振荡模式下TGF-β_1基因转染成肌诱导化ADSCs构筑工程化平滑肌的研究

Construction of Tissue-engineered Smooth Muscle by Loading ADSCs with Encapsulated TGF-β_1 Gene and Oscillating Seeding

目的:分析工程化平滑肌的体外生物学行为,评估构筑工程化平滑肌的理想方法,为修复女性尿道损伤提供新的方法。方法:转化生长因子β1腺病毒载体(Ad-TGF-β1)转染兔脂肪基质细胞(ADSCs),观察细胞形态、转染效率及细胞周期变化。接种到壳聚糖/胶原(Cs/Col)支架,构建工程化平滑肌:A组:凝胶+生长因子+振荡种植静态培养,B组:凝胶+生长因子+静态种植静态培养,C组:凝胶+基因转染+振荡种植静态培养,D组:静态种植静态培养。第1天、4天、8天、12天、16天、20天、24天、28天,电镜、共聚焦观察,检测细胞存活率、细胞增殖、Ⅲ型胶原及DNA含量。结果:C组细胞骨架发育完善,生长旺盛、细胞外基质丰富,细胞存活率高于其他组(P<0.05),增殖活力、Ⅲ型胶原及DNA含量均高于其他组(P<0.05)。结论:凝胶包埋基因与振荡模式能加速种子细胞扩增及优化工程化平滑肌构建质量。为细胞移植治疗尿道损伤开创了新的模式。

Objective：To analyze the biological behavior of tissue-engineered smooth muscle in vitro,and to evaluate an effective way in constructing tissue-engineered smooth muscle,which provide a new method for repair of women urethral injury.Methods：Adipose derived stromal cells（ADSCs）were transfected by adenovirus vector transforming growth factor-β1（Ad-TGF-β1）.The cell morphous,transfection rate and cell cycle were observed.Tissue-engineered smooth muscle was constructed by chitosan/collagen（Cs/Col）braces in the following ways：ADSCs were encapsulated with gels containing TGF-β1 plus oscillating seeding and static culture（group A）,ADSCs were encapsulated with gels containing TGF-β1 plus static culture ADSCs（group B）,and ADSCs were encapsulated with gels containing TGF-β1gene transfection plus oscillating seeding and static culture（group C）,and ADSCs were loaded in static seeding（group D）.Scan electron microscope and confocal laser microscope were used to observe cellular pattern and survival rate,as well as cell activity,collagen type Ⅲ and DNA level were detected at 1,4,8,12,16,20,24,28days.Results：New tissue showed more remarkable cell ingrowth with abundant extracellular matrix in group C,compared with other groups,the survival rate in group C was significantly higher（P0.05）.The levels of cell activity,collagen type Ⅲ,and DNA were significantly higher than other groups（P0.05）.Conclusions：gels encapsulation of TGF-β1 gene combined with oscillating seeding promotes the cell proliferation and improves the quality of tissue-engineered smooth muscle.It provides a new mode for treatment of urethral injury by cell transplantation.