Gene expression of single articular chondrocytes
Gene expression of single articular chondrocytes
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DOI：
10.1007/s00441-006-0258-5
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发表时间：
2007-01-01
影响因子：
3.6
通讯作者：
Athanasiou, Kyriacos A.
中科院分区：
文献类型：
--
作者：
Eleswarapu, Sriram V.;Leipzig, Nic D.;Athanasiou, Kyriacos A.
尽管先前在组织工程领域的研究已经提供了关于关节软骨的重要信息，但他们的结论是基于群体平均水平，并没有考虑到细胞亚群的变化。为了获得对软骨细胞的精确理解，我们研究了软骨区和播种时间对单个软骨细胞基因表达的影响，以选择组织工程的最佳区域（一期），随后评估了生长因子暴露在一期（二期）所选择的区域。在第一阶段，浅层和中/深层牛关节软骨细胞单层接种3或18小时。在第二阶段，中/深层软骨细胞（在第一阶段选择）接受100 ng/ml胰岛素样生长因子-I (IGF-I) 3小时。实时逆转录/聚合酶链反应用于量化d -甘油醛-3-磷酸脱氢酶（GAPDH）的丰度以及聚集蛋白、胶原I和II、软骨寡聚基质蛋白（COMP）、基质金属蛋白酶-1 （MMP-1）和组织金属蛋白酶-1抑制剂（TIMP-1）。GAPDH呈区向变化，但时间和igf - 1对其均无影响，这表明GAPDH是一个适合于区域内比较的内源基因，而不是跨区域比较的内源基因。在播种18 h的中/深层软骨细胞中，IGF-I增加了聚集蛋白和胶原II的表达，而在对照细胞中，TIMP-1的表达随时间增加，提示播种后软骨细胞进入基质保护状态。IGF-I减少了这种作用，表明用IGF-I治疗使软骨细胞重新聚焦于基质生成，而不是对金属蛋白酶的保护。随着TIMP-1的增加，MMP-1在表面细胞中被检测到18小时，进一步证明了基质随时间降解的趋势。所有细胞中均未检测到I型胶原蛋白，COMP未见差异，证实未发生去分化或骨关节炎变化。综上所述，这些结果建立了对单个软骨细胞行为的独特理解。
Although previous studies in the field of tissue engineering have provided important information about articular cartilage, their conclusions are based on population averages and do not account for variations in cell subpopulations. To obtain a precise understanding of chondrocytes, we investigated the effects of cartilage zone and seeding duration on single chondrocyte gene expression to select an optimal zone for tissue engineering (Phase I), followed by an evaluation of growth factor exposure on the zone selected in Phase I (Phase II). In Phase I, superficial and middle/deep bovine articular chondrocytes were seeded in monolayers for 3 or 18 h. In Phase II, middle/deep chondrocytes (selected in Phase I) received 100 ng/ml insulin-like growth factor-I (IGF-I) for 3 h. Real-time reverse transcription/polymerase chain reaction was used to quantify the abundance of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative abundances of aggrecan, collagens I and II, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). GAPDH varied zonally, but neither time nor IGF-I had an effect on it, suggesting that GAPDH is a suitable housekeeping gene for comparisons within each zone, but not across zones. IGF-I increased the expression of aggrecan and collagen II in middle/deep chondrocytes seeded for 18 h. TIMP-1 expression increased with time in control cells, suggesting that chondrocytes enter a matrix protective state after seeding. IGF-I diminished this effect, suggesting that treatment with IGF-I refocuses chondrocytes on matrix production rather than on protection from metalloproteinases. Concomitant to increasing TIMP-1, MMP-1 was detectable by 18 h in superficial cells, providing further evidence of a trend toward matrix degradation with time. Collagen I was undetected in all cells, and no differences were observed for COMP, confirming that no dedifferentiation or osteoarthritic changes occurred. Taken together, these results establish a unique understanding of individual chondrocyte behavior.