Indian Hedgehog信号通路在软骨细胞成熟及转分化过程中的调控作用

目的 探究Indian Hedgehog（IHH）信号通路对软骨内成骨过程中软骨细胞成熟以及转分化的影响。方法 取10日龄野生型小鼠的胫骨组织,采用原位杂交和免疫组织化学染色检测生长板区域IHH信号通路相关分子Ihh、Ptch1和Gli1的表达水平。构建肥大软骨细胞特异性Ihh基因敲除小鼠（Col10a1^Cre/+; Ihh^null/C）,并采用影像学检查和阿利新蓝染色评估该小鼠的骨骼发育状况。构建肥大软骨细胞IHH信号通路持续激活小鼠（Col10a1^Cre/+; R26Smo^M2/M2和 Col10a1^Cre/+; Ptch1^LacZ/C）,采用HE染色、原位杂交和TUNEL染色分别对受精15.5天胎鼠胫骨组织形态结构、Ihh（肥大软骨细胞分子标志物）和Col1a1（成骨细胞分子标志物）以及肥大软骨细胞凋亡水平进行检测;另外应用HE染色对10日龄小鼠的胫骨组织进行组织学分析。结果 肥大软骨细胞合成分泌IHH,但不表达Ptch1和Gli1。抑制肥大软骨细胞合成IHH蛋白会导致出生后小鼠出现侏儒症;X线检查结果显示小鼠出现严重的骨骼发育不良,包括胸廓狭小、球形头骨以及椎骨发育异常等表现。持续启动IHH信号通路时,胚胎早期软骨细胞成熟分化过程虽未见异常,但是出生后小鼠的骨小梁、骨内膜以及皮质骨等结构均出现一定的异常表现。结论 IHH信号通路虽然不参与肥大软骨细胞的终末分化过程,但在软骨细胞转分化的过程中起到了重要的调控作用。     

Morphometric Changes in the Epiphyseal Plate of the Growing and Young Adult Male Rat After Long-Term Salmon Calcitonin Administration

The function of the epiphyseal plate is related to the differentiation and maturation of the chondrocytes, especially of the hypertrophic zone. Salmon calcitonin exerts a positive effect on chondrocytes of different types of cartilage, e.g., articular cartilage, osteochondral callus formation, and the epiphyseal plate. In the present study, the effect of long-term daily salmon calcitonin treatment upon epiphyseal plate function was examined in 80 male Wistar rats aged 12 weeks at the beginning of the experiment. A daily dose of 6 IU of salmon calcitonin enhanced the number of the chondrocytes of the hypertrophic zone of the upper tibial epiphyseal plate, increased the mean thickness of the epiphyseal plate, and accelerated the longitudinal growth of long bones. It was found that the peripheral growth of the epiphyseal plate was delayed after calcitonin treatment in comparison with the placebo-treated animals. The most effective period for calcitonin treatment on epiphyseal plate function seems to be the late accelerated period of growth, i.e., puberty. In conclusion, long-term salmon calcitonin treatment has a beneficial effect on longitudinal skeletal growth and this effect remains throughout the adult life of the animal. Salmon calcitonin does not enlarge the surface of the epiphyseal plate.     

正常骺板及牵拉分离后胶原纤维构筑的扫描电镜观察

目的 探讨骺板受到牵拉后其胶原纤维超微构筑的变化。方法 正常对照骺板取自１１岁男童离体新鲜胫骨近端骺板１例,１３岁女童胫骨远端骺板１例;牵拉分离后的骺板取自１１岁男童截肢后残肢胫骨远端骺板。将经外固定器牵拉分离后的儿童胫骨远端骺板和未牵拉的儿童胫骨远端及近端骺板制成标本进行扫描电镜观察。结果及结论 骺板经牵拉后其胶原纤维超微构筑发生了改变,胶原纤维趋向于力的纵轴方向排列。     

