Heymanns J, Unsicker K
Department of Anatomy and Cell Biology, University of Marburg, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1987 Nov;84(21):7758-62. doi: 10.1073/pnas.84.21.7758.
Ciliary neurotrophic factor (CNTF) is a protein supporting the in vitro survival of a characteristic spectrum of embryonic chicken and rat peripheral neurons. High-speed supernatants of extracts from two neuroblastoma (NB) cell lines--the mouse C 1300 N2a and the human IMR 32--mimic the effects of CNTF on identical target neurons. Promotion of survival is dose-dependent with an ED50 of 80 micrograms (IMR 32) and 140 micrograms (C 1300 N2a) of protein per ml and saturable at plateau values for surviving neurons identical to those achieved with purified CNTF. Small amounts of a CNTF-like material are also detectable in medium conditioned by NB cells. The activity is destroyed by heat and trypsin and not blocked by antibodies to (mouse) nerve growth factor. Unlike the neurite-promoting and neuronal-survival modulating agent laminin, it cannot be depleted on poly(L-alpha-ornithine)-coated plastic surfaces. NB IMR 32 cell extracts were electrophoresed using NaDodSO4/PAGE and transferred to nitrocellulose. Ciliary ganglion neurons seeded on the blotting paper in culture medium lacking CNTF ("cell blot") exclusively survive on two distinct bands with apparent molecular masses of 24 and 48 kDa. Twenty-four kilodaltons is the molecular mass of a CNTF purified from rat sciatic nerve. These results suggest that NB cells may contain a CNTF-like protein and provide further evidence that neurons may store neurotrophic factors. Purified (chicken) CNTF failed to affect proliferation and neurite growth of NB cells. The biological relevance of CNTF for NB cells, therefore, remains to be elucidated.
睫状神经营养因子（CNTF）是一种能支持特定种类的胚胎鸡和大鼠外周神经元在体外存活的蛋白质。两种神经母细胞瘤（NB）细胞系——小鼠C 1300 N2a和人IMR 32——提取物的高速上清液可模拟CNTF对相同靶神经元的作用。存活促进作用呈剂量依赖性，每毫升蛋白质的半数有效剂量（ED50）分别为80微克（IMR 32）和140微克（C 1300 N2a），且对于存活神经元而言，在达到与纯化CNTF相同的平台值时达到饱和。在NB细胞条件培养基中也可检测到少量的CNTF样物质。该活性可被加热和胰蛋白酶破坏，且不被（小鼠）神经生长因子抗体阻断。与促进神经突生长和调节神经元存活的层粘连蛋白不同，它不能在聚（L-α-鸟氨酸）包被的塑料表面被耗尽。用十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）对NB IMR 32细胞提取物进行电泳，然后转移至硝酸纤维素膜上。接种在缺乏CNTF的培养基中的滤纸上的睫状神经节神经元（“细胞印迹”）仅在两条明显分子量分别为24 kDa和48 kDa的条带上存活。24 kDa是从大鼠坐骨神经中纯化得到的CNTF的分子量。这些结果表明NB细胞可能含有一种CNTF样蛋白，并进一步证明神经元可能储存神经营养因子。纯化的（鸡）CNTF未能影响NB细胞的增殖和神经突生长。因此，CNTF对NB细胞的生物学相关性仍有待阐明。
