骨形态发生蛋白-2对软骨寡聚基质蛋白在软骨细胞内表达的影响

Effects of bone morphogenetic protein-2 on cartilage oligomeric matrix protein expression in chondrocytes

目的 :定量研究骨形态发生蛋白 (BMP 2 )对软骨寡聚基质蛋白 (COMP)基因在人软骨细胞及ATDC5 5细胞内表达的影响。方法 :原代培养成人及胚胎的关节软骨细胞 ,经BMP 2刺激后 ,利用real timeRT PCR定量分析COMP基因的表达情况 ;采用静息期不表达COMP基因的鼠源性成软骨细胞株ATDC5 5作为研究对象 ,给以BMP 2刺激 ,通过RT PCR检测COMP表达的变化 ;将COMP基因的全长启动子克隆到报告基因Luciferase的上游并转染入ATDC5 5细胞 ,分别给以 5 0 0 μg·L-1的BMP 2和 /或 10 μg·L-1的Noggin刺激 ,测定其对Luciferase活性的影响。结果 :BMP 2使成人软骨细胞内的COMP基因表达提高近 3倍 ,而对胚胎软骨细胞的作用较弱 ,提高了1.5倍 ;COMP基因在胚胎软骨细胞内的基础表达量约是在成人软骨细胞内的 2倍 ;经BMP 2刺激后ATDC5 5细胞株明显表达COMP基因 ,并且BMP 2的这种效应能够被其拮抗剂Noggin所抑制 ;BMP 2明显促进了Luciferase的活性 ,约提高了 5倍 ,且Noggin特异性的抑制了BMP 2的这种作用。 结论 :BMP 2能够明显促进COMP基因在人关节软骨细胞及鼠源性成软骨细胞株ATDC5 5内的表达。

Objective: To quantitatively evaluate the effects of BMP 2 on the COMP gene expression in both primary human chondrocytes and chondrogenic cell line ATDC55. Methods:Human adult and fetal chondrocytes were stimulated by BMP 2.The COMP gene expression level was analyzed by real time reverse transcription PCR assay and normalized to a reference mRNA (GAPDH).Next, a full length mCOMP promoter cloned upstream of luciferase reporter gene was transfected into ATDC55 cells and stimulated by 500 μg·L -1 BMP 2 in the presence or absence of 10 μg·L -1 of Noggin. Results:We found that BMP 2 up regulated COMP gene expression by approximate 3 folds in human adult chondrocytes, and 1.5 folds in human fetal chondrocytes. The difference in magnitude of COMP gene stimulation by BMP 2 might attribute to the difference of COMP gene basal expression level in chondrocytes from different sources because it was found that the COMP gene expression was 2 folds higher in quiescent fetal chondrocytes than in adult chondrocytes. For further analysis of the effect of BMP 2 on COMP gene expression by RT PCR, a rat chondrogenic cell line of ATDC55 cells was used. While no COMP gene expression was detected in unstimulated cells, COMP expression was significantly induced after treatment with BMP 2. This induction could be specifically blocked by 10 μg·L -1 of Noggin. It was found that BMP 2 markedly increased the luciferase reporter activity by about 5 folds and again, Noggin specifically blocked the BMP 2 activity.Conclusion: BMP 2 up regulates the COMP gene expression in both primary human chondrocytes and chondrogenic cell line ATDC55.