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

doi:10.13523/j.cb.20161001

中国生物工程杂志

2016, Vol. 36

Issue (10): 1-7 DOI: 10.13523/j.cb.20161001

研究报告

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

沈鹏飞, 王斌, 谢子康, 郑冲, 瞿玉兴

常州市中医医院常州 213000

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

SHEN Peng-fei, WANG Bin, XIE Zi-kang, ZHENG Chong, QU Yu-xing

First Department of Orthopaedics, Changzhou TCM Hospital, Changzhou 213000, China

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摘要：

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。

关键词： 软骨寡聚基质蛋白; 骨髓间充质干细胞; 骨形态发生蛋白质2; 细胞分化; 基因过表达

Abstract:

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.

Key words: Gene overexpression Mesenchymal stem cells Cartilage oligomeric matrix protein Bone morphogenetic protein 2 Cell differentiation

收稿日期: 2016-04-05 出版日期: 2016-10-25

ZTFLH:

R684

基金资助:

常州市卫生计生委青年科技资助项目（QN201501）

通讯作者: 沈鹏飞,电子信箱:pfshen836@126.com E-mail: pfshen836@126.com

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引用本文:

沈鹏飞, 王斌, 谢子康, 郑冲, 瞿玉兴. 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[J]. 中国生物工程杂志, 2016, 36(10): 1-7.

SHEN Peng-fei, WANG Bin, XIE Zi-kang, ZHENG Chong, QU Yu-xing. Effects of Cartilage Oligomeric Matrix Protein Overexpression on BMP-2 Induced Cell Differentiation of Bone Marrow Mesenchymal Stem Cells. China Biotechnology, 2016, 36(10): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20161001 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I10/1

[1] 虞冀哲, 董学海, 陈海丹, 等. 间充质干细胞应用于软骨组织工程的研究进展. 生物骨科材料与临床研究, 2015, 12(4): 60-64. Yu J Z, Dong X H, Chen H D, et al. Reviewed the research progress of cartilage tissue engineering using mesenchymal stem cells. Orthop Biomech Mater Clin Study, 2015, 12(4): 60-64.
[2] Liang Y, Fu Y, Qi R, et al. Cartilage oligomeric matrix protein is a natural inhibitor of thrombin. Blood, 2015, 126(7): 905-914.
[3] Kluzek S, Bay-Jensen A C, Spector T, et al. Higher serum levels of cartilage oligomeric matrix protein (comp) are associated with self-reported knee pain. Osteoarthr Cartilage, 2014, 22(3): S74.
[4] Kluzek S, Bay-Jensen A C, Judge A, et al. Serum cartilage oligomeric matrix protein and development of radiographic and painful knee osteoarthritis. A community-based cohort of middle-aged women. Biomarkers, 2015, 20(8): 557-564.
[5] Kim M J, Lee B, Yang K, et al. BMP-2 peptide-functionalized nanopatterned substrates for enhanced osteogenic differentiation of human mesenchymal stem cells. Biomaterials, 2013, 34(30): 7236-7246.
[6] Barati D, Shariati S R, Moeinzadeh S, et al. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel. J Controll Release, 2016, 223: 126-136.
[7] Guzzo R M, Gibson J, Xu R H, et al. Efficient differentiation of human iPSC-derived mesenchymal stem cells to chondroprogenitor cells. J Cell Biochem, 2013, 114(2): 480-490.
[8] 朱瑜琪, 王金荣, 王智耀. 间充质干细胞促进关节软骨的修复与再生. 中国组织工程研究, 2015, 19(50): 8195-8200. Zhu Y Q, Wang J R, Wang Z Y. Mesenchymal stem cells promote articular cartilage repair and regeneration. Chin J Tissue Eng Res, 2015, 19(50): 8195-8200.
[9] 曲峰, 袁邦拓, 齐玮, 等. Wnt3a 对骨髓间充质干细胞成软骨分化的影响. 中国矫形外科杂志, 2016, 24(2): 155-159. Qu F, Yuan B T, Qi W, et al. Effect of Wnt3a on chondrogenic differentiation of nesenchymal stem cells. Orthop J China, 2016, 24(2): 155-159.
[10] Dharmavaram R M, Liu G, Tuan R S, et al. Stable transfection of human fetal chondrocytes with a type II procollagen minigene: expression of the mutant protein and alterations in the structure of the extracellular matrix in vitro. Arthritis Rheum, 1999, 42(7): 1433-1442.
[11] Du Y, Wang Y, Wang L, et al. Cartilage oligomeric matrix protein inhibits vascular smooth muscle calcification by interacting with bone morphogenetic protein-2. Circ Res, 2011, 108(8): 917-928.
[12] Ishida K, Acharya C, Christiansen B A, et al. Cartilage oligomeric matrix protein enhances osteogenesis by directly binding and activating bone morphogenetic protein-2. Bone, 2013, 55(1): 23-35.
[13] Guo P, Shi Z L, Liu A, et al. Effects of cartilage oligomeric matrix protein on bone morphogenetic protein-2-induced differentiation of mesenchymal stem cells. Orthop Surg, 2014, 6(4): 280-287.
[14] Wang W J, Sun C, Liu Z, et al. Transcription factor Runx2 in the low bone mineral density of girls with adolescent idiopathic scoliosis. Orthop Surg, 2014, 6(1): 8-14.
[15] Ge C, Cawthorn W P, Li Y, et al. Reciprocal control of osteogenic and adipogenic differentiation by ERK/MAP kinase phosphorylation of Runx2 and PPARγ transcription factors. J Cell Physiol, 2016, 231(3): 587-596.
[16] Liu C F, Lefebvre V. The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis. Nucleic Acids Res, 2015, 43(17): 8183-8203.
[17] Gurusinghe S, Young P, Michelsen J, et al. Suppression of differentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors. Biotechnol Lett, 2015, 37(7): 1495-1504.
[18] 王冠, 陈星星, 薛鑫, 等. Ⅱ 型胶原体外促进大鼠骨髓间充质干细胞向软骨细胞分化. 第三军医大学学报, 2015, 37(9): 886-890. Wang G, Chen X X, Xue X, et al. Collagen Ⅱ promotes differentiation of rat bone marrow mesenchymal stem cells into chondrocytes in vitro. J Third Mil Med Univ, 2015, 37(9): 886-890.
[19] Lu Y, Ding M, Li N, et al. Col10a1-Runx2 transgenic mice with delayed chondrocyte maturation are less susceptible to developing osteoarthritis. Am J Transl Res, 2014, 6(6): 736-745.
[20] Gu J, Lu Y, Li F, et al. Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation. Cell Death Dis, 2014, 5(10): e1469.

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