Physiological analysis of the promoter structiure of the rat growth hormone (GH) gene by introducing the human GH gene into sponteneous dwarf rats.

通过将人类 GH 基因导入自发性侏儒大鼠中，对大鼠生长激素 (GH) 基因的启动子结构进行生理分析。

• 批准号: 11671092
• 负责人: KATAKAMI Hideki
• 金额: $ 1.98万
• 依托单位: MIYAZAKI MEDICAL COLLEGE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671092/
• 关键词: spontenous dwarf rat (dr); isolated complete GH deficiency; human GH gene; targetted expression to the pituitary; rat GH gene promoter -1.7kbp; gene therapy; normal recovery of growth; ラット視床下部GHRH・SRIF神経細胞機能の回復; 遺伝性侏儒症ラット(dr); ヒト成長ホルモン(GH) 遺伝子導入

A strain of spontaneous dwarf rat (SDR) of isolated GH deficiency (gene symbol dr) produces characteristics similar to those of the human disorder, isolated GH deficiency Type I.To recover normal growth in SDR, we established two strains of hGH-transgenic SDR : one bore a chimeric gene (PGGB) of the 1.7 kbp 5'flanking segment of the rGH gene fused to the human GH (hGH) gene (2.1 kbp) in SDR (PGGB-dr), and the other with a chimeric hGH gene (2.1 kbp) fused to the mouse metallothionein I promoter (MT, 1.8 kbp)(MT-dr). Both Northern blotting and RT-PCR analyses showed that the transgene transcript was expressed exclusively in the pituitary of PGGB-dr, while MT-dr expressed the transgene ubiquitously except for the pituitary. In spite of the lactogenic action of expressed hGH, both the litter size and growth rate of PGGB-dr were identical to those of age- and sex-matched non-transgenic littermates. In contrast, MT-dr showed giant phenotype. Sequential blood sampling from individual PGGB-dr … More under light ether anesthesia at day 20, 40 and 50 after birth showed age-related increases in plasma hGH, as did plasma rGH levels in each animal of normal Sprague-Dawley rats (CNT), whereas plasma hGH levels were consistently high without age-related changes in MT-dr. When plasma hGH levels were measured by repeated blood sampling every 20 min from 1000h to 1800h in 3 month-old male PGGB-dr, hGH secretion was pulsatile with peaks, occurring every 3-4 hours as did rGH in male CNT.In female PGGB-dr hGH secretory peaks occurred at irregular intervals with higher basal hGH levels, as did rGH in female CNT.By contrast, MT-dr of both sexes showed high plasma hGH levels without secretory bursts throughout the experiments. Changes in the hypothalamic GHRH and SRIF gene expression in SDR were normalized in PGGB-dr, but not in MT-dr, suggesting the hypothalamic negative feedback system for GH secretion resumed in PGGB-dr. Plasma rGH levels were undetectable in any animals of both SDR and transgenic SDR.The present study indicates that the targeted hGH gene expression to the pituitary, which is under the normal hypothalamic control of GHRH and SRIF, results in normal growth with pulsatile hGH secretion in SDR. Less

一株分离GH缺乏症（基因符号dr）的自发性侏儒大鼠（SDR）产生了与人类疾病（分离GH缺乏症i型）相似的特征，为了使SDR恢复正常生长，我们建立了两株转基因hgh的SDR：一种是rGH基因5'侧翼1.7 kbp的嵌合基因（PGGB），在SDR中与人生长激素（hGH）基因（2.1 kbp）融合（PGGB-dr），另一种是与小鼠金属硫蛋白I启动子（MT, 1.8 kbp）融合（MT-dr）的嵌合hGH基因（2.1 kbp）。Northern blotting和RT-PCR分析显示，转基因转录物仅在PGGB-dr的垂体中表达，而MT-dr除垂体外普遍表达转基因。尽管表达的hGH有产乳作用，但PGGB-dr的产仔数和生长速度与年龄和性别匹配的非转基因产仔完全相同。相比之下，MT-dr表现出巨大的表型。在出生后第20,40和50天，在轻度乙醚麻醉下，更多的PGGB-dr显示血浆hGH与年龄相关的增加，正常Sprague-Dawley大鼠（CNT）的每只动物的血浆rGH水平也是如此，而MT-dr的血浆hGH水平一直很高，没有与年龄相关的变化。当在3个月大的雄性PGGB-dr中，从1000h到1800h每20分钟重复抽血测量血浆hGH水平时，hGH分泌呈脉动状，每3-4小时出现一次峰值，雄性CNT中的rGH也是如此。在女性PGGB-dr中，hGH的分泌高峰在较高的基础hGH水平的不规则间隔发生，女性CNT中的rGH也是如此。相比之下，在整个实验过程中，两性MT-dr都表现出较高的血浆hGH水平，而没有分泌暴增。下丘脑GHRH和SRIF基因在SDR中的表达变化在PGGB-dr中归一化，而在MT-dr中没有，提示下丘脑GH分泌负反馈系统在PGGB-dr中恢复。在任何SDR和转基因SDR动物的血浆rGH水平均未检测到。本研究提示，在正常下丘脑GHRH和SRIF的控制下，将hGH基因靶向表达到垂体，可导致SDR患者生长正常，并有搏动性分泌hGH。少

项目成果

Katakami H et al.: "Regulation of body fluid balance in spontaneous dwarf rats caused by isolated growth hormone deficicency."Endocrine J. 46. S93-95 (1999)

Katakami H 等人：“孤立性生长激素缺乏引起的自发性侏儒大鼠体液平衡的调节。”Endocrine J. 46. S93-95 (1999)

Katakami H, Yonekawa T, Matsukura S: "Recovery of normal growth with pulsatile human GH release in spontaneous dwarf rats by targetting expression of the human GH gene to the pituitary gland."Clin Pediatr Endocrinol. in press.

Katakami H、Yonekawa T、Matsukura S：“通过将人 GH 基因的表达靶向垂体，使自发性侏儒大鼠的脉冲式人 GH 释放恢复正常生长。”Clin Pediatr Endocrinol。

米川忠杜、片上秀喜、松倉 茂: "ヒト成長ホルモン(hGH)分泌様式のラット性周期に及ぼす影響 : トランスジェニックラット(Tg)を用いての検討"第73回日本間脳下垂体腫瘍学会内分泌学術総会抄録集. 76(1). 143 (2000)

Tadayori Yonekawa、Hideki Katakami、Shigeru Matsukura：“人类生长激素 (hGH) 分泌模式对大鼠性周期的影响：使用转基因大鼠 (Tg) 的研究”第 73 届日本垂体肿瘤学会内分泌学术会议摘要 76(1)。 143 (2000)

Tadao Yonekawa,Hideki Katakami,Masazumi Hirabayashi,Masatsugu Ueda,and Shigeru Matsukura.: "Functional expression of human growth hormone(hGH) transgene in the rat somatotroph."Endcrine J. 46S. 75-80 (1999)

Tadao Yonekawa、Hideki Katakami、Masazumi Hirabayashi、Masatsugu Ueda 和 Shigeru Matsukura.：“人生长激素 (hGH) 转基因在大鼠生长激素中的功能表达。”Endcrine J. 46S。

Katakami H: "Study on pulsatile human GH secretion in a stain of the human GH-transgenic rats which shows pituitary-specific expression of the transgene. (in Japanese)"Annual Research Report of Foundation of Growth Science. 13. 399-406 (1997)

Katakami H：“对人 GH 转基因大鼠染色中的脉动人 GH 分泌的研究，显示转基因的垂体特异性表达。（日语）”生长科学基金会年度研究报告。