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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。
OBJECTIVE: To investigate the feasibility of constructing tissue engineered complexes from human bone marrow stromal cells (BMSCs) modified by human leptin (hLEP) gene and bio-Gide complex . Methods: BMSCs were isolated and cultured by whole bone marrow adherent method. Ad-hLEP-EGFP carrying leptin gene was used to infect BMSCs. The green fluorescence was observed under inverted fluorescence microscope. The expression of hLEP was detected by enzyme-linked immunosorbent assay (ELISA) MTT assay was used to detect the proliferative activity of infected cells. BMSCs infected with Ad-hLEP-EGFP and Bio-Gide collagen membrane were cultured in vitro for 24 h, and the construction of leptin gene modified tissue engineering complex was observed by confocal laser scanning microscopy and scanning electron microscopy. Results: Using Ad-hLEP-EGFP infection, BMSCs could express high level of hLEP with no change in cell proliferation ability. Scanning electron microscopy results showed that leptin gene-modified BMSCs grew well on Bio-Gide collagen membrane and secreted extracellular matrix. Laser confocal microscopy revealed that leptin-genetically modified BMSCs migrated to different layers of the Bio-Gide membrane. CONCLUSION: Leptin gene-modified BMSCs can be complexed with Bio-Gide collagen membrane and grow well. The leptin gene-modified tissue engineering complex can be successfully constructed and is expected to be useful for periodontal tissue engineering research.