重组人生长激素对荷人胃癌裸鼠移植瘤血管内皮生长因子表达的影响

目的研究重组人生长激素（rhGH）对生长激素受体（GHR）不同表达状态裸鼠人胃癌移植瘤生长及血管内皮生长因子（VEGF）表达的影响。方法采用免疫细胞化学染色法筛选出GHR阳性和阴性表达的细胞株各1株，分别接种于24只裸鼠皮下。将两种细胞接种裸鼠均随机分为对照组（0．9％NaCl，0．2ml／d）、低剂量rhGH组（0．5U·kg^-1·d^-1，0．2ml／d）和高剂量rhGH组（2．5U·kg^-1·d^-1，0．2mL／d）3组，每组8只，各组均连续给药14d，观察并记录裸鼠体重和肿瘤体积变化；采用酶联免疫吸附法测定各组裸鼠血清VEGF含量，免疫组织化学方法检测胃癌组织中VEGF蛋白表达，RT-PCR方法检测胃癌组织VEGFmRNA水平变化。结果筛选出GHR阳性表达的人胃癌细胞株SGC-7901和阴性表达的MKN-45。对于GHR^＋SGC-7901接种裸鼠，rhGH给药组皮下移植瘤体积较对照组增大（P〈0．05），且高剂量rhGH组促增长效应最为显著（P〈0．05），3组间体重差异无统计学意义（P〉0．05）；高剂量rhGH组的血清VEGF浓度为（252．94±15．32）ng／L，明显高于对照组的（49．94±5．73）ng／L和低剂量rhGH组的（167．60±9．54）ng／L（P〈0．05）；对照组VEGF表达为中度阳性，rhGH给药组呈强阳性；高剂量rhGH组肿瘤组织中VEGFmRNA相对表达量为0．6470±0．0447，明显高于对照组的0．3230±0．0258和低剂量rhGH组的0．4120±0．0351（P〈0．05）。对于GHR—MKN-45接种裸鼠，rhGH给药组体重明显大于对照组（P〈0．05）；肿瘤体积大小、血清VEGF水平、肿瘤组织VEGF蛋白及mRNA表达，3组间差异均无统计学意义（P〉0．05）。结论rhGH能促进GHR阳性表达的SGC-7901移植瘤生长，并促进VEGF表达增高；对于GHR阴性的MKN-45移植瘤，则没有表现出明显的促肿瘤生长及促VEGF表达效应。GHR存在可能是rhGH影响VEGF分泌的关键靶点。     

Pyridinium crosslinks of collagen as a marker of bone resorption rates in children and adolescents: Normal values and clinical application

The aim of our study was to establish normal values of urinary pyridinoline (Pyr) and deoxypyridinoline (DPyr) excretion for children aged 3–18 years, examine the biological variability of the marker, and assess its clinical value for pediatric patients with growth hormone deficiency. Pyr and DPyr was measured in first void urine samples from 692 healthy subjects (340 boys, 352 girls) by high-performance liquid chromatography. At sampling, age, body height, and weight was recorded for all individuals. Short-term variability in crosslinks excretion was examined in four healthy children. The clinical value of the marker was studied in seven patients with growth hormone (GH) deficiency. In childhood, crosslinks excretion exceeded normal adult values by about fivefold and declined during puberty. In the age range of 13–18 years, gender-related differences in Pyr and DPyr levels were observed, presumably resulting from the earlier onset of puberty in girls. Urinary levels of Pyr and DPyr were highly correlated both in males and females. Pyr/DPyr ratio was significantly higher in adolescents than children, suggesting enhanced release of Pyr from extraosseous sources. In both genders, neither age nor anthropometric variables showed a linear effect on crosslinks excretion. The range of within-subject, short-term variability in urinary Pyr and DPyr was relatively high (CV: 6%–21%), indicating that single measurements of crosslinks excretion may not adequately reflect bone resorption rates in children. Pyr and DPyr levels were significantly lower in GH-deficient patients and normalized during human growth hormone (hGH) therapy. Significant correlations between growth velocity (GV) and crosslinks levels were found, but individual prediction of GV increment during hGH treatment may be inaccurate. Pyr/DPyr ratio was not related to GV. It is concluded that measurement of urinary Pyr and DPyr excretion in children may be a valuable tool to assess bone resorption rates in population-based studies. In individual patients, however, only qualitative evaluation of disease severity and response to treatment seems justified.     

