动态压力对负载胰岛素样生长因子1基因兔脂肪间充质干细胞增殖能力和机械性能的影响

Effects of dynamic pressure on the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells transfected with insulin-like growth factor-1

背景:脂肪间充质干细胞是目前公认的较为优秀的组织工程软骨种子细胞,基因转染技术可以有效诱导其成软骨分化,应用生物反应器模拟体内力学环境,是目前广大学者探索体外构建组织工程软骨的新思路。目的:探讨周期性动态压应力联合胰岛素样生长因子1基因转染对种植于壳聚糖/明胶支架中的兔脂肪间充质干细胞增殖能力和弹性模量的影响。方法:在脂质体介导下应用pcDNA3.1-IGF-1基因转染兔脂肪间充质干细胞,G418筛选,获得稳定转染的细胞株;将负载或不负载胰岛素样生长因子1基因的脂肪间充质干细胞以5×10^10 L^-1的密度接种于壳聚糖/明胶支架中培养2 d,然后分别于动态压力(2%形变,1 Hz,4 h/d)环境中或静态培养条件下培养7 d;扫描电镜、Masson三色染色及阿尔新蓝染色观察细胞/支架复合物的形态学变化,MTT法绘制细胞增殖曲线,CM-Dil荧光染料标记细胞计数评估细胞增殖效率,DMMB定量测定软骨特异性细胞外基质糖胺聚糖含量,Real time PCR法检测软骨特异性基因Ⅱ型胶原的相对表达,应用BioDynamicTM mechanical tester测量各组细胞/支架复合物不同形变时的轴线压应力,计算对应的弹性模量。结果与结论:动态压力联合胰岛素样生长因子1转染可显著提高脂肪间充质干细胞的增殖能力,细胞分布更为均匀,软骨特异性细胞外基质糖胺聚糖及胶原分泌增多,上调Ⅱ型胶原基因的表达,机械性能显著改善。单纯胰岛素样生长因子1组的细胞增殖能力和弹性模量均优于单独动态压力组,但动态压力条件下细胞在支架中分布更为均匀。结果表明,动态压力与胰岛素样生长因子1基因转染均可显著提升兔脂肪间充质干细胞的增殖能力和机械性能,二者呈协同作用。

BACKGROUND: Adipose mesenchymal stem cells are currently recognized as excellent seed cells for tissue engineering cartilage. Gene transfection technology can effectively induce them to differentiate into cartilage. The bioreactor is used to simulate the mechanical environment in vivo. It is a new idea for the majority of scholars to explore the construction of tissue engineering cartilage in vitro. OBJECTIVE: To investigate the effects of cyclic dynamic compressive stress combined with insulin-like growth factor-1 gene transfection on the proliferation and elastic modulus of rabbit adipose mesenchymal stem cells implanted in chitosan/gelatin scaffold.METHODS: Rabbit adipose mesenchymal stem cells were transfected with pcDNA3.1-IGF-1 gene mediated by liposome. The stable transfected cell lines were screened by G418. The adipose mesenchymal stem cells transfected with or without insulin-like growth factor-1 gene were inoculated in chitosan/gelatin scaffold at the density of 5×1010L-1 for 2 days, and cultured under dynamic pressure(2% at 1 Hz, 4 hours per day) or static culture conditions for 7 days, respectively. The morphological changes of the cell/scaffold complex were observed by scanning electron microscope, Masson trichrome staining and alcian blue staining. The cell proliferation curve was drawn by MTT assay. The cell proliferation efficiency and distribution were evaluated by CM-Dil fluorescencelabeling method, and the content of total glycosaminoglycan was quantitatively determined by DMMB. The differences of type Ⅱ collagen among different groups were compared with real time PCR. Compressive mechanical properties of the cell/scaffold constructs were assessed using a BioDynamicTM mechanical tester, and the corresponding elastic modulus was calculated.RESULTS AND CONCLUSION: Dynamic pressure combined with insulin-like growth factor-1 transfection could significantly improve the cell proliferation ability of the cell/scaffold complex;the cell distribution was more uniform;glycosaminoglycan and collagen secretion in the cartilage-specific extracellular matrix were increased;the expression levels of type Ⅱ collagen were up-regulated;and the mechanical properties were significantly improved. The cell proliferation and elastic modulus of insulin-like growth factor-1 group were better than those of single pressure group, but the distribution of cells in scaffolds was more uniform under dynamic pressure. The results indicate that both dynamic pressure and insulin-like growth factor-1 gene transfection can significantly improve the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells;the two have synergistic effect.