兔髂骨骺板软骨细胞体外构建骺板样软骨组织

目的　利用组织工程学技术体外构建骺板样软骨组织。方法　从 4～ 5周龄兔髂骨骺板软骨处获取软骨细胞 ,在离心管内轻微离心后 ,体外培养。行组织学观察。结果　培养至第 7天时 ,细胞呈现定向分化 ,形态与体内骺板软骨细胞相类似 :肥大软骨细胞体积较大、呈圆形或椭圆形 ;增殖、成熟软骨细胞体积小 ,呈圆形或扁圆形 ;细胞周围充满大量的细胞外基质。这些不同分化阶段的细胞形成了分化区带 ,肥大软骨细胞位于上侧 ,增殖、成熟细胞位于中间 ,其次是散在静止软骨细胞。培养第 14天 ,分化区带更加明显 ,增殖、成熟细胞和肥大软骨细胞呈现纵向定向排列。培养第 2 1天 ,组织表面出现膜样的结构。结论　体外构建的骺板样软骨组织与天然骺板的组织学形态极为相似。从髂骨处骺板处获取肥大软骨细胞进行体外构建骺板软骨材料 ,更具有临床实用性     

Carboxypeptidase Z (CPZ) Links Thyroid Hormone and Wnt Signaling Pathways in Growth Plate Chondrocytes

Carboxypeptidase Z (CPZ) removes carboxyl‐terminal basic amino acid residues, particularly arginine residues, from proteins. CPZ contains a cysteine‐rich domain (CRD) similar to the CRD found in the frizzled family of Wnt receptors. We have previously shown that thyroid hormone regulates terminal differentiation of growth plate chondrocytes through activation of Wnt‐4 expression and Wnt/β‐catenin signaling. The Wnt‐4 protein contains a C‐terminal arginine residue and binds to CPZ through the CRD. The objective of this study was to determine whether CPZ modulates Wnt/β‐catenin signaling and terminal differentiation of growth plate chondrocytes. Our results show that CPZ and Wnt‐4 mRNA are co‐expressed throughout growth plate cartilage. In primary pellet cultures of rat growth plate chondrocytes, thyroid hormone increases both Wnt‐4 and CPZ expression, as well as CPZ enzymatic activity. Knockdown of either Wnt‐4 or CPZ mRNA levels using an RNA interference technique or blocking CPZ enzymatic activity with the carboxypeptidase inhibitor GEMSA reduces the thyroid hormone effect on both alkaline phosphatase activity and Col10a1 mRNA expression. Adenoviral overexpression of CPZ activates Wnt/β‐catenin signaling and promotes the terminal differentiation of growth plate cells. Overexpression of CPZ in growth plate chondrocytes also removes the C‐terminal arginine residue from a synthetic peptide consisting of the carboxyl‐terminal 16 amino acids of the Wnt‐4 protein. Removal of the C‐terminal arginine residue of Wnt‐4 by site‐directed mutagenesis enhances the positive effect of Wnt‐4 on terminal differentiation. These data indicate that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by modulating Wnt signaling pathways through the induction of CPZ and subsequent CPZ‐enhanced activation of Wnt‐4.     

The Domain of Hypertrophic Chondrocytes in Growth Plates Growing at Different Rates

In this study, we tested the hypotheses that (a) both the domain volume (volume of the cell and the matrix it has formed) and matrix volume of juxtametaphyseal hypertrophic chondrocytes in the growth plate is tightly controlled, and that (b) the domain volume of juxtametaphyseal hypertrophic chondrocytes is a strong determinant of the rate of bone length growth. We analyzed the rate of bone length growth (oxytetracycline labeling techniques) and nine stereologic and kinetic parameters related to the juxtametaphyseal chondrocytic domain in the proximal and distal radial and tibial growth plates of 21- and 35-day-old rats. The domain volume increased with increasing growth rates, independent of the location of the growth plate and the age of the animal. Within age groups, the matrix volume per cell increased with increasing growth rates, but an identical growth plate had the same matrix volume per cell in 21- and 35-day-old rats. The most suitable regression model (R ²= 0.992) to describe the rate of bone length growth included the mean volume of juxtametaphyseal hypertrophic chondrocytes and the mean rate of cell loss/cell proliferation. This relationship was independent of the location of the growth plate and the age of the animal. The data suggest that the domain volume of juxtametaphyseal hypertrophic chondrocytes, as well as the matrix volume produced per cell, may be tightly regulated. In addition, the volume of juxtametaphyseal hypertrophic chondrocytes and the rate of cell loss/rate of cell proliferation may play the most important role in the determination of the rate of bone length growth. Received: 2 December 1996 / Accepted: 24 March 1997     

