Effects Of Exosomes Derived From Rat Bone Marrow Mesenchymal Stem Cells On Degenerative Nucleus Pulposus Cells

ObjectivesNew research shows that neck and shoulder pain,low back and leg pain in the population are increasing,placing a heavy burden on individuals,families and society.The main cause of the disease is intervertebral disc degeneration.The exact mechanism of the disease is not clear.In the field of basic research,it is recognized that the main mechanism of intervertebral disc degeneration is the decrease in the activity and number of nucleus pulposus cells,and the decrease in extracellular matrix secretion,leading to dehydration and degeneration of the nucleus pulposus,protrusion of the nucleus pulposus from the intervertebral disc.It causes the nerve roots compressed,coupled with the corresponding disc height loss,biomechanical changes,leading to clinical symptoms.The treatments program are mainly conservative treatments and surgical treatments.Although in recent years the surgical treatments have made great progress in alleviating symptoms while reducing surgical trauma to the patients,the treatments are still destructive and have no fundamental therapeutic effect on the degenerative disc itself.Even after surgery,recurrence,accelerated degeneration of adjacent intervertebral discs,changes in spinal physiological curvature,and so on.In order to treat intervertebral disc degeneration disease fundamentally,experts and scholars turn their attention to biological therapy,and ha ve obtained many research results.In our previous study,we found that after non-contact co-culture of bone marrow mesenchymal stem cells and nucleus pulposus cells,stem cells differentiated into nucleus pulposus-like cells and expressed the phenotype of nucleus pulposus cells.Moreover,the nucleus pulposus cells showed stronger function than those before the experiment.In co-culture system,the main form of information exchange between cells is exosomes.Considering the widespread characteristics of exosomes,Our team supposed that bone marrow mesenchymal stem cells can secrete exosomes,and the exosomes can improve the function of degenerative nucleus pulposus cells and delay the process of degeneration.Research methods1.The extraction and identification of BMSCs:1)The bone marrow adherent cells of the SD rat are extracted by the full-bone marrow method,and the culture solution for removing the exosomes was used for culturing and passage;2)The extracted adherent cells were identified by three-line differentiation including Osteogenesis,lipogenesis and cartilage formation and by flow cytometry.2.The collection,extraction and identification of the exosomes of BMSCs :1)Collecting the culture solution in the process of culturing and passage the cells that had been identified successfully;2)Carrying out centrifugation on the collected supernatant by differential centrifugation method,and preserving the obtained precipitate after the concentration was measured;3)Observing the size and the form of the obtained precipitate under a TEM.4)The exosomes marker proteins were detected by WB method.3.The extraction of NPCs and the establishment of NPCs degeneration model: 1)SD rat NPCs were extracted and cultured;2)Observing the morphological changes of the cells with the passage under microscope,and selecting the appropriate generations for subsequent experiments;3)Comparing the second and sixth generations after staining by?-galactosidase,and confirming whether the cells produced degeneration by passage.4.The observation on the exosomes of BMSCs uptaked by degenerative NPCs: The exosomes of BMSCs were stained with CM-Dil and the degenerative NPCs were stained with CM-Dio.co-cultured for 24 hours,the exosomes of BMSCs uptaked by degenerative NPCs were observed under laser confocal microscope.5.The effects of the exosomes of BMSCs on degenerative NPCs: 1)Three groups were set up in this experiment: Control group: Degenerate NPCs(P6),Co-culture group: Degenerate NPCs(P6)and BMSCs(P3)were cultured in Transwell system;Exosome group: Degenerated NPCs(P6)were induced by the medium containing 50?g/ml exosomes of BMSCs.2)After induced culture for 14 days,the morphological changes of exosome group cells and their morphological differences with P2 NPCs were observed.3)Induced culture for 3 days,7 days,10 days,and 14 days later,three groups of NPCs marker genes ACAN,COL2,SOX9,TIMP1 and MMP1 were detected by PCR method.The relative expression of mRNAs were statistically analyzed.4)Induced culture for 3 days,7days,10 days,and14days later,the expression of NPCs marker genes ACAN,COL2,SOX9,TIMP1 and MMP1 corresponding proteins were detected by WB method.Result1.The adherent cells obtained from bone marrow of SD rats by whole bone marrow method: 1)the cells were fusiform,the density of cells were uniform,the size were uniform,and the cells grew like vortex;2)The expression of CD29,CD44 and CD90 were 99.8%,95.4% and 99.8%,the expression ofCD34 and CD45 were 6.16% and 6.08%.3)After osteogenic differentiation and culture,the formation of calcium nodules was observed.After adipogenic differentiation culture,the formation of fat droplets was obvious.After chondrogenic differentiation and culture,the accumulation of glycosaminoglycan was observed;2.The precipitates obtained by differential centrifugation:1): The disk-shaped vesicles with diameter of 30-100 nm were observed under transmission electron microscope(TEM),and the size of the vesicles was not uniform.2):Detected by WB method,the CD63,CD81 and Tsgl01 were highly expressed,Calnexin was not expressed.3.The P1 and P2 NPCs were spindle-like and single in shape.With the increase of algebra,the morphology of cells varied gradually,most of them were fusiform,and when they were passed on to P6,the morphology of most cells changed greatly.Because there were much impurities in P1 NPCs,we selected P2 and P6 NPCs for follow-up experiments.P6 and P2 NPCs were stained by cell senescence ?-galactosidase staining method.The staining color degree and staining cell proportion of P6 NPCs(75%)were significantly higher than those of P2 NPCs(20%).4.After the exosomes of BMSCs and degenerated NPCs were stained and co-cultured for 24 hours,all exosomes of BMSCs were observed in P6 NPCs under laser confocal microscope.5.1): After experiment,the volume of the NPCs were smaller and the pseudopod were smaller,but they were bigger than the P2 NPCs.2): The relative expression of ACAN,COL2,SOX9 and TIMP1 gene mRNA in co-culture group were significantly higher than that in control group,while that in exosome group were significantly higher than that in co-culture group(P<0.05).With the increase of time,the relative expression of the co-culture group and the exosome group increased gradually(P<0.05).The relative expression of MMP1 mRNA in co-culture group was significantly lower than that in control group,while that in exosome group was significantly lower than that in co-culture group(P<0.05).In the co-culture group and exosome group,the relative expression level decreased gradually with the increase of time(P<0.05).3): The corresponding proteins of ACAN,COL2,SOX9,TIMP1 and MMP1 gene were detected by WB method.The results showed the same trend as the PCR results.Conclusion1.The adherent cells extracted by whole bone marrow method were successfully identified as stem cells,and the precipitated by differential centrifugation were successfully identified as exosomes.2.Compared with P2 NPCs,P6 NPCs produced obvious degeneration in morphology and function,and successfully established degeneration model.3.The exosomes of BMSCs could be absorbed by degenerative NPCs and could improve the morphology and function of degenerative NPCs.This study provides a new idea for the fundamental treatment of intervertebral disc degeneration disease.