过表达BDNF基因重组慢病毒转染MSCs的实验研究
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摘要
目的观察BDNF基因重组慢病毒修饰MSCs过表达BDNF的情况。方法构建携带BDNF基因的慢病毒载体;分离培养大鼠MSCs;将基因重组慢病毒转染MSCs;RT-PCR、Western blot检测各组BDNF基因及蛋白表达水平。结果绿色荧光试验证实BDNF基因重组慢病毒转染MSCs成功;RT-PCR、Western blot验证MSCs-EGFP-BDNF组BDNF基因及蛋白表达明显高于MSCs组及MSCs-EGFP组,MSCs组与MSCs-EGFP组比较差异无统计学意义。结论 BDNF基因重组慢病毒修饰的MSCs基因及蛋白表达均增高,MSCs成功过表达BDNF。
Objective To observe over expression of BDNF in MSCS modified by recombinant lentivirus.Methods We established the lentiviral vector carrying BDNF gene;MSCs from rat brain was cultured;the lentiviral vector carrying BDNF was transfected into MSCs;RT-PCR and Western blot were used to detect the expression of BDNF gene and protein in each group.Results The transfection of BDNF recombinant lentiviral vector into MSCs was verified through green fluorescent assa.RT-PCR and Western blot showed that the expression of BDNF gene and protein in MSCs-EGFP-BDNF group was higher than that in MSCs group and MSCs-EGFP group.There was no statistical difference between the MSCs group and the MSCs-EGFP group.Conclusion BDNF gene and protein were successfully over expressed by transfection of BDNF recombinant lentiviral vector into MSCs.
Objective To observe over expression of BDNF in MSCS modified by recombinant lentivirus.Methods We established the lentiviral vector carrying BDNF gene;MSCs from rat brain was cultured;the lentiviral vector carrying BDNF was transfected into MSCs;RT-PCR and Western blot were used to detect the expression of BDNF gene and protein in each group.Results The transfection of BDNF recombinant lentiviral vector into MSCs was verified through green fluorescent assa.RT-PCR and Western blot showed that the expression of BDNF gene and protein in MSCs-EGFP-BDNF group was higher than that in MSCs group and MSCs-EGFP group.There was no statistical difference between the MSCs group and the MSCs-EGFP group.Conclusion BDNF gene and protein were successfully over expressed by transfection of BDNF recombinant lentiviral vector into MSCs.
引文
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[26]CUI X,CHEN L,REN Y,et al.Genetic modification of mesenchymal stem cells in spinal cord injury repair strategies[J].Biosci Trends,2013,7(5):202-208.
[27]VAN VELTHOVEN C T,BRACCIOLI L,WILLEMEN H L,et al.Therapeutic potential of genetically modified mesenchymal stem cells after neonatal hypoxicischemic brain damage[J].Mol Ther,2014,22(3):645-654.
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[29]BAYATI V,HASHEMITABAR M,GAZOR R,et al.Expression of surface markers and myogenic potential of rat bone marrow-and adipose-derived stem cells:a comparative study[J].Anat Cell Biol,2013,46(2):113-121.
[30]TAN S L,AHMAD T S,SELVARATNAM L,et al.Isolation,characterization and the multi-lineage differentiation potential of rabbit bone marrow-derived mesenchymal stem cells[J].J Anat,2013,222(4):437-450.
[2]FERNANDEZ V V B,ROMANIUK M A,CHOI H,et al.Mesenchymal stem cells and their use in therapy:what has been achieved[J].Differentiation,2013,85(1-2):1-10.
[3]ISKANDAR M E,CIPRIANO A F,LOCK J,et al.Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces[J].Conf Proc IEEE Eng Med Biol Soc,2012,2012:5 666-5 669.
[4]LEE J,BAEK J H,CHOI K S,et al.Cyclin-dependent kinase 4signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells[J].Cell Cycle,2013,12(3):442-451.
[5]SALGADO A J,SOUSA J C,COSTA B M,et al.Mesenchymal stem cells secretome as a modulator of the neurogenic niche:basic insights and therapeutic opportunities[J].Front Cell Neurosci,2015,9:249.
[6]YILMAZ G,ALEXANDER J S,ERKURAN Y C,et al.Induction of neuro-protective/regenerative genes in stem cells infiltrating post-ischemic brain tissue[J].Exp Transl Stroke Med,2010,2(1):11.
[7]MARKHAM A,BAINS R,FRANKLIN P,et al.Changes in mitochondrial function are pivotal in neurodegenerative and psychiatric disorders:how important is BDNF[J].Br J Pharmacol,2014,171(8):2 206-2 229.
[8]GOMES C,SMITH S C,YOUSSEF M N,et al.RNA polymer-ase 1-driven transcription as a mediator of BDNF-induced neurite outgrowth[J].J Biol Chem,2011,286(6):4 357-4 363.