Effect of Metal Ions on Calcifying Growth Plate Cartilage Chondrocytes

The effects of the trace metals zinc (Zn), manganese (Mn), and cadmium (Cd) on the metabolism of growth plate chondrocytes was examined using a mineralizing culture system. Supplementation of serum-free primary cultures of growth plate chondrocytes with 10–100 μm Zn resulted in an increase in cell protein and greatly increased alkaline phosphatase (AP) activity; however, above 25 μm Zn mineralization of the cultures was reduced. The effects of Zn on cellular protein and AP activity were enhanced by the addition of the albumin to the culture media. Removal of Zn from basal culture media resulted in recoverable reductions in cellular protein and AP activities. Cadmium was acutely toxic to chondrocyte cell cultures at concentrations above 5 μm. Even at very low concentrations (0.25 μm) Cd caused significant reductions in DNA, cellular protein, and matrix protein synthesis. In contrast, Cd had negligible effects on AP activity or culture mineralization. Manganese treatment (50 μm) resulted in reduced levels of proteoglycan, cell protein, DNA synthesis, and collagen synthesis, although AP specific activity did not change. At 10 μm, Mn significantly reduced mineralization but had only minor influence on other culture parameters. Both Zn (200 μm) and Cd (0.1 μm), but not Mn, induced the synthesis of metallothionein. The physiological and biochemical effects of specific metal ions is largely dependent on their physicochemical properties, especially their ligand affinities. Knowledge of these properties allows predictions to be made regarding whether the organic or the mineral phase are most likely to be affected in a mineralized tissue. Received: 13 March 1997 / Accepted: 12 August 1997     

血小板源性生长因子对体外兔关节软骨细胞的生物学行为的影响

目的　了解血小板源性生长因子 (PDGF)对体外生长的兔关节软骨细胞的生物学效应 ,为组织工程构件软骨提供理论基础。方法　取第 3代兔关节软骨细胞体外单层培养 ,培养液DMEM中以终浓度分别为 3、 10、 30、 10 0、 30 0 μg .L 1的PDGF各作用细胞 2 ,4及 6d ,以MTT法检测细胞的增殖情况 ,并在 3μg .L 1PDGF作用下 ,采用流式细胞技术进行细胞周期亚时相分析。同时 ,检测基质中糖醛酸的变化反映蛋白多糖含量的变化。结果　结果显示在较低浓度 (3μg .L 1)PDGF即能明显促进培养软骨细胞的增殖 ,且以第 2d刺激效果最明显 ;增加因子浓度不能进一步促进细胞增殖。在 3～ 30 0 μg .L 1浓度下糖醛酸的含量变化不显著。结论　PDGF对培养软骨细胞以剂量时间依赖性方式刺激其增殖但对细胞的分泌功能代谢无明显影响     

Growth Differentiation Factor-5 Stimulates the Growth and Anabolic Metabolism of Articular Chondrocytes

Objective： To observe the effect of growth differentiation factor-5 （GDF-5） on the growth and anabolic metabolism of articular chondrocytcs. Methods： The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱ collagen by RT-PCR, the collagen phenotypic expression of chondrocytes detected by immunofluorescence. Results： After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2al mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/ml, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Chondrocytes were cultured with GDF-5 for 21 days, immunofluorescent staining of type Ⅱ collagen was clear, the type Ⅰ and X collagen were negative. Conclusion： GDF-5 enhanced the growth of mature articular chon- drocytes, and stimulated the cellular cartilage matrices formation, but did not change the collagen phenotypic ex- pression of chondrocytes in mono-layer culture.