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北京大学学报（医学版） ›› 2015, Vol. 47 ›› Issue (2): 219-225. doi: 10.3969/j.issn.1671-167X.2015.02.006

• 论著 • 上一篇    下一篇

骨形成蛋白-7对多孔钽/软骨细胞复合物分泌功能以及Col-Ⅱ、AGG和Sox9基因表达的影响

张辉1,2，李亮3，王茜1,4，甘洪全3，王辉1,5，毕成1,3，李琪佳6，王志强1,3△   

  1. （1. 南方医科大学研究生学院，广州510515； 2. 唐山市第二医院关节一科，河北唐山063000； 3. 河北联合大学附属医院骨科，河北唐山063000； 4. 河北联合大学基础医学院人体解剖系，河北唐山063000； 5. 唐山市第二医院手一科，河北唐山063000； 6. 河北联合大学实验中心，河北唐山063000）
  • 出版日期:2015-04-18 发布日期:2015-04-18

Influence of BMP-7 on chondrocyte secretion and expression of Col-Ⅱ，AGG and Sox9 mRNA in porous tantalum-chondrocyte composites in vitro

ZHANG Hui1,2, LI Liang3, WANG Qian1, 4, GAN Hong-quan3, WANG Hui1, 5, BI Cheng1, 3, LI Qi-jia6, WANG Zhi-qiang1,3△   

  1. (1. Graduate School, Southern Medical University, Guangzhou 510515, China; 2. Department of Joint Surgery, the Second Hospital of Tangshan, Hebei Tangshan 063000, China; 3. Department of Orthopaedics, Hebei United University Affiliated Hospital, Hebei Tangshan 063000, China; 4. Department of Anatomy, Basic Medical College of Hebei United University, Hebei Tangshan 063000, China; 5. Department of Hand Surgery, the Second Hospital of Tangshan, Hebei Tangshan 063000, China; 6. Experimental Center, Hebei United University, Hebei Tangshan 063000, China)
  • Online:2015-04-18 Published:2015-04-18

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摘要： 目的：研究人重组骨形成蛋白-7（bone morphogenetic protein-7，BMP-7）对国产多孔钽/软骨细胞复合物中软骨细胞分泌功能以及Ⅱ型胶原（type Ⅱ collagen，Col-Ⅱ）、蛋白聚糖（aggrecan，AGG）和SRY相关高迁移率组基因9（SRY-related high mobility group-box gene 9，Sox9）mRNA表达的影响。方法：3周龄新西兰幼兔，取双膝关节软骨细胞分离培养及鉴定，将生长状态良好的第2代细胞以1×106/mL浓度接种于多孔钽并给予不同浓度BMP-7，分为对照组（多孔钽/软骨细胞组）、50 μg/L BMP-7/多孔钽/软骨细胞组、100 μg/L BMP-7/多孔钽/软骨细胞组及200 μg/L BMP-7/多孔钽/软骨细胞组。CCK-8法检测不同浓度BMP-7对多孔钽/软骨细胞生长及增殖的影响，扫描电镜观察各组软骨细胞在多孔钽表面及内部生长情况，二甲基亚甲蓝（dimethyl methylene blue，DMMB）比色定量法对BMP-7/多孔钽/软骨细胞复合物糖胺多糖（glycosaminoglycan，GAG）进行定量检测，实时荧光定量PCR检测Col-Ⅱ、AGG和Sox9 mRNA的表达。结果：原代软骨细胞培养24 h后呈梭形生长，4 d后细胞呈多角形，胞浆丰富。阿辛蓝（alcian blue）、番红O（safranin O）、Col-Ⅱ免疫细胞化学染色均呈阳性反应。CCK-8法细胞增殖检测结果显示，100 μg/L BMP-7/多孔钽/软骨细胞组细胞增殖水平高于其他各组（P<0.05）。扫描电镜示各组软骨细胞在多孔钽表面生长状态良好，细胞有多个突起、伸展、叠层生长并覆盖于多孔钽表面。GAG定量检测显示，100 μg/L BMP-7/多孔钽/软骨细胞组GAG含量比其他各组均明显升高（P<0.05）；实时荧光定量PCR检测显示，各实验组Col-Ⅱ、AGG和Sox9 mRNA表达量均高于对照组，以200 μg/L BMP-7/多孔钽/软骨细胞组升高最为明显（P<0.05）。结论：BMP-7/多孔钽/软骨细胞复合体能促进体外软骨细胞增殖及细胞外基质的分泌，促进软骨基因的表达。

关键词: 骨形态发生蛋白7, 钽, 软骨细胞, 胶原Ⅱ型, 葡糖氨基聚糖类

Abstract: Objective： To study the influence of bone morphogenetic protein-7 (BMP-7) on chondrocyte secretion and expression of type Ⅱ collagen (Col-Ⅱ), aggrecan (AGG) and SRY-related high mobility group-box gene 9 (Sox9) mRNA in porous tantalum-chondrocyte composites. Methods: The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified. The 2nd generation of chondrocytes with 1×106/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group (tantalum/chondrocyte), 50 μg/L BMP-7 group (50 μg/L BMP-7/tantalum/chondrocyte), 100 μg/L BMP-7 group (100 μg/L BMP-7/tantalum/chondrocyte), and 200 μg/L BMP-7 group (200 μg/L BMP-7/tantalum/chondrocyte). The proliferation of chondrocytes was measured by CCK-8 assay. The chondrocyte growth and morphology were observed by scanning electron microscopy (SEM). The synthesis of glycosaminoglycan (GAG) in chondrocytes was tested by dimethyl methylene blue (DMMB) colorimetric quantification method. Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were detected by realtime PCR. Results: The chondrocytes were spindle--shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days. The chondrocytes were affirmed by alcian blue, safranin O and Col-Ⅱ immunocytochemistry staining. The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups (P<0.05). The chondrocytes implanted into porous tantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tantalum by SEM observation. DMMB quantitative determination of GAG showed that GAG amount of chondrocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups (P<0.05). The expressions of Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were up-regulated in the experimental groups, compared with the control group and the best effect appeared when concentration of BMP-7 was 200 μg/L. (P<0.05). Conclusion: BMP-7/tantalum/chondrocytes composites enhanced in vitro chondrocyte proliferation and extracellular matrix greatly, and can promote chondrogenic gene expression.

Key words: Bone morphogenetic protein 7, Tantalum, Chondrocytes, Collagen type Ⅱ, Glycosaminoglycans

中图分类号: 

  • R684

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ISSN 1671-167X
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