膜细胞在无血清、特定培养基中至少存活一周，但长出的突起很少，这可通过活性染料5,6 - 羧基荧光素二乙酸酯（5,6 - CFDA）观察到。添加AF-1可使培养物中高达25%的细胞在6天时伸出长度超过75微米的突起；AF-2的作用较小但非常显著。为了验证神经突生长是否源自视网膜神经节细胞本身，我们将亲脂性染料4 - Di - 10 - ASP应用于视顶盖，并在培养视网膜前让其沿视神经扩散几天。含有该染料的细胞长出轴突的比例远高于从总体细胞群体中观察到的比例，这表明神经节细胞是这些因子的选择性靶点。AF-1或AF-2的作用并非继发于活力增强，因为因子的存在既不影响总体细胞存活，也不影响培养6天后剩余的视网膜神经节细胞数量。在广泛的浓度范围内测试的几种特定因子，例如神经生长因子（NGF）、脑源性神经营养因子（BDNF）、神经营养因子-3（NT-3）、睫状神经营养因子（CNTF）、牛磺酸、视黄酸、酸性或碱性成纤维细胞生长因子，均不能模拟AF-1和AF-2的活性。AF-1在条件培养基（CM）中的浓度比在视神经匀浆中高得多，这表明它是被主动分泌的；AF-2在细胞内和细胞外的浓度相似。鉴于AF-1和AF-2源自视神经细胞并作用于视网膜神经节细胞，它们可能在体内诱导视神经再生中起重要作用。
