Werther G A, Haynes K, Waters M J
Department of Endocrinology and Diabetes, Royal Children's Hospital, Parkville, Victoria, Australia.
J Clin Endocrinol Metab. 1993 Jun;76(6):1638-46. doi: 10.1210/jcem.76.6.7684746.
The role of GH in the fetus remains unclear, in spite of high circulating levels. In order to determine potential sites of action of GH in the human fetus, we have used a combination of immunocytochemistry and northern blotting to examine human fetal tissues for GH receptor or binding protein and its messenger RNA. Human fetal abortus tissues of 15-20 weeks gestation were obtained for sternum and skin. Decalcified paraffin sections, were prepared for immunostaining with a monoclonal antibody to the GH receptor or control antibodies. Chondrocytes were digested from sternum and costal cartilage for primary culture in monolayers for up to 14 days. Skin fibroblasts were similarly cultured and studied at passages 5-10. Polyadenylated mRNA was prepared from cultured chondrocytes and fibroblasts for subsequent northern blotting with a complementary DNA probe to the hGH receptor. Positive immunostaining for GH receptor was seen in growth plate, including chondrocytes in the proliferative and hypertrophic zones, perichondrium, osteoblasts, and erythroid precursor cells. GH receptor immunostaining was also seen in skin sections throughout epidermis, including sebaceous and sweat glands, and vessels and fibroblasts in the dermis. Cultured chondrocytes showed patchy staining, with colonies of immunopositive cells proliferating in culture. Skin fibroblasts showed uniform GH receptor immunostaining. Control sections and cultured cells did not stain. Cultured chondrocytes showed specific binding of 125I-hGH(1-3%), with a typical displacement curve for GH receptor. As for immunostaining, binding increased with time in culture. Finally, northern blotting revealed a single 5.1 kilodalton band representing GH receptor mRNA in both chondrocytes and skin fibroblasts. This study has demonstrated the presence of GH receptor protein and mRNA on human fetal tissues including growth plate chondrocytes and osteoblasts, as well as skin epidermal structures and dermal fibroblasts. These receptors are capable of binding hGH, raising the possibility that, in contrast to current dogma, GH receptors play a functional role in the human fetus by 15 weeks gestation.
尽管胎儿体内生长激素（GH）的循环水平很高，但其在胎儿中的作用仍不清楚。为了确定GH在人类胎儿中的潜在作用位点，我们结合免疫细胞化学和Northern印迹法，检测人类胎儿组织中的GH受体或结合蛋白及其信使核糖核酸（mRNA）。获取妊娠15至20周的人类流产胎儿的胸骨和皮肤组织。制备脱钙石蜡切片，用抗GH受体单克隆抗体或对照抗体进行免疫染色。从胸骨和肋软骨中消化软骨细胞进行单层原代培养，培养时间长达14天。皮肤成纤维细胞以同样方式培养，并在第5至10代进行研究。从培养的软骨细胞和成纤维细胞中制备聚腺苷酸化mRNA，随后用针对人GH受体的互补DNA探针进行Northern印迹分析。在生长板中可见GH受体的阳性免疫染色，包括增殖区和肥大区的软骨细胞、软骨膜、成骨细胞和红系前体细胞。在整个表皮的皮肤切片中也可见GH受体免疫染色，包括皮脂腺和汗腺，以及真皮中的血管和成纤维细胞。培养的软骨细胞呈斑点状染色，免疫阳性细胞集落可在培养物中增殖。皮肤成纤维细胞呈均匀的GH受体免疫染色。对照切片和培养细胞未染色。培养的软骨细胞显示出125I-hGH的特异性结合（1-3%），具有典型的GH受体置换曲线。与免疫染色情况一样，结合随培养时间增加。最后，Northern印迹分析显示，在软骨细胞和皮肤成纤维细胞中均出现一条代表GH受体mRNA的5.1千道尔顿的条带。本研究证明了在人类胎儿组织包括生长板软骨细胞、成骨细胞以及皮肤表皮结构和真皮成纤维细胞中存在GH受体蛋白和mRNA。这些受体能够结合hGH，这增加了一种可能性，即与当前的观点相反，GH受体在妊娠15周时就在人类胎儿中发挥功能作用。
