EFFECTS OF GROWTH-HORMONE AND INSULIN-LIKE GROWTH FACTOR-I ON COLONY FORMATION OF RABBIT EPIPHYSEAL CHONDROCYTES AT DIFFERENT STAGES OF MATURATION
EFFECTS OF GROWTH-HORMONE AND INSULIN-LIKE GROWTH FACTOR-I ON COLONY FORMATION OF RABBIT EPIPHYSEAL CHONDROCYTES AT DIFFERENT STAGES OF MATURATION
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DOI：
10.1677/joe.0.1150263
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发表时间：
1987-11-01
影响因子：
4
通讯作者：
ISAKSSON, OGP
中科院分区：
文献类型：
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作者：
LINDAHL, A;NILSSON, A;ISAKSSON, OGP
采用悬浮培养的方法，研究了人生长激素（hGH）和胰岛素样生长因子I（IGF-I）对不同成熟阶段兔胫骨骺软骨细胞集落形成的影响。解剖8周龄雄性家兔胫骨近端骺生长板，将其分为近端、中间和远端三个不同的区域，每个区域分别代表来自萌发、增殖和肥大细胞层的软骨细胞的富集。通过胶原酶消化分离来自这三个区域的软骨细胞，并在10%（v/v）新生小牛血清存在下在用0.5%（w/v）琼脂糖稳定的悬浮液中培养14天。根据直径对菌落进行分类，并根据菌落大小（克隆效率分布）估计菌落数。直径为56 μ m或更大的细胞簇被分类为集落。克隆效率（每1000个接种的细胞形成的集落数）为10.1 ± 0.1。0.7和6.0 .+-。0.8对于分别从近端区和中间区分离的软骨细胞。胰岛素样生长因子I（25-200 ng/ml）增加了从近端区分离的软骨细胞中的集落数（122 ± 100 ng/ml）。9.0-156 .+-. 8.4%;对照值，100%）和来自中间区（136 . 14.0-191 .+-. 29%）。低浓度的hGH（10-40 ng/ml）刺激从近端区分离的软骨细胞中的集落形成（125 ± 100 ng/ml）。6.4-137 .+-. 7.9%），而高浓度的hGH（160 ng/ml）无效。从中间区分离的软骨细胞对低浓度的hGH（10-20 ng/ml）无反应。高浓度的hGH（40-160 ng/ml）减少了来自该区域的软骨细胞的集落形成（81 ± 100 ng/ml）。9.1-53 .+-.对照值的4.2%）。在不存在或存在hGH或IGF-I的情况下，从远端区分离的软骨细胞没有形成集落或形成数量不显著的集落。IGF-I和hGH均引起从近端区分离的软骨细胞中克隆效率分布的显著变化。IGF-I增强了小集落（集落直径64-176 μ m）的形成，而hGH增加了大集落（集落直径112-320 μ m）的数量。事实上，非常大的菌落（菌落直径> 272 μ m）仅在含有hGH的培养物中观察到。在从中间区分离的软骨细胞中，IGF-I刺激了各种大小的集落，并且克隆效率的分布没有观察到变化。人生长激素在160 ng/ml显着改变了软骨细胞中的克隆效率的分布，从中间区由于对小尺寸的集落形成的抑制作用。这些结果表明，悬浮培养的骺软骨细胞的克隆效率取决于它们在完整生长板中的先前空间位置。GH刺激生长板近端部分形成大尺寸细胞集落的发现表明GH与有限数量的表现出高增殖能力的细胞相互作用，例如与干细胞软骨细胞或早期增殖软骨细胞相互作用。
The effect of human GH (hGH) and insulin-like growth factor I (IGF-I) on colony formation of rabbit epiphyseal tibial chondrocytes at different stages of maturation was studied in suspension culture. The epiphyseal growth plate from the proximal tibia of 8-week-old male rabbits was dissected and divided into three different (proximal, intermediate and distal) zones, each zone representing an enrichment of chondrocytes from the germinative, proliferative and hypertrophic cell layers respectively. Chondrocytes from these three zones were isolated by collagenase digestion and cultured in the presence of 10% (v/v) newborn calf serum in suspension stabilized with 0.5% (w/v) agarose for 14 days. The colonies were classified according to diameter and the number of colonies was estimated as a function of colony size (distribution of cloning efficiency). Cell clusters with a diameter of 56 .mu.m or more were classified as colonies. The cloning efficiency (number of colonies formed per 1000 seeded cells) was 10.1 .+-. 0.7 and 6.0 .+-. 0.8 for chondrocytes isolated from the proximal an intermediate zones respectively. Insulin-like growth factor I (25-200 ng/ml) increase the number of colonies in chondrocytes isolated from the proximal zone (122 .+-. 9.0-156 .+-. 8.4%; control value, 100%) and from the intermediate zone (136 .+-. 14.0-191 .+-. 29%). Low concentrations of hGH (10-40 ng/ml) stimulated colony formation in chondrocytes isoalted from the proximal zone (125 .+-. 6.4-137 .+-. 7.9%) whereas a high concentration of hGH (160 ng/ml) was ineffective. Chondrocytes isolated from the intermediate zone showed no response to low concentrations of hGH (10-20 ng/ml). High concentrations of hGH (40-160 ng/ml) reduced colony formation of chondrocytes from this zone (81 .+-. 9.1-53 .+-. 4.2% of control value). Chondrocytes isolated from the distal zone formed either none or an insignificant number of colonies in the absence or presence of hGH or IGF-I. Both IGF-I and hGH caused a significant change in distribution of cloning efficiency in chondrocytes isolated from the proximal zone. IGF-I potentiated the formation of small colonies (colony diameter 64-176 .mu.m) while hGH increased the number of large colonies (colony diameter 112-320 .mu.m). In fact, very large colonies (colony diameter > 272 .mu.m) were only seen in cultures containing hGH. Among chondrocytes isolated from the intermediate zone, IGF-I stimulated colonies of all sizes and no change was seen in the distribution of cloning efficiency. Human GH at 160 ng/ml significantly altered the distribution of cloning efficiency in chondrocytes from the intermediate zone due to an inhibitory effect on the formation of colonies of small size. These results show that the cloning efficiency of epiphyseal chondrocytes in suspension culture is dependnet upon their previous spatial location in the intact growth plate. The finding that GH stimulated the formation of colonies of large size cells in from the proximal part of the growth plate indicates that GH interacts with a limited number of cells which exhibit a high proliferative capacity, e.g. with stem cell chondrocytes or early proliferative chondrocytes.