Study on abnormal GH gene expression in spontaneous dwarf rats

最近在日本发现了一种自发性侏儒鼠（dr）。遗传类型为常染色体隐性遗传。成年纯合子（dr +/+）大鼠的体重为杂合子（dr +/-）大鼠的1/3~1/4。利用免疫细胞化学技术的研究迄今表明，该菌株有一个孤立的垂体GH缺乏症。在雌性动物中，用人GRF（1 - 29）NH 2（10 μ g，bid，sc x5 d）引发垂体GH不能刺激身体生长、GH分泌（血清GH水平：ND）或增加垂体GH含量（对照的0.06%）。当给予hGH（200 mIU，bid，sc x 5d）时，雄性侏儒动物的体重增加（52 g vs. 3g，生理盐水）。这些动物中的GRH含量是治疗前对照组的45%。GH处理部分恢复了降低的GRH含量（对照的70%）。DR +/+组GRH和SRIF的HPLC分析图谱与DR +/-组相同。侏儒动物下丘脑GRH阳性细胞形态和分布正常。结果表明，下丘脑总RNA的分析显示正常大小的GRH mRNA增加（dr +/-增加2 - 4倍）。GH和PRL的肝DNA Southern分析（EcoRI或Hind III消化）显示正常。与此相反，Nonhem分析总垂体RNA显示正常大小的GH mRNA条带几乎完全丢失，而PRL mRNA则显示正常。对GH基因dr +/+区的cDNA和基因组DNA进行序列分析，发现两个点突变，一个位于GH基因第3内含子与第4外显子的连接处，另一个位于第5外显子。建立了一种灵敏的HGRF酶免疫分析方法，用于筛选携带金属硫蛋白偶联HGH/GRF基因的转基因侏儒大鼠。

A strain of spontaneous dwarf rats (dr) was recently discovered in Japan. The type of inheritance is an autosomal recessive. The body weight of adult homozygous rats (dr +/+) is from one third to one fourth of that of heterozygotes (dr +/-). Studies utilizing immunocytochemical techniques have so far shown that the strain has an isolated pituitary GH deficiency. Priming of the pituitary GH with human GRF (1-29) NH2 (10 mug, bid, sc x5 d) in female animals failed to stimulate body growth, GH secretion (serum GH levels : ND) or to increase the pituitary GH content (0.06% of control). When hGH (200 mIU, bid, sc x 5d) was given, animals showed increase in body weight (52 g vs. 3g with saline) in male dwarf animals. GRH content in these animals was 45 % of that in controls before treatment. GH treatment partially restored the decreased GRH content (70% of control). The patterns of HPLC analysis of the GRH and SRIF in dr +/+ were identical to those in dr+/-. GRH-containing cells of the hypothalamus in dwarf animals appeared normal in shape and distribution. Nonhem, analysis of total hypothalamic RNA showed an increase in the GRH MRNA of normal size (2-4folds increase of dr+/-). Southem analysis of hepatic DNA (EcoRl or Hind III digest) for GH and PRL appeared normal. In contrast, Nonhem analysis of total pituitary RNA showed an almost total loss of the GH MRNA band of normal size while the PRL MRNA appeared normal. The sequence analysis of both CDNA and genomic DNA of GH in the dr+/+ disclosed two point mutations, one at the junction between 3th intron and the 4th exon, the other within the 5th exon of GH gene. A sensitive HGRF EIA is established for the screening of transgenic dwarf rats which bear metallothinein-coupled HGH/GRF genes.