载基因pcDNA-EGFP-TGFβ1壳聚糖微球转染滑膜间充质干细胞的初步研究

Preliminary Study of Chitosan/pcDNA-EGFP-TGFβ1 Nanoparticles Used in the Transfection of Synovial-derived Mesenchymal Stem Cells

本研究目的是探讨载基因壳聚糖／pcDNAEGFP—TGF~I纳米微球转染滑膜间充质干细胞（SDMSCs）的能力。通过离子交联法制备壳聚糖／pcDNA—EGFP—TGFβ1复合物，扫描电镜检测复合物的形貌和粒径。获取兔膝关节滑膜组织，分离培养SDMSCs至第3代，用壳聚糖／pcDNA—EGFPTGFβ1复合物转染，荧光显微镜和流式细胞仪检测转染情况。结果显示，制备的壳聚糖／pcDNA—EGFP—TGFβ11复合物溶液均匀透亮，复合物为大小形态较为一致的球形，粒径约50nm；转染48h的SDMSCs约8％～10％转染成功，绿色荧光颗粒主要分布在胞浆和胞核内；流式细胞仪检测结果显示转染后的SDMSCs活性较好。因此，离子交联法制备的壳聚糖／pcDNA—EGFPTGFβ1纳米微球可以转染SDMSCs，但其转染效率有待进一步提高。

The objective of this study is to explore the application possibility of chitosan/pcDNA-EGFP-TGFβ1 nano- particles in the transfection of synovial-derived mesenchymal stem cells （SDMSCs）. Chitosan/pcDNA-EGFP TGFβ1 nanoparticies were fabricated through method of ionic crosslinking. The SDMSCs were harvested from rabbit joints and cultured to passage 3. The SDMSCs were then transfected with chitosan/pcDNA-EGFP TGFβ1 nanopartieles. Scanning electronic microscope （SEM） was employed to detect the shape and diameter of the nanoparticles. The transfected SDMSCs were examined under the fluorescence microscope and detected through the flow cytometry （FCM）. The SEM examination showed that the contour of the fabricated chitosan/pcDNA-EGFP-TGFβl nanoparti cles was round and its average diameter was 50nm. After being cultured for 48h, the SDMSCs transfected by chi- tosan/pcDNA-EGFP TGFβ1 nanoparticles could be detected under the fluorescence microscope, and the live SDM- SCs could also be examined through FCM. The transfection rate was 8% - 10%. Therefore, it suggested that the chitosan/pcDNA-EGFP TGFI31 nanoparticles fabricated through the method of ionic crosslinking could transfect the SDMSCs, but the transfection rate should be improved.