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(typeⅡcollagen,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/m L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)、50μg/L BMP-7/多孔钽/软骨细胞组、100μg/L BMP-7/多孔钽/软骨细胞组及200μg/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,100μg/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,100μg/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200μg/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。
Objective：To study the influence of bone morphogenetic protein-7 （ BMP-7 ） on chondro-cyte 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） , 50μg/L BMP-7 group （50μg/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 chondro-cytes 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-Ⅱimmunocytochemistry 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 tan-talum by SEM observation.DMMB quantitative determination of GAG showed that GAG amount of chon-drocytes 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 group
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2015年第2期219-225,共7页
Journal of Peking University:Health Sciences
基金
"十二五"国家科技支撑计划项目(2012BAE06B03)资助~~
