Pellet培养与纤维蛋白凝胶支架体外成软骨能力的比较  

Comparison of in vitro chondrogenic ability between two culture modes:pellet culture system and fibrin gel scaffold

作　　者：黄亮节[1] 翁土军[2] 张春丽[2] 刘彦[2] 钱隆[2] 侯树勋[2] HUANG Liang-jie WENG Tu-jun ZHANG Chun-li LIU Yan QIAN Long HOU Shu-xun(Xijing Hospital, the fourth Military Medical University, Xi＇an, Shanxi, 710032, China)

机构地区：[1]第四军医大学西京医院,西安710032 [2]解放军总医院第一附属医院骨科、北京市骨科植入医疗器械工程技术研究中心,北京100048

出　　处：《中国骨与关节杂志》2017年第3期198-203,共6页Chinese Journal of Bone and Joint

摘　　要：目的比较Pellet培养和纤维蛋白凝胶支架两种培养方式诱导脂肪干细胞向软骨细胞分化及合成细胞外基质的差异。方法人脂肪组织酶消化分离脂肪干细胞,培养基为DMEM含10%胎牛血清,利用Pellet培养和纤维蛋白凝胶支架两种培养体系将P3代脂肪干细胞诱导成软骨后21天取材进行苏木精-伊红染色,番红O染色,DMMB法测定胞外基质中GAG含量,荧光定量PCR测定Ⅱ型胶原基因表达。结果苏木精-伊红染色与番红-O染色显示,纤维蛋白凝胶实验组软骨细胞外基质大量分泌,并融合成片,说明纤维蛋白凝胶支架可更好促进干细胞成软骨分化、维持软骨细胞表型。GAG/DNA结果显示,纤维蛋白凝胶组促GAG分泌的能力优于其余各组(P<0.05),其Ⅱ型胶原m RNA表达的能力也最强(P<0.05)。结论利用纤维蛋白凝胶支架诱导人脂肪干细胞成软骨分化的能力优于无支架的Pellet三维培养体系。Objective To compare the effects of 2 different culture modes： pellet culture system and fibrin gel scaffold on the chondrocytic differentiation of adipose-derived stem cells（ ADSCs） and the synthesis of cartilage extracellular matrix. Methods Adipose tissues were used to isolate human ADSCs and grown in Dulbecco＇s modified Eagle＇s medium（ DMEM） containing 10% fetal bovine serum（ FBS）. After the human ADSCs were cultured to the 3rd generation, they were induced into cartilage by 2 kinds of culture modes respectively. At the 21 st day, the cells were observed by hematoxylin-eosin（ HE） staining and safranine-O staining. The glycosaminoglycan（ GAG） content in extracellular matrix was determined by content dimeth-ylmethylene blue（ DMMB） method. The expression of type Ⅱ collagen gene was assessed by real-time polymerase chain reaction（ PCR）. Results HE staining and safranine-O staining showed that the extracellular matrix of chondrocytes was strongly secreted in the fibrin gel scaffold group with transforming growth factor β3（ TGFβ3） and fused into a sheet. It＇s indicated that the fibrin gel scaffold could better promote cartilage differentiation of stem cells and maintain chondrocyte type. The results of GAG / deoxyribonucleic acid（ DNA） showed that the ability to secrete GAG in the fibrin gel scaffold group with TGFβ3 was better than that of the other group（ P〈0.05）, and the expression of type Ⅱ collagen messenger ribonucleic acid（ m RNA） was the strongest（ P〈0.05）. Conclusions The ability of fibrin gel scaffold to differentiate ADSCs into cartilage is better than that of scaffold-free pellet culture system.

关 键 词：纤维蛋白 干细胞 组织工程 软骨 Pellet培养 脂肪源干细胞 

分 类 号：R684[医药卫生—骨科学]