Identification of dopaminergic neuron trophic factor using subtraction hybridization and bioassay

本实验采用生物测定和差减杂交的方法，对去甲肾上腺素(DA)去神经支配的大鼠纹状体多巴胺能神经元的营养因子进行了鉴定。研究结果如下：1.神经移植：移植的DA能神经元在DA缺乏的纹状体比在正常的纹状体生长得更好。结果：1.在DA缺乏的纹状体，酪氨酸羟化酶免疫反应较强(x 1.3)，轴突延伸较强(x 1.3-1.8)。生物测定：在许多组分中，DA缺乏的纹状体组织提取物中分子量为13-45kD的组分对培养的DA能神经元的存活和轴突伸展的促进作用最强。无论有无星形胶质细胞饲养层，这一效应都是稳定的。通过增强慢激活/长时程钙电流，促进PC12D神经突起的形成。经神经生长因子和成纤维细胞生长因子抗体治疗后，其疗效降至50%左右。抗体：目前尚未获得效价较高的抗体。基因表达：以lambdagt10为载体，从去多巴胺的纹状体组织中获得了cDNA文库。将这些cDNA与完整纹状体和DA缺失纹状体的mRNAs进行差异杂交后，我们鉴定出54个斑块，它们在DA缺失纹状体中的mRNA表达明显高于正常纹状体。经Sourthernblotting和原位杂交证实，至少有6个斑块的mRNA表达增强。对这些DNA进行测序后，鉴定出三个亚基分别为细胞色素氧化酶亚基-I、α-珠蛋白和核糖体蛋白S-8。另外三个被发现是未知的物质。因此，我们得到了几个DAR能神经元营养因子的候选者。目前正在对这些物质的提纯和表征进行调查。

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.