Ultramicrostructure Observation Of The Spinal Cord Injure Rat Tissue Repair After Transplanted NSCs Modified With TrkB

Spinal cord injury (SCI) is a kind of extremely severe damage that having high disability rate. The traditional treatment to SCI usually included spine fracture-dislocation stabilization, relieve spinal cord compression, symptom therapy and rehabilitative treatment. On recent, following development of study of neuropathophysiology and auxology, conventional idea that nervous tissue do not regenerative had challenged, the transplantation of nervous tissue and cells as a new therapy method is gradually being applied to the treatment of SCI, and some satisfactory results were found. Nerual stem cells (NSCs), especially the cells derived from bone marrow stromal cells(BMSCs) become study emphasis.The BMSCs are a part of bone marrow remove hemopoietic stem cells. It was confirmated recently that the BMSCs in under definite culture condition can differentiate into a variety of cell, for instance mesenchymal cell, fibroblast, osteoblast, chondrocyte, adipocyte muscle Cells, endothelial cell and neuron, glial cell and so on, passing clone proliferation. Detected of BMSCs pluripotency and succeed of toward nerve cell directional differentiation, increase new pathway for NSCs source. Meanwhile, it possess convenience harvested, adequacy resource, autografting, avoidance ethics dispute and immunological rejection that applying embryonic stem cell (ESCs), also provide a new idea for solve "self-NSCs" transplantation project cell quantity short of problem.Multitude study exterior and interior were indicated that transplant multi-origin NSCs into SCI animal model could promoted tissue repair and function improvement. However, it was not found that the synapse contact between grafted cells and host cells, to say in other words, nerve circuit loop interrupted not found to get reablement and rebuild. We could observe neuraxon or dendric under electron microscope when we observe ultramic-structure, but we did not distinguished it derived from grafted cells or host cells. Why? It was the chief reason that we could not labeled the neuraxon or dendric notwithstanding we could labeled transplanted cells applied many sign methods.Ferumoxides(FE), take as labeled marker of grafting cells, were obtained extensive attention. It could effective labeled transplanting cells when it combined with poly 1 lysine(PLL) formed FE-PLL compound. Because its bead small, average diameter is 80nm, diameter of ferric oxide lied in cores is 20nm, it can possible diffusing to extremity of neuraxon and bendritic of labeled cells following endochylema transportation. Tyrosine kinase receptor B(TrkB), as specificity receptor of brain-derived neurotrophic factor(BDNF) lied in cellular membrane, have a small quantity disposition in common nerve cells and can distribute extremity of neuraxon and bendritic even synapse following cellular membrane extension. If labeled grafting cells applying FE-PLL or exogenous TrkB carried marker do not lied in host cells, we could distinguish the source of neuraxon and bendritic under electron microscope.Based on these, we take NSCs derived from BMSCs as transplantation cells, established rat SCI model, and transfect pEGFP-C2-TrkB into the grafting cells, supplied symbol protein-EGFP to labeling exogenous TrkB receptor, labeled cells with FE-PLL before transplanted. Harvested patho-tissue at 4 weeks after cells transplanted, observing cells survival, differentiation, and observing neuraxon and bendritic of grafted cells and synapse contact between host cells and transplanted cells by Ferumoxides or EGFP immunohistochemistry labeled by colloidal gold under electron microscope, and observing nerve circuit loop interrupted did or not get reablement and rebuild applying biotinylated dextran amine(BDA) tracing corticospinal tract and to explore ultrastructural evidence of tissue repair of SCI after NSCs transplanted.Part one: the induction and differentiation of BMSCs into NSCs in ratObjective: To study the feasibility of induction and differentiation of BMSCs into NSCs in 9L/fisher homologous genes rat in vitro.Methods: Under the sterile condition, BMSCs, which isolated from bone marrow stromal of 9L/fisher homologous genes rat and were separated by gradient density centrifugation, were cultured, induced and differentiated into neural stem cells and neuron-like cells with cytokines consisted of retinoic acid(RA, 0.5μg/ml) and special culture medium confected by our lab(patent number: ZL02134314.4), and then evaluated by immunocytochemistry (SABC, first antibodys were Rat anti-Nestin, Rat anti neuron-specific enolase (NSE), Rat antineuron specific nuclear protein (NeuN). Second antibodys were biotinylated Goat anti Rat IgG and streptavidin peroxidase.).Results: We can see division and proliferation in adherent cells in culture, which can form cell clones like islands. When continuous cultured, and give appropriate differentiated condition, these cells can differentiate into poly-form cells with some