[9]CHEN B Y,WANG X,WANG Z Y,et al.Brain-derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells,possibly by triggering the Wnt/beta-catenin signaling pathway[J].J Neurosci Res,2013,91(1):30-41.
[10]JEONG C H,KIM S M,LIM J Y,et al.Mesenchymal stem cells expressing brain-derived neurotrophic factor enhance endogenous neurogenesis in an ischemic stroke model[J].Biomed Res Int,2014,2014:129145.
[11]SHARMA A D,BRODSKIY P A,PETERSEN E M,et al.High throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies[J].J Vis Exp,2015,(95):e52 242.
[12]SONG M S,LEARMAN C R,AHN K C,et al.In vitro valida-tion of effects of BDNF-expressing mesenchymal stem cells on neurodegeneration in primary cultured neurons of APP/PS1mice[J].Neuroscience,2015,307:37-50.
[13]TANNA T,SACHAN V.Mesenchymal stem cells:potential in treatment of neurodegenerative diseases[J].Curr Stem Cell Res Ther,2014,9(6):513-521.
[14]HAN Z M,HUANG H M,WANG F F.Brain-derived neurotrophic factor gene-modified bone marrow mesenchy-mal stem cells[J].Exp Ther Med,2015,9(2):519-522.
[15]LI L,LI B,ZHANG H,et al.Lentiviral vector-mediated PAX6overexpression promotes growth and inhibits apoptosis of human retinoblastoma cells[J].Invest Ophthalmol Vis Sci,2011,52(11):8 393-8 400.
[16]NASRI M,KARIMI A,ALLAHBAKHSHIAN F M.Production,purification and titration of a lentivirusbased vector for gene delivery purposes[J].Cytotechnology,2014,66(6):1 031-1 038.
[17]SUN X Z,LIU G H,WANG Z Q,et al.Over-expression of VEGF165in the adipose tissue-derived stem cells via the lentiviral vector[J].Chin Med J(Engl),2011,124(19):3 093-3 097.
[18]BARDE Y A,EDGAR D,THOENEN H.Purification of a new neurotrophic factor frommammalian brain[J].EMBO J,1982,1(5):549-553.
[19]SHARMA A D,BRODSKIY P A,PETERSEN E M,et al.High throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies[J].J Vis Exp,2015(95):e52242.
[20]SYGNECKA K,HEIDER A,SCHERF N,et al.Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model[J].Stem Cells Dev,2015,24(7):824-835.
[21]DE MIGUEL M P,FUENTES-JULIAN S,BLAZQUEZMARTINEZ A,et al.Immunosuppressive properties of mesenchymal stem cells:advances and applications[J].Curr Mol Med,2012,12(5):574-591.
[22]SOLEYMANINEJADIAN E,PRAMANIK K,SAMADIAN E.Immunomodulatory properties of mesenchymal stem cells:cytokines and factors[J].Am J Reprod Immunol,2012,67(1):1-8.
[23]KHALILI M A,SADEGHIAN-NODOUSHAN F,FESAHAT F,et al.Mesenchymal stem cells improved the ultrastructural morphology of cerebral tissues after subarachnoid hemorrhage in rats[J].Exp Neurobiol,2014,23(1):77-85.
[24]LIU W,LU G,WANG B,et al.Transfection of BDNF gene promotes bone mesenchymal stem cells to differentiate into neuron-like cells[J].Zhong Nan Da Xue Xue Bao Yi Xue Ban,2012,37(5):441-446.
[25]ZHOU S.From bone to brain:human skeletal stem cell therapy for stroke[J].Cent Nerv Syst Agents Med Chem,2011,11(2):157-163.
[26]CUI X,CHEN L,REN Y,et al.Genetic modification of mesenchymal stem cells in spinal cord injury repair strategies[J].Biosci Trends,2013,7(5):202-208.
[27]VAN VELTHOVEN C T,BRACCIOLI L,WILLEMEN H L,et al.Therapeutic potential of genetically modified mesenchymal stem cells after neonatal hypoxicischemic brain damage[J].Mol Ther,2014,22(3):645-654.
[28]ZAMINY A,SHOKRGOZAR M A,SADEGHI Y,et al.Mesenchymal stem cells as an alternative for Schwann cells in rat spinal cord injury[J].Iran Biomed J,2013,17(3):113-122.
[29]BAYATI V,HASHEMITABAR M,GAZOR R,et al.Expression of surface markers and myogenic potential of rat bone marrow-and adipose-derived stem cells:a comparative study[J].Anat Cell Biol,2013,46(2):113-121.
[30]TAN S L,AHMAD T S,SELVARATNAM L,et al.Isolation,characterization and the multi-lineage differentiation potential of rabbit bone marrow-derived mesenchymal stem cells[J].J Anat,2013,222(4):437-450.