Preventive effects of intermittent administration of human parathyroid hormone on steroid-induced osteopenia in rats

The purpose of this study was to determine the preventive effect of intermittent administration of human parathyroid hormone (h-PTH) on bone change in steroid-treated rats; this was done by histomorphometric and biochemical analysis. Seven-month-old female Wistar rats were divided into four groups; in-each of the four groups one subgroup was treated for 4 weeks and one for 8 weeks. The groups consisted of: untreated controls, a steroid group (receiving prednisolone), a steroid + PTH group (predniso-lone and h-PTH administered simultaneously), and a steroid + PTH vehicle group. Prednisolone (2.5 mg/kg) and h-PTH (1–34) (6.0 μg/kg) were administered six times a week during the experimental period. At necropsy, bilateral tibiae were collected: one was used for preparing undecalcified sections after Villanueva bone staining, and the other for decalcified tartrate-resistant acid phosphatase (TRAP) stained sections. Biochemical analysis showed that steroids increased urinary calcium at the 8th week; however, such bone metabolic markers as serum 1,25-(OH)₂D and urinary deoxypyridinoline did not change in any treatment group. Histomorphometrically, steroid-induced osteopenia was established at the 8th week by inhibition of both bone formation and bone resorption. The simultaneous intermittent administration of PTH plus steroid, however, increased both bone formation and bone resorption, resulting in increases in bone volume beginning at 4 weeks. These results suggest that the simultaneous intermittent administration of PTH with steroid prevents steroid-induced low-turnover osteopenia by stimulating bone turnover.     

Secretogranin I (chromogranin B) mRNA accumulation is hormonally regulated in GH3B6 rat pituitary tumor cells

Secretogranin I (SgI; chromogranin B) belongs to a class of acidic tyrosine-sulfated secretory proteins believed to play a role in the secretory process of endocrine cells. Our aim here was to compare the levels of SgI mRNA to that of prolactin (PRL) and growth hormone (GH), using rat pituitary cell lines. As far as the constitutive expression is concerned, we found a positive correlation between SgI mRNA and PRL mRNA levels. However, the neuropeptide TRH (50 nM) inhibited the accumulation of SgI mRNA in GH3B6 cells whereas, as expected, it induced a rapid and sustained increase in PRL mRNA accumulation. By contrast, 17β-estradiol (1 nM) stimulated the accumulation of both SgI and PRL mRNAs, with the same EC₅₀ (18–59 pM). Reciprocally, treatment with dexamethasone (100 nM) reduced the level of SgI and PRL mRNAs to 23% and 29% of control, respectively, but led to a 2.1-fold increase in the GH mRNA level. Altogether, the present work shows that SgI gene expression is subject to multiple hormonal regulations and occasionally parallels the regulation of the PRL gene but never that of the GH gene, under the conditions tested.     

Interactions between growth hormone and dexamethasone in skeletal growth and bone structure of the young mouse

Summary The present study investigated the interactions of growth hormone (GH) and glucocorticoid on skeletal growth and bone structure in young mice. The purpose of this study was to examine the possible prevention by GH of the damage inflicted by dexamethasone (Dex) at sites of skeletal growth and ossification. Dex (1 mg/kg) with or without rat GH (rGH) or bovine GH (bGH), 1 mg/kg, was given for 4 weeks, from age 3–7 weeks, to female ICR mice. Tibiae, humerus, and vertebrae were analyzed morphometrically and biochemically. Growth, as determined by the mouse weight, tibial length, and humerus protein content was found to be compromised by dexamethasone. This was prevented by rGH or bGH. The epiphyseal growth plate width, trabecular bone volume, cortical bone width, mineral bone content, and alkaline and acid phosphatase activity were decreased by dexamethasone. These were prevented by rGH or by bGH. The findings of the present study suggest that in the mouse, GH can decrease or even avoid some of the pathological features in growing bones inflicted by high-dose glucocorticoid treatment.     

Selective modification at the N-terminal region of human growth hormone that shows antagonistic activity

A new analogue of recombinant human growth hormone (hGH), hGH des(1–6,14) was expressed in Escherichia coli, refolded and purified to homogeneity. The mutation decreased the hormone's ability to bind lactogenic and somatogenic receptors through its site 1, and almost completely abolished its ability to bind these receptors through site 2, as evidenced by both binding and gel-filtration experiments. More specifically, the binding to prolactin receptors (PRLRs) from various species or their soluble recombinant extracellular domains (ECDs) was decreased 1.5–4-fold, whereas the binding to hGH receptor (hGHR) was decreased 10–85-fold. These changes caused an almost total loss of hormone agonistic activity in several in vitro bioassays and subsequently, the hGH des(1–6,14) analogue acquired antagonistic properties. This antagonistic activity was dependent upon modification of site 1. In those cases in which the binding was reduced only slightly, e.g. binding to rabbit PRLRs, hGH des(1–6,14) acted as a strong antagonist, whereas in others in which the binding of site 1 was reduced to a higher degree, such as other PRLRs and, in particular, hGHR, the antagonistic activity was correspondingly weaker. Circular dichroism spectra of the analogue suggested that these changes do not result from a decrease in overall -helix content, but rather from minor local structural modifications at the N-terminus.     

