兔脂肪干细胞体外构建组织工程软骨：负载胰岛素样生长因子1基因的作用

背景：前期研究证实在二维培养条件下，转染胰岛素样生长因子1基因能够明显促进脂肪间充质干细胞的增殖。 目的：在前期研究基础上，利用壳聚糖明胶复合物为立体培养的载体，观察三维立体培养条件下胰岛素样生长因子1基因转染对兔脂肪间充质干细胞增殖能力的影响，体外初步构建组织工程软骨。 方法：分离、培养成年新西兰大耳白兔脂肪间充质干细胞，构建稳定表达pcDNA3.1-IGF-1的细胞株，鉴定后接种于壳聚糖三维支架材料上，分组培养：空白对照组接种未转染基因的脂肪间充质干细胞，空载体组接种胰岛素样生长因子1诱导条件下培养的脂肪间充质干细胞，基因转染组接种稳定转染胰岛素样生长因子1基因的脂肪间充质干细胞，培养1周。扫描电镜观察各组细胞形态，MTT法绘制细胞增殖曲线，CM-DiL荧光标记后观察细胞增殖比率及细胞分布，DMMB法测定糖胺聚糖含量。 结果与结论：成功构建稳定表达pcDNA3.1-IGF-1的细胞株，转染后的细胞株在mRNA水平上获得胰岛素样生长因子1的表达，并成功翻译为蛋白。扫描电镜示各组细胞在支架上贴附、伸展良好，基因转染组细胞生长最为旺盛。MTT 及GAG检测结果提示，基因转染组增殖能力最强、糖胺聚糖最高(P < 0.01)，荧光标记显示细胞分布均匀，存活率高(P < 0.05)。提示胰岛素样生长因子1基因转染联合三维立体培养能够有效促进兔脂肪间充质干细胞的增殖和软骨细胞外基质糖胺聚糖的分泌。

In vitro construction of tissue-engineered cartilage using rabbit adipose-derived mesenchymal stem cells: Effects of transfected insulin-like growth factor-1 gene

BACKGROUND: The preliminary study confirmed that transfection of insulin-like growth factor-1 (IGF-1) gene can significantly promote the proliferation of adipose-derived mesenchymal stem cells (ADSCs) in two-dimensional culture conditions. OBJECTIVE: On the basis of preliminary study, to investigate the proliferation capacity of rabbit ADSCs transfected with IGF-1 gene in three-dimensional culture conditions using chitosan gelatin complex scaffold as carrier, and to construct tissue-engineered cartilage initiatively. METHODS: ADSCs were harvested from the posterior subcutaneous adipose tissue of adult New Zealand white rabbits. After being transfected with pcDNA3.1-IGF-1, the cells were seeded onto Chitosan-gelatin scaffolds, and divided into groups: Blank control group, non-transfected ADSCs was incubated; Empty vector group: ADSCs induced by IGF-1 was incubated; Gene transfection group was transfected ADSCs/scaffold composite. After culture for 1 week, cell morphology was observed by scanning electron microscope (SEM), and the growth curve was measured by MTT, the proliferation and distribution of cells were labeled by CM-DiL, and the contents of glycosaminoglycan in each group were determined by using dimethylmethylene blue dye-binding assay. RESULTS AND CONCLUSION: The stable expression of pcDNA3.1-IGF-1 cell lines were established successfully and showed stable expression of IGF-I in mRNA and protein level. SEM showed that the cells exhibited a good attachment and stretch in the scaffold, in which gene transfection group grew the most vigorously. MTT and GAG results suggested that gene transfection group displayed strongest proliferate ability with highest glycosaminoglycan content (P < 0.01). CM-DiL fluorescence showed that the cells distributed well with the highest survival rate (P < 0.05). Those revealed that transfected rabbit ADSCs with IGF-1 gene can promote the proliferation of ADSCs/scaffold composite and the secretion of chondral extracellular matrix such as glycosaminoglycan.