Study on the development of pituitary adenoma in human GRF-transgenic spontaneous dwarf rats.

用常规方法将由小鼠金属硫蛋白I启动子和人生长激素释放激素基因(外显子1、内含子1和外显子2-5融合基因)组成的2.2kbp的基因结构注入SD雌性大鼠的雄性原核，建立了2#和3号人生长激素释放激素(HGHRH)转基因大鼠。HGHRH-TG SD雄性大鼠与雌性矮小大鼠(DR+/+)交配。将雄性hGHRH-TG侏儒大鼠(+/-)与雌性DR(+/+)杂交，最终获得两株hGHRH-TG DR(+/+)。对其中一株hGHRH-TG DR(#2)的垂体激素、GHRH受体和c-fos的表达进行了分析。所有hGHRH-TG DR和高水平的血浆hGHRH(&GT；5,000 pg/ml)都表现为矮小，但有垂体腺瘤，其hGHRH免疫染色阳性，PRL染色阳性，ACTH染色很少，GH、TSH-β、LH-β和FSH-β均不表达。与生长激素基因表达下降(对照组的1/20)相比，大鼠GHRH受体和c-fos的mRNA分别显著增加10倍和4倍。Northern印迹分析和HE染色结果显示，hGRF基因在垂体、肝、脾、肾和卵巢中均有表达，而GRF-RC基因转录本和腺瘤形成则仅在垂体中表达。这些结果表明：1)GH非依赖性腺瘤形成；2)GRF作用于其RC，通过自分泌机制刺激生长激素腺瘤的形成。尽管由于GH缺乏，hGHRH-TG DR的数量很少，不足以广泛研究细胞因子的基因表达，但腺瘤中PTHrP基因的表达增加。

Two strains of human growth hormone (GH)-releasing hormone (hGHRH) transgenic rats (2#and#3) were first established by microinjection of a 2.2 kbp-gene constract which consisted of mouse metallothionein I-promoter and human GHRH minigene (exon 1, intron 1, and fusion gene of exon 2-5) into male pronucleus of the oocytes from SD female rats by the standard method. HGHRH-Tg SD male rats were, next, mated with female dwarf rats (dr+/+). Two strains of hGHRH-Tg dr (+/+) were finally obtained by crossing male hGHRH-Tg dwarf rats (+/-) with female dr (+/+). One strain of hGHRH-Tg dr (#2) were analyzed for expression of pituitary hormones, GHRH receptor and c-fos. All hGHRH-Tg dr with high plasma hGHRH levels (>5,000 pg/ml) showed dwarf, but had pituitary adenomas which showed positive immunostaining for hGHRH with some staining for PRL and little staining for ACTH.They showed no immunostaing for GH,TSH-beta, LH-beta or FSH-beta. In contrast to decreased GH gene expression (1/20 of controls), mRNA for rat GHRH receptor and c-fos were markedly increased by 10 and 4 folds, respectively. Expression of hGRF gene was found in the pituitary, liver, spleen, kidney and ovary, while GRF Rc gene transcripts and adenoma formation were exclusively found in the pituitary by northern blot analysis and HE staining, respectively. These results suggest that 1) GH-independent adenoma formation can occur and 2) GRF could act on its Rc and stimulate adenoma formation of the somatotroph by the autocrine mechanism.Although the numer of hGHRH-Tg dr was small due to GH deficiency and not enough to allow extensive survey for gene expression for cytokines, there was an increase in PTHrP gene expresion in adenomatous pituitary glands.