Identification of dopaminergic neuron trophic factor using subtraction hybridization and bioassay

采用生物测定法和减法杂交技术，对大鼠多巴胺失神经纹状体中多巴胺能神经元的营养因子进行了鉴定。结果如下：神经移植：与正常纹状体相比，缺乏da的纹状体中移植的DAergic神经元生长得更好。酪氨酸羟化酶免疫反应性较高（x 1.3），神经突延伸较强（x 1.3-1.8）。生物测定：在许多组分中，从缺乏da的纹状体中提取的分子量为13至45 kD的组分对培养的da能神经元的存活和神经突的延伸有最强烈的促进作用。无论是否有星形胶质细胞喂养层，这种效果都是稳定的。它还通过增强缓慢激活/持久的Ca电流刺激PC12D的神经突发生。用NGF和FGF抗体治疗后，效果降至50%左右。抗体：到目前为止还没有获得强效度的抗体。基因表达：以lambdagt 10为载体，从da缺失的纹状体组织中获得cDNA文库。在将这些cdna与来自完整和缺乏da的纹状体的mRNA进行差异杂交后，我们鉴定出54个斑块，其mRNA表达在缺乏da的纹状体中要高得多。通过southern印迹和原位杂交，在至少6个斑块中再次证实了mRNA表达的增强。对这些dna进行测序后，鉴定出其中3个为细胞色素氧化酶亚基- 1、α -珠蛋白和核糖体蛋白S-8。另外三种被发现是未知物质。由此，我们得到了几个能神经元的营养因子候选体。目前正在研究这些物质的纯化和特性。

Using bioassay and subtraction hybridization, we tried to identify the trophic factor for the dopaminergic (DAergic) neurons in the DA-denervated striatum in the rat. Results are following.1. Neural transplantation : Grafted DAergic neurons grew better in the DA-depleted striatum than in the normal striatum. Tyrosine hydroxylase immunoreactivity was higher (x 1.3) and the neurite extension was stronger (x 1.3-1.8) in the DA-depleted striatum.2. Bioassay : Among many fractions, a fraction with molecular weight of 13 to 45 kD in the tissue extract from the DA-depleted striatum enhanced most strongly the survival and neurite extension of the cultured DAergic neurons. This effect was stable with or without astrocyte feeder layers. It also stimulated the neurite genesis in PC12D with the enhancement of the slow activating/long-lasting Ca current. By the treatment of antibodies for NGF and FGF the effect was reduced down to about 50%.3. Antibody : So far antibodies with strong titers have not been obtained yet.4. Gene expression : Using lambdagt 10 as a vector, we got cDNA library from the DA-depleted striatal tissue. After the differential hybridization of these cDNAs with the mRNAs from the intact and DA-depleted striatum we identified 54 plaques whose mRNA expression was much higher in the DA-depleted striatum. The enhancement of mRNA expression was reconfirmed in at least 6 plaques by Sourthern blotting and in situ hybridization. After sequencing these DNAs, three were identified as cytochrome oxidase subunit-I, alpha-globin and ribosomal protein S-8. Other three were found to be unknown substances.Thus, we got several candidates for trophic factor for DAergic neurons. The purification and characterization of these substances are now under the investigation.