Department of Orthopaedics, Dongguk University Ilsan Hospital, Republic of Korea.
Osteoarthritis Cartilage. 2011 Apr;19(4):449-57. doi: 10.1016/j.joca.2011.01.005. Epub 2011 Jan 18.
The aim of the present study was to determine if the electroporation-mediated gene transfer of SOX trio enhances the chondrogenic potential of adipose stem cells (ASCs).
ASCs were transfected with SOX trio genes using an electroporation technique and cultured for 3 weeks. The pellets were analyzed for DNA and glycosaminoglycan (GAG) analysis, and the gene and protein expression of SOX-5, SOX-6, SOX-9, type 1 collagen (COL1Al), type 2 collagen (COL2Al) and type 10 collagen (COL10A1) using real-time PCR and Western blot analysis. Further in vivo studies were carried out by subcutaneous transplantation of pellets in severe combined immunodeficiency (SCID) mice for 3 weeks.
The gene transfer efficiency was high (approximately 70%). Transfected ASCs showed high expression of corresponding genes after 21 days, and each SOX protein was detected in ASCs transfected with the corresponding gene. The chondrogenic differentiation of ASCs, as demonstrated by GAG levels and Safranin-O staining, showed significant enhancement when SOX trio were co-transfected, while subsets with single gene transfer of SOX-5, -6, or -9 did not show significant elevation. SOX trio co-transfection enhanced COL2A1 mRNA, but did not increase COL1A1 and COL10A1 mRNA. Type II collagen protein dramatically increased, and type X collagen decreased with co-transfection of the SOX trio. When pellets were implanted in the subcutaneous pouch of SCID mice for 3 weeks, ASCs co-transfected with SOX trio demonstrated abundant proteoglycan, significantly reduced mineralization.
The electroporation-mediated transfection of SOX trio greatly enhances chondrogenesis from ASCs, while decreasing hypertrophy.
本研究旨在探讨电穿孔介导的 SOX 三因子基因转染是否能增强脂肪干细胞（ASCs）的软骨形成潜力。
使用电穿孔技术将 SOX 三因子基因转染入 ASCs 并培养 3 周。分析细胞球的 DNA 和糖胺聚糖（GAG）分析，以及实时 PCR 和 Western blot 分析 SOX-5、SOX-6、SOX-9、I 型胶原（COL1Al）、II 型胶原（COL2Al）和 X 型胶原（COL10A1）的基因和蛋白表达。进一步通过将细胞球皮下移植到严重联合免疫缺陷（SCID）小鼠中进行 3 周的体内研究。
基因转染效率较高（约 70%）。转染后的 ASCs 在第 21 天表现出高表达相应基因的特征，并且在转染相应基因的 ASCs 中检测到每种 SOX 蛋白。通过 GAG 水平和番红 O 染色显示，当共转染 SOX 三因子时，ASCs 的软骨分化显著增强，而单独转染 SOX-5、-6 或 -9 的亚群则没有显著提高。SOX 三因子共转染增强了 COL2A1 mRNA，但没有增加 COL1A1 和 COL10A1 mRNA。II 型胶原蛋白显著增加，X 型胶原减少，与 SOX 三因子共转染。当细胞球植入 SCID 小鼠的皮下囊袋中 3 周时，共转染 SOX 三因子的 ASCs 表现出丰富的蛋白聚糖，显著减少矿化。
电穿孔介导的 SOX 三因子转染极大地增强了 ASCs 的软骨形成能力，同时减少了肥大。