long and thin apophysis. Evaluate in immunocytochemical: in earlier period, the cultured cells without induce and differentiate condition can express Nestin positive. It is indicated the cells had some character of stem cell; when these cells differentiated, there were some cells express NSE and NeuN positive.Conclusions: BMSCs of rat could proliferate and differentiate into the cells expressing Nestin and NSE and NeuN antigen. Considering its source convenience, the BMSCs could be considered as one of the ideal seed cells of the NSCs.Part two: Transfection of exogenous TrkB into rat neural stem cellsObjective: To obtain the rat NSCs expressing exogenous TrkB(tyrosine kinase receptor B) gene stably and in long term and to evaluate the transfection efficiency with liposomes-mediated transfection method in vitro.Methods: The eukaryotic expression vector pEGFP-C₂-TrkB (plasmid-enhanced green fluorescent protein-C₂-TrkB) was transfect into NSCs with LIPOEECTAMINE. 2000 liposomes-mediated transfect method. The transfect efficiency was calculated via expression of EGFP, which was detected with fluorescent microscope at 24h after transfect. After G418 screening, the TrkB mRNA expression of positive NSCs were analyzed with semi-quantitative RT-PCR. The NSCs were cultured and either transfect with pEGFP-C₂-TrkB or pEGFP-C₂ by LIPOEECTAMINE. 2000 or uninfected in vitro respectively, analyzed the protein level expression information after plasmid pEGFP-C₂-TrkB transfected by means of TrkB immunocytochemistry Cy3 fluorescent staining.Results: EGFP were successfully expressed 24 hours after nucleofection, the rate of positive EGFP expression was 18.124±0.63%. The positive EGFP expression was enhanced gradually alone with the prolonged culture time, and showed the strongest one week after marked, with about40.194±0.70%. The expression intensity of EGFP did not attenuate even one month after marked. Cell culture till one week, 500ng/L G418 was added into medium. Five days later, all of the untransfect NSCs came to death and some of NSCs contained exogenous pEGFP-C₂-TrkB or pEGFP-C₂ were fall off from the culture bottle and dead. The concentration of G418 was changed to 300ng/L at 5th day and its effect was maintained over 20 days. The expression of TrkB in pEOFP-C2-TrkB was higher significantly than those in pEGFP-C₂ NSCs and untransfeted cells with half-quantitation RT-PCR determine TrkB mRNA. The expression of TrkB in protein level in NSCs transfect plasmid pEGFP-C₂-TrkB was higer significantly than in NSCs transfect pEOFP-C2 and untransfeted cells under confocal microscopy after TrkB immunocytochemistry Cy3 fluorescent staining and protein expression most concentrates in cellular membrane.Conclusions: The study obtain anti-G418 cell clone that stabilize express ectogenous TrkB by means of transfection pEOFP-C₂-TrkB into NSCs with LIPOEECTAMINE. 2000 liposomes-mediated transfect method, and confirmed NSCs transfect could multiplicity express TrkB in mRNA and protein level, and TrkB expression most concentrate in cellular membrane in protein level. LIPOEECTAMINE. 2000 is a convenient and efficient gene transfect method for the introduction of the genes into rat NSCs, the pEGFP-C₂-TrkB gene transferred could stabilize expression, EGFP plays an symbol role in the expression of the pEGFP-C²-TrkB gene.Part three: Cytobiology study on intracellular labeling of neural stem cells derived from bone marrow of rat with FerumoxidesObjective: The ability to track NSCs transplanted into host by neuroimaging in vivo will undoubtedly aid our understanding of how these cells mediate functional recovery after cells transplantation. Ferumoxides is a class of dextran-coated superoparamagnetic iron oxides (SPIO) nanoparticle used as MRI contrast agent for hepatic imaging. The purpose of this study is to explore effect of Ferumoxides on NSCs biological abilities to survive and proliferate and to observing distribution information of iron partical laid in NSCs successfully labeled by Ferumoxides, and provide a basis of clinical and lab study application for MRI tracing labeled cells and to observeing and distinguishing neuraxon and cytodendrite were derived from transplanted cells or host cells.Methods: The culture, induction and differentiation of NSCs derived from the rat BMSCs in vitro were completed. Admixture and vibrate 25μg/ml final concentration Ferumoxides and 0.75μg/ml final concentration Poly-1-lysine (PLL) 30 minutes so as to form Ferumoxides and PLL compound(FE-PLL), and applying FE-PLL compound labeled NSCs. The efficiency of FE-PLL labeled NSCs were evaluated by Prussian blue staining and electron microscopy. Cellular viability of labeled NSCs was investigated by methyl thiazolyl tetrazolium (MTT) test. The cellular apoptosis of labeled NSCs were studied by flow cytometry. The study eventually divided into four groups: A group, single NSCs labeled group; B group, 0.75μg/ml final concentration poly-1-lysine labeled group; C group, 25μg/ml final concentration Ferumoxides labeled group; D group, 0.75μg/ml final concentration