Experimental Study On Sinerem Labeling And MRI Tracking Of Newborn Rats Schwann Cells In Vitro

BackgroundThe spinal cord is easily injured by different biological or physical reasons.Unfortunately,it is not so easy to recover for its poor ability of regeneration.Therefore,it was generally considered the loss of function which including part or complete palsy,gatism and lost senses caused by spinal cord injury(SCI) was permanent.the afunction following SCI is caused by disorganization of neuro and glia cells,disintegrating neurite and demyelination.Up to now,clinically, there is no effective treament of SCI.when Internists administrate a large dose of steroid hormone and take other supported treatment,surgeons suggests rebuilding the stability of spinal cord,take the depression operation for example.Recently,cell transplantion provide a new chance of recovering SCI.In present,the experiments shows that cell transplantion may be an effective way to cure SCI.The cells transplante into the spinal cord not only replace the injured cells,but also secret the factors that is necessary for cell growing and glial scar inhibiting.Therefore,seed cells are cultured and amplified in vitro and then transplanted into spinal cord seems to be the most attractive and effective way of recovering SCI in future.Schwann cells(SCs) makes up the myelin of peripheral nerveous system(PNS) and has been proved to play the key role of the regeneration of PNS.As one of the most important seed cells,SCs are early used in cell transplantation for having the advantages of easy obtaining,abount resource,low immunology,as well as stable genetics.A part of experiments has verified that SCs will spontaneously transplant into spinal cord following SCI and take part in the SCI recovering.ln addition,SCs still have other contributions,including:â‘ the secretion of neurotrophic factors such as nerve growth factor,brain-derived neurotrophic factor;â‘¡the production and release of extracellular matrix molecules such as laminin;â‘¢promoting axon regeneration after injury to guide axons progress and myelin of the central beam;â‘£protecting neuronal damage and improving microcirculation.On the other hand,in order to figure out cells survival,distribution,migration, differentiation after transplantation in vivo,we need to kill the animals and comfirm by means of immunology.There is no way of non-invasive effective and real-time monitoring.Therefore,the evaluation of cell transplantation can not be used in clinical.At present,thanks to the rapid development of the molecular imaging study of magnetic resonance imaging(MRI),non-invasive tracing can possibly be used after cell transplantation in vivo.Ultra-small superparamagnetic iron oxide nanoparticles(USPIO) Sinerem as a new type of magnetic resonance contrast agent, has been approved by Food and Drugs Agent(FDA),and successfully be used in a variety of cells.We has applied to(Schwann cells)SCs research,and and proved it can be used in SCs.Presently,there is no series of related reports of the biological changes of Sinerem labeling SCs.By a variety of methods,our issues designed to study the cell viability,proliferation and apoptosis before and after Sinerem labeling of SCs in vitro,in order to explore the appropriate concentration of Sinerem to label SCs and provide experimental and technical basis for transplantation treatment of SCI models in vivo. PARTâ… Experimental study of purification of newborn rat Schwann cells by trypsin with low concentration after several rounds of differential detachment procedures.Objective:To observe the culture,purification and passage of SCs in vitro.To master cell culture technology and pave way for further experiments.Methods:After 3-5d of newborn Sprague-Dawley(SD) rats were dissected under sterile conditions,the bilateral brachial plexus and sciatic nerve were removed to produce a single cell suspension of 3×10⁵ per milliliter,then the cell suspension were seeded in 25cm² culture flask which has been paved by PLL overnight.After purification of newborn rat Schwann cells by trypsin with low concentration after several rounds of differential detachment procedures,the shape and the growth of SCs were observed in the inverted phase contrast microscope.On the same time,the specific identification of SCs was confirmed by immunofluorescence of P75^NTR,a cell surface molecules of identification of SCs.Results:after being seeded,SCs are mainly dispersal round and floating in the culture medium,and the other,a small number of cells aggregated a mass;After 24 h,the majority of cells which have cohered on the culture flasks.We can easily observe that most of cohered cells stretch out ecphyma,which looks small, bipolar-shaped,three-dimensional.Keep on cultivating,the axons of SCs get longer and longer.After 72h,axons are found connecting and winding like a net with a lot of intersection.At the same time,we can see another cells with different shape,which looks large,irregular-shaped,three-dimensional;After being purified by low concentrations of digestive enzymes,SCs arrange closely and orderly,and show linear, parallel,palisade-like,spindle-shaped or whirlpool-shaped;most of cells are same slim,and only a very small number of cells are large and flat.Under high power microscope,we can see that SCs are slim and around whose round cell body has a bright halo can be seen,and whose both sides are sharp and extend 2 to 3 long axons, whose axons are connectted. P75^NTR can specifically identify SCs on the fluorescence microscope.We can find out positive cells were with red fluorescence after excitation.Our cultured cells were confirmed to be SCs,which express P75 and can be seen under the fluorescence microscope.Conclusion:The neonatal rat SCs can be cultured and amplified in vitro,which with vigorous growth and proliferation ability and can be specifically identified by P75^NTR.At,present,it is one of the seed cells for repairing SCI..We purify newborn rat Schwann cells by trypsin with low concentration after several rounds of differential detachment procedures.It is not necessary to add reagents and special drugs,and takes simple steps,and can be easily repeated.It is a safe,fast,easy and effective way of SCs purification,ant its purity can reach more than 95 percent.Partâ…¡Experimental study on cytobiological characteristics of Feridex labeled rabbit bone marrow stroma cellsObjective:To label neonatal rat Schwann cells by complex the of ultra-small superparamagnetic iron oxide(USPIO) Sinerem and transfection reagent poly-L-lysine(PLL).to evaluate preliminarily the safety and appropriate concentration of Sinerem labeling for future research.Methods:SD rat sciatic nerve and brachial plexus were taken and digested with collagenase in primary culture,and purified by ultra-low concentrations of trypsin,the preparation of different concentrations of Sinerem-PLL complex were incubated with Schwann cells for 48h;Cellular morphological change observed by inverted phase contrast microscope.Iron particles are identified in cell by prussian blue staining and transmission electron microscope.The comparison of cell cycle and apoptosis was carried out by flow cytometry.The cell growth and proliferation were detected CCK-8. The cells scan sequence were processed by 4.7T MR in vitro.Results:Prussian blue staining and transmission electron microscopy imaging indicate that there is iron particles within the cytoplasm of the labeling SCs.CCK-8 assay results show that there is no statistically significant between 100μg/ml-800μg/ml range of different concentrations of labeling SCs on cell growth and proliferation.The results of flow cytometry shows that there is no different impact between labeling and unlabeling groups of cycle life and apoptosis.Conclusion:1.Different concentrations of Sinerem-PLL complex can be used to label neonatal rat SCs in vitro.2.We proved that high sinerem concentration can increase the efficiency of labeling, and make the T2 WI signal change obviously.It is safe and effective for SCs labeling simerem-PLL complex below the concentrations of 800μg/ml.3.200μg/ml Sinerem are appropriate,safe,effective,and sparing concentration of labeling cells.Therefore,It is feasible to apply labeling SCs with Sinerem-PLL complex and in the appropriate labeling concentration,we can harvest the good imaging in vitro.