瘦素基因修饰组织工程化复合物的体外构建

Construction of leptin gene modified tissue engineered composites in vitro

目的 :探讨人瘦素(human leptin,hLEP)基因修饰的大鼠骨髓基质细胞(bone marrow stromal cells,BMSCs)与引导组织再生胶原膜(Bio-Gide)复合体外构建组织工程化复合物的可行性。方法:全骨髓贴壁法分离培养大鼠BMSCs,将携带瘦素基因的腺病毒Ad-hLEP-EGFP感染BMSCs后,倒置荧光显微镜下观察绿色荧光,酶联免疫吸附法检测感染细胞hLEP的表达,MTT法检测感染后细胞的增殖活性。将经Ad-hLEP-EGFP感染后的BMSCs与Bio-Gide胶原膜体外复合培养24 h后,激光共聚焦显微镜和扫描电镜观察瘦素基因修饰组织工程复合物的构建情况。结果:采用Ad-hLEP-EGFP感染,可使BMSCs高表达hLEP,且细胞的增殖能力不变。扫描电镜结果显示,瘦素基因修饰的BMSCs在Bio-Gide胶原膜上生长良好,并分泌胞外基质。激光共聚焦显微镜显示瘦素基因修饰的BMSCs可迁移到Bio-Gide膜的不同层面。结论 :瘦素基因修饰的BMSCs能与Bio-Gide胶原膜复合,生长良好,可成功构建瘦素基因修饰的组织工程化复合物,该复合物有望用于牙周组织工程研究。

PURPOSE： To evaluate the feasibility of constructing tissue engineered composites in vitro by combining human leptin （hLEP） gene modified rat bone marrow stromal cells （BMSCs） and guided tissue regeneration collagen membrane （Bio-Gide）. METHODS： BMSCs of SD rats were isolated and cultured by whole bone marrow adherent method. BMSCS were transfected with adenovirus carrying hLEP gene （Ad-hLEP-EGFP） and observed under inverted fluorescence microscope. Enzyme linked immunosorbent assay （ELISA） was used to detect the expression of hLEP. The proliferation activity of transfected cells was assessed by MTY assay. Ad-hLEP-EGFP transfected BMSCs were cultured for 24 h in combination with Bio-Gide collagen membrane, hLEP modified tissue engineered composite was observed under laser scanning confocal microscope （LSCM） and scanning electron microscope （SEM）. RESULTS： Through Ad- hLEP-EGFP transfection, hLEP was overexpressed in BMSCs, which didn＇t affect the proliferation of cells. SEM showed hLEP modified BMSCs grew well on Bio-Gide collagen membrane and secreted extracellular matrix. LSCM suggested BMSCs could migrate to different scales of Bio-Gide collagen membrane. CONCLUSIONS： hLEP modified BMSCs can be combined with Bio-Gide collagen membrane and grow well, suggesting that hLEP modified tissue engineered composite can be successfully constructed. The composite might be suitable for periodontal tissue engineering.