poly-1-lysine and 25μg/ml final concentration Ferumoxides (FE-PLL) labeled group.Results: The observe results under inverted phase contrast microscope after FE-PLL labeled NSCs were demonstrated that the NSCs labeled by FE-PLL compound were yellow, contradictory, the NSCs labeled by single FE and by single PLL and did not labeled were not yellow, it was obviously difference among four groups cells. The observe results under inverted microscope were demonstrated that NSCs could be effectively labeled and labeling efficiency were above 90%. Prussian blue staining showed numerous blue stained fine particles in the cytoplasm of NSCs labeled FE-PLL, the labeled cells were show blue, but the NSCs labeled by single FE and by single PLL and did not labeled were not show blue. Transmission electron microscopy of FE-PLL labeled NSCs revealed the presence of numerous vesicles which are spreaded in intracytoplasm and filled with the electron-dense magnetic iron particles, these vesicles diameter were from 0.8 to 1.5μm, these vesicles principal concentrate in cell body. The results of MTT are demonstrated that labeled cell viability was not affected by FE-PLL when the concentration of ferumoxides is 25μg/ml and PLL is 0.75μg/ml, it was not significant distinguish among the FE-PLL groups and the groups labeled by single FE and by single PLL and did not labeled. Results of flow cytometry were suggested that the cells apoptosis would not be affected when the concentration of Ferumoxides is 25μg/ml and PLL is 0.75μg/ml, it was not significant distinguish among the FE-PLL groups and the groups labeled by single FE and by single PLL and did not labeled.Conclusion: The results of Prussian blue and electron microscope were show that numerous blue stained fine particles in chief lied in cell body in vitro. Results of MTT and flow cytometry were suggested that the cells viability ability and apoptosis would not be affected when the concentration of Ferumoxides is 25μg/ml and PLL is 0.75μg/ml. The iron particles main spread into intracytoplasm, we could possible observe and distinguish the neuraxon and cytodendrite were derived from the transplanted cells after cells labeled by FE-PLL transplanted into host, however, it need further observation in vivo. The above results suggested that FE-PLL labeled NSCs is a feasible, efficient and safe method. It could be make use of tract transplanted cells in vivo. We are possible to distinguish the neuraxon and dendritic derived from transplanted cells or host cells by means of FE-PLL labeling cells.Part four: Ultramicrostructure observation of the spinal cord injury rat after transplanted neural stem cells modified with protein-tyrosine kinase receptor B geneObjective: The 9L/fisher homologous genes rat model of spinal cord injury was set via transsection under microscopy. Transfected pEGFP-C2-TrkB plasmid to rat NSCs derived from BMSCs, applying FE-PLL labeled NSCs before it transplanted. Investigate the cells survival, migration, differentiation and incorporation, especially synapse present information after transplanted NSCs transacted by above-mentioned into host tissue with single cell suspension by autologous micro-transplantation method. Taken cells carried pEGFP-C2-TrkB or pEGFP-C2 or no gene transplanted as control group. Search ultramicrostructure of cell contact and connection between transplanted and host cells so as to provided a ultramicrostructure witness for incorporation between transplanted and host cells, establish a foundation for cells transplantation treatment to central nervous system disease.Methods: The 9L/fisher homologous genes rat model of spinal cord injury was set via transsection under microscopy. The rats were divided into 5 groups as follows: Group A: normal control; Group B: simple spinal cord injury; Group C: spinal cord injury and simple NSCs transplantation; Group D: spinal cord injury and NSCs carried pEGFP-C2 transplantation; Group E: spinal cord injury and NSCs carried pEGFP-C2-TrkB transplantation. 4 weeks after cells transplantation, tracing transplanted cells in vivo applying magnetic resonance imaging (Group C,D,E), took A and B groups as control. Meanwhile, transplanted cells survival and differentiation in D and E groups: Choose GFP positive cells to carry out anti-NSE and anti-GFAP immunocytochemistry fluorescent staining(second antibodys were cy3), and record express NSE or GFAP positive cells, compared NSE or GFAP positive express rate between D and E groups, and compared NSE or GFAP positive cells in D and E groups, and compared GFAP positive cells among A,B,C,D,E groups to get the message about glial scar forming. Detect and observe iron particles in transplanted by prussion blue staining and transmission electron microscope. Tracing corticospinal tract cured off by means of BDA method so as to observe neurofibra whether could recanalization and rebuild or not. The emphasis of this exprement on searching ultramicrostructure of cell contact and connection between transplanted and host cells, especially synapse structure, so