Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro.
Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro.
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DOI：
10.1186/1471-213x-3-4
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发表时间：
2003-04-24
影响因子：
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通讯作者：
Kappen, Claudia
中科院分区：
文献类型：
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作者：
Cormier, Stephania A;Mello, Maria Alice;Kappen, Claudia
背景技术背景：Hox基因编码的转录因子参与了骨骼中的模式形成，最近的证据表明，它们也在软骨内骨化的调节中发挥作用。为了更详细地分析Hoxc-8在这一过程中的作用，我们应用在体外培养系统，使用高密度培养的原代软骨细胞从新生小鼠ribs.RESULTS：培养细胞的特征在于形态学（光学显微镜）和软骨特异性细胞外基质（硫酸蛋白多糖和II型胶原）的生产的基础上。肥大表现为细胞体积增大、碱性磷酸酶活性增加和X型胶原免疫组化。通过BrdU摄取和流式细胞术评估增殖。出乎意料的是，来自Hoxc-8转基因小鼠的软骨细胞，在体内表现出延迟的软骨成熟1，能够在我们的培养系统中正常增殖和分化。这是即使新鲜分离的Hoxc-8转基因软骨细胞表现出显着的分子差异，通过实时定量PCR.CONCLUSIONS：结果表明，主要肋骨软骨细胞的行为类似于已发表的报告，从其他来源的软骨细胞，验证在体外方法的研究Hox基因的调节endochondriossification。我们对来自Hoxc-8转基因小鼠的软骨生成细胞的分析提供了证据，证明Hoxc-8在体内过表达诱导的细胞表型在体外是可逆的。
BACKGROUND: Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs.RESULTS: Cultured cells were characterized on the basis of morphology (light microscopy) and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen). Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo 1, were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR.CONCLUSIONS: The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro.