绿荧光蛋白基因修饰的大鼠骨髓间充质干细胞复合聚丙交酯-乙交酯膜的形态学观察

Morphological observation of GFP gene-modified mesenchymal stem cells cultured with PLGA substrate

目的 观察绿荧光蛋白（GFP）基因修饰的大鼠骨髓间充质干细胞（rMSCa）与聚丙交酯-乙交酯（PLGA）膜材料复合培养,为后续种子细胞.基因-支架材料-神经营养因子复合方法构建组织工程化脊髓的可行性提供依据.方法 GFP的过表达慢病毒载体（lv-GFP）转染的rMSCs-GFP按8000个细胞/cm2与PLGA膜材料复合培养,以普通培养板上的rMSC-GFP作为对照;荧光显微镜下观察细胞的形态学特性,噻唑蓝法测定每天细胞的生长增殖情况;流式细胞仪测定PLGA膜材料上第3天rMSCs-GFP的细胞周期,用FTTC标记的抗体CD34、CD90和用PE标记的抗CD44、CD106、CD45、CD11b对在膜材料上培养的第3天rMSCs-GFP进行流式细胞鉴定.结果 rMSCs-GFP在PLGA膜上贴壁生长,绝大多数MSCs可见绿色荧光,为成纤维细胞样形态,散在分布,第3天时细胞增多,开始变为多角形,第7天时细胞基本铺满膜,多为多角形和短梭形,与普通培养板上细胞相似,细胞的生长增殖在PLGA膜上与培养板上大致相同,细胞周期也大致相同[G1期细胞占89.7%,132期细胞占3.3%,S期细胞占7.0%,差异无统计学意义（P〉0.05）],细胞在PLGA膜上高表达CDg0（99.48%）、CD44（95.25%）、CDl06（77.12%）.结论 骨髓间充质干细胞是脊髓组织工程适宜的种子细胞,重组GFP的过表达慢病毒感染的后的MSCs与PLGA膜材料具有良好的组织相容性,可进一步复合方法体外构建组织工程脊髓.

Objective To investigate the feasibility of using PLGA loaded with SD rats＇ mesenchymal stem cells（rMSCs） transfected with green fluorescent protein （GFP） gene as scaffolds for combinations of molecular, cellular, and tissue-level treatments of spinal cord tissue engineering. Methods rMSCs infected with lentiviral vectors （lv-GFP） were seeded onto PLGA at 8000 cell/cm2, rMSCs-GFP grown under similar conditions on tissue culture plastic as control. The morphology of rMSC-GFP was examined by fluorescence microscopic. The activity of MSCs was detected by MTT assay everyday. Cell cycle analysis was performed after a 3-day culture on PLGA using flow cytometry. The rMSCs-GFP seeded on PLGA was identified by FITC-anti CD34,CD90 and PE-anti CD44, CD106,CD45,CDllb at the third day. Results Fluorescence microscopic examination revealed adherence of the cells to the PLGA surface within 24 h of initial plating. After 3 days, GFP cells were spindle shaped. The difference disappeared at 7 days when cells under both conditions had become confluent Cells proliferated at the same rate on the PLGA surface compared to tissue culture plastic. And cell＇s cycle was unaffected by the transduction process and seeded on PLGA. Cells maintained their stem cell phenotype as judged by expression of CD90, CD44, CD106 markers,and absence of the hematopoietic marker CD45, CD34. This demonstrated that the transduction and the PLGA surface were not adversely affecting the cells. Conclusions MSCs are a good candidate for spinal cord tissue engineering. Cells continued to express green fluorescent protein（GFP）on a long-term basis,and are compatible with polymer surfaces. Morphology,viability,and growth kinetics were maintained when cells were grown on a poly-lactic-co-glycolic acid（PLGA）polymer scaffold. Therefore,they could make further efforts for combinations of molecular, cellular,and tissue-level treatments of spinal cord tissue engineering.