Relationship between growth hormone and Somatomedin-C levels in untreated acromegaly, after surgery and radiotherapy and during medical therapy with Sandostatin (SMS 201–995)

Abstract. Several conflicting reports have been published with regard to the relationships between circulating growth hormone (GH), Somatomedin-C (SM-C) levels and clinical activity during different stages of therapy of acromegaly. We did not find a significant correlation between (fasting, post-prandial and mean 24-h) plasma GH and SM-C concentrations in twenty-two untreated acromegalic patients. There was a statistical significant correlation, however, if only the GH levels below 100 μg l^-1 were considered (n=18 patients, P<0·01). The distribution of molecular forms of GH (‘little’, ‘big’ and ‘big-big’) did not differ between the four patients with GH levels above 100 μg l^-1 and in four patients with levels between 40 μg l^-1 and 80 μg l^-1. Therefore, it is suggested that GH levels of 80–100 μg l^-1 maximally activate Somatomedin-C production in man and that further increases in GH in general will not result in a further increase in SM-C generation. There was a significant correlation between GH and SM-C levels in forty-nine acromegalic patients after surgery and/or radiotherapy (P<0·001). In twenty-three of thirty-one patients with elevated SM-C levels the disease was subjectively still active, while this was the case in none of the patients with normal SM-C levels. In eight patients the disease was considered not to be clinically active any more, despite slightly increased SM-C levels. During long-term therapy of ten acromegalic patients for 16–108 weeks (mean 66±10) with 200–300 μg Sandostatin subcutaneously, clinical activity of the disease disappeared well before mean 24–h GH and SM-C levels reached the normal levels. There was a close correlation between mean 24-h GH and SM-C levels during Sandostatin therapy (P<0·001). ‘Clinical cure’ during this medical treatment was reached in five patients, as reflected by disappearance of subjective complaints, normalization of SM-C levels and 24-h mean GH levels of 2·8±0·2 μg l^-1. Conclusions: (i) in untreated acromegaly, circulating GH and SM-C levels correlate well up to GH concentrations of 100 μg l^-1. A further increase in GH does not result in a corresponding further increase in SM-C levels, suggesting a maximally activated production, without further GH-dependent capacity. (ii) Clinical ‘cure’ of acromegaly often occurs before normalization of the circulating SM-C levels. (iii) The measurement of plasma SM-C concentrations can be used well to adjust the dose and frequency of Sandostatin administration in acromegaly. This avoids the need of measuring extensive 24-h GH profiles.     

荔枝果实发育期间内源激素含量动态

本研究测定了荔枝果实发育期间内源激素含量的动态。结果表明果实发育前期细胞分裂素(CTK),生长素(IAA)含量较高;中期乙烯(Eth),赤霉素(GA),的含量较高,这与果实的生长发育及落果有密切相关。同时,果实发育前期应用外源CTK、中期应用GA、处理果穗可显著提高产果率。     

The effects of intermittent administration of human parathyroid hormone (1–34) on streptozotocin-induced diabetic rats

Intermittent administration of low doses of human parathyroid hormone (h-PTH) has been reported to exhibit an anabolic effect on bone, increasing its mass. We investigated the effects of intermittent administration of h-PTH on bone changes in streptozotocin- (STZ-) induced diabetes mellitus (DM) rats by measuring bone mineral density and bone mineral contents and by bone histomorphometry. Wistar rats, 7–8 months old, were used. Osteoporosis was induced by diabetes mellitus, which was established by an intraperitoneal injection of STZ. Rats were separated into five groups: sham-injected, baseline control, vehicle-only-administered, and low-dose (6.0μg/kg) or high-dose (60.0μg/kg) h-PTH-administered groups. h-PTH or vehicle was injected subcutaneously six times a week for 4 weeks beginning 9 weeks after STZ administration. Bone mineral density and mineral contents were significantly lower in the baseline control and vehicle groups than in the control group. The PTH-administered groups showed higher values compared with both vehicle and baseline control groups. In bone histomorphometry, both bone volume and bone formation in the STZ group were markedly reduced. The h-PTH-administered rats showed increase in both bone volume and bone formation, which are related parameters, but administration of h-PTH did not alter the extent of eroded surface. Our results suggest that intermittent administration of h-PTH is effective in activating bone formation and in preventing further bone loss in osteoporosis developed by STZ-induced DM.