The Research Of The Differentiation When Mesenchymal Stem Cells (MSCs) Were Transplanted Into The Intervertebral Discs Of The Rabbit

PrefaceDegenerative disc disease ( DDD) often cause lower back pain by the way of nucleus pulposus herniation, spinal stenosis and segmental instability. There are few effical methods to cure DDD in clinic. Some scholars attempt to use many methods such as cell factor,gene therapy and cell transplantation to cure the degeneration of the disc. MSCs have the potency to differentiate to many sort of the cells,now it had been proved that MSCs could be induced into osteocyte, chon-drocyte, hepatocyte, tendon, skeletal muscle cell etc. To find out what kind of the cell will the MSCs differentiate to when MSCs was transplanted into the interver-tebral disc was the purpose of my research.Methods1. The source of animals.1-2 month old (weight 0. 5 - 1Kg) and 1 year old (weight 3 -5 Kg) healthy Japanese rabbits (no female or male limited) , which are provided and raised by China Medical University.2. MSCs culture in vitroSeparating and harvesting immature rabbit femur MSCs by density gradient centrifugation,seeding to culture flask by 1 ×10⁶/ml,three days later ,changing culture fluid for the first time and changing culture fluid on alternate days after the first time. When primary culture cells grow into monolayer, 1:3 subcultu-ring.3. Animal models constructionWe expose the intervertebral disc in surgery,every rabbit's L_4-5 and L_5-6 in-tervertebral disc were used for experiment. Culture fluid without cell was put into the L4₅ intervertebral disc as control group, the celliferous culture fluid was put into the L5₆intervertebral disc as experimental group.4. ImmunohistochemistryEight weeks later the experimental animals were put to death, the specimens were took out and used for Immunohistochemistry. When a cells endochyl-ema was red and its nucleus was atropurpureus we confirmed that It is a positive staining cells.Result1. MSCs culture in vitroPrimary culture cells survival ratio can reach 95% by trypan blue dyeing examination. Inverted microscope observation shows primary cells form colonies 3 days after culture, cells grow into momolayer 12 -14 days later. Subculturing cells proliferate more rapidly, reaching monolayer 9-10 days later.2. The result of the animal modelAll of the experimental animals survived well. There were hyperplasia phenomenon in experimental groupfc intervertebral discs and control groups intervertebral discs in naked eyes.3. Immunohistochemistry resultIt was homogeneous pink in control group and positive staining cells were not find. In experimental group we could find some positive staining cells.Discussion1. Choose and separation, culture, amplification of seed cellsMSCS have many conditions which the ideal seed cells should have. Those are in favor of making MSCS cultivated to be the seed cells. Density gradient centrifugation is a good method to separate MSCs.2. Immunohistochemistry analysisType II collagen protein was found in the endochylema of the transplantedMSCs. As Type II collagen protein was the characteristic secretion we can conclude that the transplanted MSCs has differentiated to chondrocyte, and could secrete some functional matrix.ConclusionThe transplanted MSCs will differentiated to chondrocyte which can secrete type II collagen protein.