兔骨髓间充质干细胞在壳聚糖支架上的诱导培养  

In vitro induced culture of the rabbit bone marrow mesenchymal stem cells on chitosan scaffolds

作　　者：张海龙[1] 傅强[1] 赵鑫[1] 陈元贵[1] 吴大江[1] 侯铁胜[1] 

机构地区：[1]第二军医大学附属长海医院骨科,上海市200433

出　　处：《中国脊柱脊髓杂志》2008年第5期385-388,I0001,共5页Chinese Journal of Spine and Spinal Cord

摘　　要：目的:观察在壳聚糖(Chitosan)三维支架上诱导兔骨髓间充质干细胞(rMSCs)向髓核样细胞分化的情况,探讨应用其制作组织工程髓核的可能性。方法:将体外培养的第3代rMSCs接种于经冷冻干燥法构建的壳聚糖三维支架上,在主要含有转化生长因子β1(TGF-β1)、胰岛素+转铁蛋白+亚硒酸钠(ITS)、地塞米松、丙酮酸钠的培养液中培养(实验组),同时设立对照组(非诱导组)。在培养7d、14d、21d时取标本行组织学检测,观察三维支架上rMSCs的生长及分化情况,对支架细胞复合物行Ⅱ型胶原免疫组化检测,测定培养液中糖胺聚糖(GAG)含量及Ⅱ型胶原的表达量,计算细胞外基质GAG含量。结果:rMSCs在壳聚糖三维支架上生长状态良好,体外培养7d、14d、21d时,实验组培养液中细胞外基质GAG含量分别为:55.8±4.6、86.7±5.8、83.2±3.4μg/cm2,对照组培养液中细胞外基质GAG含量分别为:34.2±5.6、42.6±4.8、40.8±4.4μg/cm2,两组间相同时间点比较有显著性差异(P<0.05);Ⅱ型胶原蛋白电泳及Western-blot检测实验组在培养14d与21d时可见到Ⅱ型胶原电泳条带,21d时Ⅱ型胶原蛋白含量多于14d时;对照组未见Ⅱ型胶原表达。结论:rMSCs在壳聚糖三维支架上培养生长良好,在特定的诱导培养液诱导下可以向髓核样细胞分化。Objective：To explore the characteristics of mesenchymal stem cells （rMSCs） derived from rabbit bone marrow which were cultured on three-dimensional scaffolds of chitosan,and discuss the feasibility of nucleus pulposus （NP） tissue-engineering regeneration.Method：The rMSCs were isolated from the rabbit bone marrow and then subcultured to the third passage.The rMSCs were seeded on the chitosan scaffolds and induced into NP-like cells by adding transforming growth factor β1（TGF-β1）,dexamethasone,ITS（insulin/transferring/sodiumselenite/ascorbic acid） into the medium.At the end of the every culture period（7d,14d,21d）,the compound of scaffolds and rMSCs were processed for histological study,and the expression of collagen type Ⅱ was analyzed with immunohistochemical study,the contents of GAG and collagen type Ⅱ in culture medium were detected as well.Then the glycosaminoglycan（GAG） content in the extracellular matrix（ECM） was calculated.Result：The third passage cells grew well on the chitosan scaffolds.At the end of the every culture period （7d,14d,21d）,the GAG contents in the ECM were 55.8±4.6μg/cm^2,86.7±5.8μg/cm^2,83.2±3.4μg/cm^2 in induced culture group vs. 34.2±5.6μg/cm^2,42.6±4.8μg/cm^2,40.8±4.4μg/cm^2 in the control group.At each time point, there was significant difference in the GAG content in the ECM between control group and induced culture group（P〈0.05）.The differentiation of rMSCs towards NP-like cells on the scaffolds was verified by the positive results of with sodium dodecyl sulfate polyacryl-amide gel electrophoresis （SDS-PAGE） and Western blot.The content of collagen type Ⅱ was higher in the induced group cultured for 21 days than 14 days. While there was no expression of collagen type Ⅱ had been detected in the control group.Conclusion： Under certain conditions,the rMSCs growing on the scaffolds of chitosan may differentiate towards the NP-like cells.

关 键 词：骨髓间充质干细胞 组织工程 壳聚糖 支架 兔 

分 类 号：Q813.1[生物学—生物工程]