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
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摘要
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.
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.
引文
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[19] 刘明,刘杨,邓颖,等.蓝布正提取物对血管性痴呆大鼠学习记忆能力及海马NT-3,BDNF蛋白表达的影响[J].中国实验方剂学杂志,2017,23(17):154-158.
[20] 严灿,刘银伟,吴丽丽,等.加味四逆散调控抑郁症大鼠海马BDNF、NR1表达及促进海马DG区神经再生的研究[J].中国药理学通报,2016,32(4):569-574.
[2] 岳春彦.长骨骨折患者合并脊髓损伤对骨折愈合和血清血小板衍生因子、血管内皮生长因子含量的影响[J].中国医学工程,2018,26(3):23-26.
[3] JI W,ZHANG Y,HU S,et al.Biocompatibility study of a silk fibroin-chitosan scaffold with adipose tissue-derived stem cells in vitro[J].Exp Ther Med,2013,6(2):513-518.
[4] 孙经淞,周雪颖,曲淑贤,等.骨髓间充质干细胞移植对丙烯酰胺中毒大鼠脊髓组织细胞凋亡的影响[J].中国组织工程研究,2018,22(5):680-685.
[5] 姬文晨,蒋婉婷,张党锋,等.甲基强的松龙联合过表达BDNF的ADSCs在治疗大鼠急性脊髓损伤中的作用[J].山西医科大学学报,2017,48(9):908-912.
[6] JI W,ZHANG X,JI L,et al.Effects of brain derived neurotrophic factor and neurotrophin3 on the neuronal differentiation of rat adipose-derived stem cells[J].Mol Med Rep,2015,12(4):4981-4988.
[7] 蒋华玉,王永刚.血清脑源性神经营养因子水平与脑卒中后抑郁关系的meta分析[J].郑州大学学报(医学版),2014,49(5):626-628.
[8] 冯珂,纪立金.健脾益智胶囊对MCAO大鼠海马NT-3、GAP-43、SYP-P38、PKA表达的影响[J].中华中医药杂志,2017,32(7):2923-2927.
[9] 姬文晨,张越林,张永涛,等.大鼠来源脂肪干细胞的分离培养及其分化能力的检测[J].西安交通大学学报(医学版),2012,33(6):693-697.
[10] 王会仁,曹露,江立波,等.稳定过表达生长分化因子5基因大鼠脂肪干细胞系的建立[J].中国临床医学,2017,24(2):181-187.
[11] 刘芳,陈佳,苏华.不同时间窗高压氧干预对大鼠脊髓损伤后细胞凋亡及脊髓功能恢复的影响[J].吉林大学学报(医学版),2015,41(3):578-583.
[12] 刘雯雯,程田,马珊珊,等.曲古霉素A对脊髓损伤小鼠的神经保护作用[J].郑州大学学报(医学版),2018,53(2):147-150.
[13] 王骅,王静成,王永祥,等.MP联合BDNF转染对脊髓损伤细胞模型凋亡机制的作用[J].中国骨与关节损伤杂志,2018,33(1):43-46.
[14] 吴朗,黄成,冯新民,等.胶质细胞源性神经营养因子修饰的骨髓间充质干细胞移植治疗脊髓损伤的效果[J].实用临床医药杂志,2017,21(9):94-98,102.
[15] SONG Z,YE Y,ZHANG Z,et al.Noninvasive,targeted gene therapy for acute spinal cord injury using LIFU-mediated BDNF-loaded cationic nanobubble destruction[J].Biochem Biophys Res Commun,2018,496(3):911-920.
[16] MARTINEZ-GALVEZ G,ZAMBRANO JM,DIAZ SJ,et al.TrkB gene therapy by adeno-associated virus enhances recovery after cervical spinal cord injury[J].Exp Neurol,2016,276:31-40.
[17] ISLAMOV RR,SOKOLOV ME,BASHIROV FV,et al.A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs[J].Neurosci Lett,2017,644:67-75.
[18] ISLAMOV RR,IZMAILOV AA,SOKOLOV ME,et al.Evaluation of direct and cell-mediated triple-gene therapy in spinal cord injury in rats[J].Brain Res Bull,2017,132:44-52.
[19] 刘明,刘杨,邓颖,等.蓝布正提取物对血管性痴呆大鼠学习记忆能力及海马NT-3,BDNF蛋白表达的影响[J].中国实验方剂学杂志,2017,23(17):154-158.
[20] 严灿,刘银伟,吴丽丽,等.加味四逆散调控抑郁症大鼠海马BDNF、NR1表达及促进海马DG区神经再生的研究[J].中国药理学通报,2016,32(4):569-574.