稳定过表达BDNF和NT-3基因的脂肪干细胞系的建立及其意义

Construction of adipose-derived stem cells line that can stably express brain-derived neurotrophic factor and neurotrophin-3 genes and its significance

目的建立稳定过表达脑源性神经营养因子(BDNF)和神经营养因子(NT-3)的脂肪干细胞系并探讨其意义。方法采用胶原酶消化联合差速贴壁法获取大鼠脂肪干细胞,向成骨方向诱导2周、4周后,行碱性磷酸酶染色和茜素红染色。采用Lenti-BDNF-GFP重组慢病毒液和Lenti-NT-3-RFP重组慢病毒液转染第3代脂肪干细胞,确定最佳感染复数值,按照最佳感染复数值感染脂肪干细胞,建立稳定过表达BDNF和NT-3基因的脂肪干细胞系。结果成功分离和培养的大鼠脂肪干细胞具有多向分化潜能,向成骨方向诱导后,碱性磷酸酶和茜素红染色呈阳性。Lenti-BDNF-GFP重组慢病毒液和Lenti-NT-3-RFP重组慢病毒液转染脂肪干细胞的最佳感染复数值为100,感染时间72 h,共转染组BDNF和NT-3在基因水平和蛋白水平均呈稳定高表达。结论成功建立稳定过表达BDNF和NT-3基因的脂肪干细胞系,这对脊髓损伤的治疗有重要意义,可以解决脂肪干细胞移植治疗脊髓损伤过程中神经因子分泌数量少和半衰期短等问题,为进一步研究脊髓损伤的修复机制提供了实验基础。

Objective To construct adipose-derived stem cells (ADSCs) line that can stably express brain-derived neurotrophic factor (BDNF) and neurotrophin-3(NT-3) genes and elucidate its significance. Methods ADSCs were obtained by collagenase digestion along with differential adhesion method.After 2 and 4 weeks of osteogenic induction,alkaline phosphatase staining and alizarin red staining were performed.The third generation of ADSCs were transfected with Lenti-BDNF-GFP and Lenti-NT-3-RFP recombinant lentivirus solution.The ADSCs line that stably expressed BDNF and NT-3 genes were obtained by the optimal infection value determined before. Results The ADSCs isolated and cultured successfully had the potential to differentiate in varous directions.After being induced to osteogenesis,alkaline phosphatase and alizarin red staining both showed positive.The best infection value for Lenti-BDNF-GFP and Lenti-NT-3-RFP recombinant lentivirus transfection was 100 while the infection duration was 72 hours.Expressions of BDNF and NT-3 in co-transfection group remained stable and high at both gene and protein levels. Conclusion Establishment of ADSCs with stable and over-expressed BDNF and NT-3 genes is of great significance for treatment of spinal cord injury (SCI).It can solve the problem of low amount of neurotrophin secreted and short half-life during the treatment of SCI by ADSCs transplantation,which has great significance for further studies on the repair mechanism of SCI.