| PART 1 EFFECT OF TETRAMETHYLPYRAZINE ONNEURAL STEM CELLS GENETICALLY MODIFIEDObjective:To explore the protective effects of TMP on neural stem cells(NSCs)genetically modified.To investigate the mechanism of tetramethy-lpyrazine(TMP)on protecting NSCs and prompting neural repair.Methods The second passage of primary NSCs isolated from neonatal SD rat was examined and characterized through immunohistochemistry at first.The second passage of primary NSCs were genetically modified(GM)by retrovirus with EGFP gene in vitro.Then,its were sequently cultured for 72 hours in the presence of TMP(0,50,100,200 or 300μg/ml)or 0.1%DMSO(vehicle control)in normal conditions in vitro,meanwhile,culturing second passage of primary NSCs as the normal control.Cell viability and cell death was determined through the 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheny-ltetrazolium bromide(MTT)assay,flow cytometry,and trypanblau dyeing count.The expression of Bax,Bcl-2,and Caspase-3 were investigated by western immunoblot analysis.Results:After genetically modified,the Cell viability of NSCs in vehicle control and GM control significantly decreased,while cell death and apoptosis dramatically increased(P<0.05).It also induced a significant and protein expression levels of Bcl-2 and an notable i protein expression levels of Bax and Caspase-3(P<0.05).But there was no statistical differences between vehicle control and GM control(P>0.05).Notably,compared with GM control,TMP treatment reduced cell death,ameliorated cell viability losses,suppressed cells apoptosis in a concentration-dependent manner.Moreover,the down-regulation of Bcl-2 and up-regulation of Bax and Caspase-3 were attenuated by TMP in a concentration-dependentmanner.But there were no statistical difference among GM control,50μg/ml TMP and 100μg/ml TMP group(P>0.05),likewise,no statistical difference were found between 200μg/ml TMP and 300μg/ml TMP group(P>0.05).Conclusions:TMP treatment can reduced cell death,ameliorated cell viability losses,suppressed cells apoptosis in a concentration-dependent manner after NSCs genetically modified.Moreover,it’s relative with the down-regulation of Bcl-2 and up-regulation of Bax and Caspase-3 in the mRNA and protein expression levels.It may is one of mechanism of TMP providing protective effects in peripheral nervous system injury and regeneration.PART 2 THE CONSTRUCTION OF THE CULTIVATION OF NEURAI STEM CELLS IN TISSUE ENGINEERING NERVE GRAFTObjective:To construct composite grafts of gene modified neural stem cells,acellular nerve and its morphological characteristics and immune detection as the primary graft,provide a ideal bridging allograft for repairing peripheral nervous defection.Methods:(1)Take 24 nerve segments from both-side sciatic nerves of 12 healthy adult male SD rats,20 nerve segments of them get a chemical treatment as following:①Strip overlying connective tissue,being shaken 12 hours in sterile distilled water.② After being shaken 12 hours in 3%TritonX-100,wash them 3 times with sterile distilled water.③After being shaken 24 hours in 4%sodium deoxycholate and room temperature,wash them 3 times with sterile distilled water.④ Reserve the acellular nerves in the DMEM/F12 medium at 4℃.(2)12 hydrated acellular nerves segments,after being injected 10ul(1×106/ml)genetically modified neural stem cell suspension with a micro pipette under the microscope,divided into Ⅰ、Ⅱ、Ⅲ groups(5 samples in I groups,5 samples in II groups,2 samples inⅢ groups),Ⅰ and Ⅱ groups were cultured 1 weeks in conventional DMEM/F12 medium(Ⅰ group)or adding 200ug/ml TMP(II group).Then,observe cells distribution and framed construction through the line fluorescence microscope,HE staining and immunohistochemical(Nestin,NF-200,GFAP)staining.(3)Take 24 healthy adult male SD rats equally divide into A、B、C、D 4 groups,4 sorts(24 sticks)nerves segments(sciatic nerves reserved in 4℃ for 1 week of SD rat,I group tissue-engineered nerve,II group tissue-engineered nerve,sciatic nerves of homogeneous SD rats were subcutaneously embed in order.After SD rats bred 7 days,take out all nerves segments for microscope observation and count of CD4+and CD8+lymphocyte after HE dyeing.Results:After being chemically extracted,on intersected slices,epineurium and perineurium of acellular nerve graft formed a circular structure,to be filled with loose collagen structure inside,having no myelin or cell components.The nerve membrane and cellular basement membrane kept complete.On the longitudinal slices,many pipe-like structures neatly arranged without cell element.After injected the neural stem cells,Conclusions:Acellular nerve graftchemicallyextracted have no cells and myelin components,while remaining complete neural basement membrane and framework,moreover,the main element of basement membrane-laminin almost keep virgin. |
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