A型激酶锚定蛋白2基因表达对生长板软骨细胞功能的影响

Effects on the function of growth plate chondrocytes by A-kinase anchoring protein 2 gene expression

中文关键词:  A型激酶锚定蛋白2  软骨细胞  增殖  分化  ERK1/2信号通路

英文关键词:A-kinase anchoring protein 2  Chondrocytes  Proliferation  Differentiation  ERK1/2 signal path?鄄way

中文摘要:

  【摘要】 目的：探讨A型激酶锚定蛋白2（A-kinase anchoring protein 2，AKAP2）基因表达对生长板软骨细胞（growth plate chondrocytes，GPCs）增殖、分化及细胞外基质代谢的影响及作用机制。方法：设计AKAP2过表达和基因敲除质粒转染GPCs对AKAP2进行过表达或干扰，构建AKAP2过表达阴性对照组（Vector组）、AKAP2过表达组（AKAP2 OE组）、AKAP2干扰阴性对照组（si-NC组）、AKAP2干扰组（si-AKAP2组）和AKAP2 OE+U0126组[U0126：细胞外信号调节蛋白激酶1/2（extracellular signal regulated kinase 1/2，ERK1/2）通路阻滞剂]，记录各组GPCs细胞增殖活力及钙盐沉积情况，检测细胞外基质中Ⅱ型胶原α1（collagen type Ⅱ alpha 1，COL2A1）、碱性磷酸酶（alkaline phosphatase，ALP）、Ⅱ型胶原（collagen type Ⅱ，COLⅡ）、增殖细胞核抗原（pro?鄄liferating cell nuclear antigen，PCNA）和性别决定区Y框蛋白9（SRY-related high-mobility group box gene 9，SOX9）以及ERK1/2表达和磷酸化ERK1/2（p-ERK1/2）磷酸化水平，并进行统计学分析。结果：与Vector组相比，AKAP2 OE组细胞内AKAP2、ALP、COL2A1基因表达与AKAP2、PCNA、SOX9、ALP、COLⅡ蛋白表达及48h细胞活力、p-ERK1/2磷酸化水平均升高，橘红色钙结节显著增多，差异均有统计学意义（P<0.05）；与si-NC组相比，si-AKAP2组上述相应检测指标均呈降低趋势（P<0.05）。与AKAP2 OE组相比，AKAP2 OE+U0126组ALP、COLⅡA1基因表达和PCNA、SOX9、ALP、COLⅡ蛋白表达及48h细胞活力、p-ERK1/2磷酸化水平均降低，橘红色钙结节减少，差异均有统计学意义（P<0.05）；而ERK1/2基因表达均无显著性差异（P>0.05）。结论：AKAP2基因可以通过调控ERK1/2信号通路影响软骨细胞增殖、分化和细胞外基质代谢，并可能进一步改变正常的生长板软骨内成骨模式。

英文摘要:

  【Abstract】 Objectives: To investigate the effect and mechanism of A-kinase anchoring protein 2 (AKAP2) gene expression on the proliferation, differentiation and extracellular matrix metabolism of growth plate chon?鄄drocytes(GPCs). Methods: Designing AKAP2 overexpression and knockdown plasmids to transfect GPCs for overexpression or interference with AKAP2. The negative control of overexpression group(Vector group), the AKAP2 overexpression group (AKAP2 OE group), the negative control of interference group(si-NC group), the AKAP2 interference group(si-AKAP2 group) and AKAP2 OE+U0126 group[U0126: a blocker of the extracellu?鄄lar signal regulated kinase 1/2(ERK1/2) pathway] were constructed. GPCs cell proliferation and calcium depo?鄄sition were recorded. The expression of collagen type Ⅱ alpha 1 (COL2A1), alkaline phosphatase (ALP), col?鄄lagen type Ⅱ(COLⅡ), proliferating cell nuclear antigen(PCNA), SRY-related high-mobility group box gene 9(SOX9) and ERK1/2, phosphorylation level of phosphorylated ERK1/2(p-ERK1/2) were tested. The data were statistically analyzed. Results: Compared with the Vector group, the gene expression of AKAP2, ALP, and COL2A1, and the protein expression level of AKAP2, PCNA, SOX9, ALP, COLⅡ, p-ERK1/2, and cell via?鄄bility were significantly higher, and calcium nodules significantly increased in the AKAP2 OE group (P<0.05). Compared with the si-NC group, the si-AKAP2 group showed the opposite downtrend. Compared with the AKAP2 OE group, the expression of ALP, COLⅡA1, the protein expression level of PCNA, SOX9, ALP, and COLⅡ, p-ERK1/2, cell viability, and protein deposition of COLⅡ were significantly lower, and significantly decreased in the AKAP2 OE+U0126 group (P<0.05). No difference was found in the expression of ERK1/2 (P>0.05). Conclusions: AKAP2 could affect the proliferation, differentiation and extracellular matrix metabolism of chondrocytes through the ERK1/2 signaling pathway, and it may further change the normal growth plate endochondral bone formation pattern.