软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein，COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化，通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP，采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化；通过茜素红染色观察成骨终末阶段矿化结节的生成情况，阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后，成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05），RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05），而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05），SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组，而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05），结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化，促进骨髓间充质干细胞成软骨分化，并抑制软骨细胞的成熟肥大，为软骨组织工程研究提供新的方向。

Effects of Cartilage Oligomeric Matrix Protein Overexpression on BMP-2 Induced Cell Differentiation of Bone Marrow Mesenchymal Stem Cells

Objective: To study the effects of cartilage oligomeric matrix protein (COMP) overexpression on bone morphogenetic protein 2 (BMP-2) induced cell osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). Methods: MSCs, transfected with plasmid DNA encoding recombinant human COMP, were induced to differentiate into osteocytes and chondrocytes by BMP-2. Western blotting and real-time PCR were used to detect the overexpression of COMP, the expression level changes of osteogenesis related genes collagen type Ⅰ, RUNX2, osteocalcin and chondrogenesis related genes collagen type Ⅱ, SOX9, aggrecan, collagen type X, respectively. Alizarin red staining for osteogenic differentiation and alcian blue staining for chondrogenic differentiation were conducted to evaluate the tendency of cell differentiation. Results: Results of RT-PCR and Western blotting showed that the expression levels of COMP mRNA and protein were significantly increased in MSCs, which were transfected with plasmid DNA encoding recombinant human COMP (P<0.05). The mRNA expression levels of RUNX2, Col1a1 and the protein expression levels of RUNX2, osteocalcin in overexpressed group all decreased significantly compared with that of control group (P<0.05). However, the mRNA expression levels of SOX9, aggrecan and the protein expression levels of SOX9, Col2a1 in overexpressed group all increased significantly compared with that of control group (P<0.05). Alizarin red staining were weakened while alcian blue staining was enhanced. The expression level of Col10a1 gene in overexpressed group was higher than that in control group (P<0.05). Conclusion: Overexpression of COMP gene in MSCs could inhibit BMP-2 induced osteogenic differentiation, promote BMP-2 induced chondrogenic differentiation and supress the chondrocyte hypertrophy and maturation, which may provide new insight for cartilage tissue engineering.