CAJ | 학술논문

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／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）、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。目的：研究人重組骨形成蛋白-7（bone morphogenetic protein-7，BMP-7）對國產多孔鐽／軟骨細胞複閤物中軟骨細胞分泌功能以及Ⅱ型膠原（ typeⅡcollagen，Col-Ⅱ）、蛋白聚糖（ aggrecan，AGG）和SRY相關高遷移率組基因9（SRY-related high mobility group-box gene 9，Sox9）mRNA錶達的影響。方法：3週齡新西蘭幼兔，取雙膝關節軟骨細胞分離培養及鑒定，將生長狀態良好的第2代細胞以1×106／mL濃度接種于多孔鐽併給予不同濃度BMP-7，分為對照組（多孔鐽／軟骨細胞組）、50μg／L BMP-7／多孔鐽／軟骨細胞組、100μg／L BMP-7／多孔鐽／軟骨細胞組及200μg／L BMP-7／多孔鐽／軟骨細胞組。 CCK-8法檢測不同濃度BMP-7對多孔鐽／軟骨細胞生長及增殖的影響，掃描電鏡觀察各組軟骨細胞在多孔鐽錶麵及內部生長情況，二甲基亞甲藍（ dimethyl methylene blue，DMMB）比色定量法對BMP-7／多孔鐽／軟骨細胞複閤物糖胺多糖（ glycosaminoglycan，GAG）進行定量檢測，實時熒光定量PCR檢測Col-Ⅱ、AGG和Sox9 mRNA的錶達。結果：原代軟骨細胞培養24 h後呈梭形生長，4 d後細胞呈多角形，胞漿豐富。阿辛藍（ alcian blue）、番紅O（ safranin O）、Col-Ⅱ免疫細胞化學染色均呈暘性反應。 CCK-8法細胞增殖檢測結果顯示，100μg／L BMP-7／多孔鐽／軟骨細胞組細胞增殖水平高于其他各組（ P＜0．05）。掃描電鏡示各組軟骨細胞在多孔鐽錶麵生長狀態良好，細胞有多箇突起、伸展、疊層生長併覆蓋于多孔鐽錶麵。 GAG定量檢測顯示，100μg／L BMP-7／多孔鐽／軟骨細胞組GAG含量比其他各組均明顯升高（ P＜0．05）；實時熒光定量PCR檢測顯示，各實驗組Col-Ⅱ、AGG和Sox9 mRNA錶達量均高于對照組，以200μg／L BMP-7／多孔鐽／軟骨細胞組升高最為明顯（ P＜0．05）。結論：BMP-7／多孔鐽／軟骨細胞複閤體能促進體外軟骨細胞增殖及細胞外基質的分泌，促進軟骨基因的錶達。
목적：연구인중조골형성단백-7（bone morphogenetic protein-7，BMP-7）대국산다공단／연골세포복합물중연골세포분비공능이급Ⅱ형효원（ typeⅡcollagen，Col-Ⅱ）、단백취당（ aggrecan，AGG）화SRY상관고천이솔조기인9（SRY-related high mobility group-box gene 9，Sox9）mRNA표체적영향。방법：3주령신서란유토，취쌍슬관절연골세포분리배양급감정，장생장상태량호적제2대세포이1×106／mL농도접충우다공단병급여불동농도BMP-7，분위대조조（다공단／연골세포조）、50μg／L BMP-7／다공단／연골세포조、100μg／L BMP-7／다공단／연골세포조급200μg／L BMP-7／다공단／연골세포조。 CCK-8법검측불동농도BMP-7대다공단／연골세포생장급증식적영향，소묘전경관찰각조연골세포재다공단표면급내부생장정황，이갑기아갑람（ dimethyl methylene blue，DMMB）비색정량법대BMP-7／다공단／연골세포복합물당알다당（ glycosaminoglycan，GAG）진행정량검측，실시형광정량PCR검측Col-Ⅱ、AGG화Sox9 mRNA적표체。결과：원대연골세포배양24 h후정사형생장，4 d후세포정다각형，포장봉부。아신람（ alcian blue）、번홍O（ safranin O）、Col-Ⅱ면역세포화학염색균정양성반응。 CCK-8법세포증식검측결과현시，100μg／L BMP-7／다공단／연골세포조세포증식수평고우기타각조（ P＜0．05）。소묘전경시각조연골세포재다공단표면생장상태량호，세포유다개돌기、신전、첩층생장병복개우다공단표면。 GAG정량검측현시，100μg／L BMP-7／다공단／연골세포조GAG함량비기타각조균명현승고（ P＜0．05）；실시형광정량PCR검측현시，각실험조Col-Ⅱ、AGG화Sox9 mRNA표체량균고우대조조，이200μg／L BMP-7／다공단／연골세포조승고최위명현（ P＜0．05）。결론：BMP-7／다공단／연골세포복합체능촉진체외연골세포증식급세포외기질적분비，촉진연골기인적표체。
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 groups, compared with the control group and the best effect appeared when concentration of BMP-7 was 200μg/L.(P<0.05).Conclusion:BMP-7/tantalum/chondrocytes composites enhanced in vitro chon-drocyte proliferation and extracellular matrix greatly, and can promote chondrogenic gene expression.