as to provided a ultramicrostructure witness for incorporation between transplanted and host cells applying two methods. The first, applying transmission electron microscope to observe the cells containing iron particles and whether this particles could dispersed into neuraxon and cytodendrite. According the method, we will observe ultramicrostructure of cell contact and connection between transplmated and host cells. The second, provided a specificness tumor marker, EGFP, to exogenous TrkB that in chief express in cellular membrane, applied GFP immunocytochemistry staining(second antibodys labeled by gold colloid), we can observe ultramicrostructure of cell contact and connection, esprislly synapse contact, between transplanted and host cells though observe gold colloid dispersion information in cellular membrane and neuraxon and cytodendrite.Results: We could found the transplanted cells labeled by can clear visualization under with MRI detection of animal model: FE-PLL labeled transplants clearly demarcated the injection site on SET1- and SET2-weighted MR images 4 weeks postgrafting. The high density of labeled cells in injury spinal cord in a strong signal and the dark appearance in do not injection site in all scans. Surrounding the cells injection site, in the T1-weighted scans, a hyperintense border was apparent around the dark transplant region. MRI in T2- weighted scans sequence showed remarkable low signal change in the cells injection site. Among T2-weighted scans, the most significant change occurred in GRE T2-weighted scans sequence. The tissue and cells visualization under MRI were contain iron particles confirmed by prussion blue staining and transmission electron microscope. The cells transplanted could survival and differentiated into cells expressing NSE or GFAP, quantity of GFAP positive cells were more than the NSE positive cells. It is profit to promote the cells survival and differentiated into NSE positive cells and decrease differentiated into GFAP positive cells though transfect TrkB into transplanted cells, and lighten glial scar after SCI in some extent, and educe indirectly biological effect of BDNF so as to promote injury tissue regeneration and reparation. Results of BDA tracing corticospinal tract were indicated neurofibra breakdown could recanalization and nerve ring rebuild. It could strengthen this action though transfect TrkB. Results of ultramicrostructure observe were indicated we could effective distinguish transplanted cells and host cells by means of labeled cells with FE-PLL and observe the contact between transplanted cells body and host cells body, however, we did not distinguish whether neuraxon and cytodendrite were derived from transplanted cells or host cells because the iron particle did not spread into neuraxon and cytodendrite follow as endochylema transportation. Meanwhile, we could effective distinguish transplanted cells and host cells and their neuraxon and cytodendrite though immunocytochemistry staining(second antibody labeled by gold colloid) evaluated exogenous TrkB specificness tumor marker, EGFP, gold colloid particles can distributed into neuraxon and cytodendrite and presynaptic membrane. We found the high density gold particle uniformity distributed in cellular membrane and cells ecphyma and presynaptic membrane. There were contact between transplanted cells body or ecphyma and host cells body or ecphyma, the contact between transplanted cells ecphyma and body more than it between transplanted cells ecphyma and host cells body, however, these contact were not posses typical synaptic structure.Conclusion:1,The FE-PLL compound could effective labeled NSCs, and we could observe and tract transplanted NSCs labeled by FE-PLL with MRI,but,we did not distinguish whether neuraxon and cytodendrite were derived from transplanted cells or host cells so that did not effective observe contact between transplanted cells and host cells; 2,Provided a specificness tumor marker, EGFP, to exogenous TrkB, we could effective distinguish transplanted cells and host cells and their neuraxon and cytodendrite though immunocytochemistry staining(second antibody labeled by gold colloid);3,The transplanted NSCs could survival and differentiated into cells expressing NSE or GFAP in host vivo, quantity of GFAP positive cells were more than the NSE positive cells, could promote the cells survival and differentiated into NSE positive cells, decrease differentiated into GFAP positive cells, lighten glial scar after SCI in some extent and educe indirectly biological effect of BDNF so as to promote injury tissue regeneration and reparation;4,The transplanted NSCs could formed contact with host cells, these contact were not posses typical synaptic structure. BDA tracing corticospinal tract were indicated neurofibra breakdown could recanalization and nerve ring rebuild, could strengthen this action though transfect TrkB to transplanted NSCs.5,there were a lot of question need to solve notwithstanding it had invariably therapeutic effect applying NSCs transplanted treat SCI.