The Research Of Rabbit Adipose Stem Cells Labelled With Spio And The Construction Of Rabbit Model Lumbar Intervertebral Disc Degeneration And MR Quantitative Analysis

PART1THE CULTURE AND IDENTIFICATION OF RABBITADIPOSE STEM CELLS AND ITS MAGNETICRESONANCE IMAGING IN VITROObjective:To study the method of culture and identification of rabbitadipose stem cells, labeling the cells by superparamagnetic iron oxideparticles(SPIO) combined with poly-l-lysine(PLL) in virto,and to discussthe feasibility of SPIO-PLL to trace ADSCs.Methods:ADSCs were obtained by means of isolation, purificationand culture,and the cell surface antigens CD44, CD90and CD34wereidentified through flow cytometry(FCM). The intracellular iron particleswere detected by Prussian blue staining and transmission electronmicroscopy (TEM).1×107ADSCs unlabeled and labeled with25,50,75μg/ml SPIO-PLL respectively were performed with3.0T MR scanning in vitro,meanwhile,1×107ADSCs (labeled1d),1×107(labeled3d) and5×106((labeled1d) were also scanned, including T1WI, T2WI and T2*WIarrays, the signal intensity (SI) and relaxation time of each tube weremeasured.Results:The third generation ADSCs has a high degree ofpurity,arranged regularly,and presented whirlpool-like.The expression rateof CD44and CD90were98.7%,99.2%respectively, but CD34expressednegatively.The results of Prussian blue staining and TEM showed thatparticles existed in each cytoplasm,and the rate of labeling was nearly100%. T2*WI and T2WI show a more obvious decrease in SI than that ofT1WI with the increasing concentration of SPIO-PLL.The percentagechange of SI of1×107ADSCs (labeled1d) was significantly higher thanthat of1×107(labeled3d) and5×106(labeled1d). The relaxation time ofT2*and T2compared with T1was statistically significant (F=161.47,P<0.05),but the relaxation time between T2*and T2was not statisticallysignificant(F=5.88,P>0.05).Conclusion:sufficient ADSCs can be obtained through isolation，purification and culture. ADSCs can be labeled effectively by SPIO-PLLand detected through MR scanning in vitro,T2*WI and T2WI arrays aremore sensitive. PART2THE CONSTRUCTION OF A RABBIT MODEL OFLUMBAR INTERVERTEBRAL DISC DEGENERATIONBY NUCLEUS PULPOSUS SUCTION AND MRQUANTITATIVE STUDYObjective:To build a rabbit model of lumbar intervertebral discdegeneration and improve the recognition of its mechanism.Methods:The nucleus pulposus of L2/3,L3/4,L4/5discs of12rabbitswere aspirated by using a18G needle and the normal L1/2, L5/6discs ofeach rabbit and the discs between L2-5vertebral body of6rabbits acted asthe normal intra and inter control groups, respectively. The rabbits wereperformed with sagittal MR T2WI and T2mapping sequence scanning atthe pre-and-post-operative4th,8th,12th week, respectively. The signalintensity of each disc was evaluated and T2relaxation time was measuredcarefully.The discs between L1-6were dissected and then performed withHE and Masson staining to evaluate the form of discs,content change ofnucleus pulposus cells and matrix between normal and experimentalgroups. Results:Intervertebral disc presented like a bump in size of a bean,and its nucleus pulposus could be punctured successfully with a18G needlein the root of transverse process via spinal bypass.The normal discsmanifested with uniform high signal in T2WI,and the signal intensity ofexperimental group reduced gradually after operation.The signal intensitydecreased sharply at the8th week and became completely low signalintensity at the12th week post-operation.The significant difference of T2relaxation time was found between normal and experimental at the8thweek groups(p <0.05).The results of HE and Masson staining showed thatthe cells and matrix decreased gradually,moreover,the boundary betweenannulus fibrosus and nucleus pulposus became obscure.The nucleuspulposus displayed the process of fibrosis and was almost replaced byfibrous tissue at8th week.The discs was fibrocartilage and local cartilageand osteophyte can be observed at the12th week.Conclusion:The rabbit model of lumbar intervertebral discdegeneration can be established successfully by nucleus pulposussuction,and it can be dynamically monitored and quantitatively analyzed byMRI.The study provides a reliable experimental method to improve therecognition of the mechanism of intervertebral disc degeneration.