| Purpose:To investigate the differences of the effects between nucleus pulposus stem cells and nucleus pulposus cells in repairing degenerated intervertebral disc,which provides experimental evidence for further application of mesenchymal stem cells to the degenerated intervertebral disc repair treatment.Experimental method:1.Isolation,culture and bioactivity detection of nucleus pulposus mesenchymal stem cells:We used flow cytometry screening method to isolate and culture the mesenchymal stem cells,by which the osteogenic,chondrogenic and adipogenic induced differentiation assays were respectively taken.Osteogenic differentiation assay contains alkaline phosphatase and alizarin red staining.Chondrogenic and adipogenic differentiation assays were taken by the appraisal of toluidine blue staining and the oil red O dye staining.2.Isolation and culture,bioactivity detection of nucleus pulposus cells:Nucleus pulposus cells were obtained through cell adherent method.The proliferation ability of the collected cells was detected.The collagen II and toluidine blue staining were respectively taken.3.Experimental grouping:All of the 32 rabbits were randomly divided into four groups(A、B、C、D):Blank,NPCs,NPSCs and DMEM group.4.Imageological examination:Lateral lumbar X-ray detections were taken to assess intervertebral disc height index(DHI%)1D before and 12W after the injecting cell transplantation of the treatment.The relative signal intensity(RSI)of nucleus pulposus was calculated through MRI evaluation.5.PCR test:By extracting total RNA,the expression of collagen protein II and proteoglycans divided by the expression of GAPDH to get the relative expression level of collagen protein Ⅱ and protein.6.Quantitative detection of proteoglycan and collagen protein II.7.Statistical analysis:Use spss20.0 software for the statistical analysis.The differences of the measurement data between groups were compared by one-way analysis of variance,which were shown significant different(P<0.05).There were no significant statistical difference(P>0.05).Main result:1.Osteogenic differentiation and detection of the nucleus pulposus mesenchymal stem cells showed positive staining in alizarin red and Alkaline phosphatase dye.The cells were blue and vary sizes of lipid droplets were formed by toluidine blue staining in the induced chondrogenesis and adipogenic differentiation and detection.2.Primary nucleus pulposus cells of rabbit were in uniform size and roundness shape.The cell proliferation activity was low at the early stage,and was markedly enhanced as the time increased to 5 to 13 days.Toluidine blue staining and type II collagen immunohistochemical staining showed positive staining.3.12W after the transplantation,the DHI%of group B was significantly increased(P<0.05),compared with other groups.The relative signal intensity index(RSI)of the nucleus pulposus of group B was significantly more intensive than that of group C(P<0.05).4.PCR and quantitative detection of proteoglycan and type Ⅱ collagen protein tests showed that the genetic expression level of proteoglycan and type Ⅱ collagen protein of group B was significantly higher than other groups.The protein expression level of proteoglycan in group B was statistical higher,compared to group C.However,there were no significant differences among 4 groups in the expression of type II collagen(P>0.05).Conclusion:Both nucleus pulposus cells and the nucleus pulposus mesenchymal stem cells can improve the genetic expression level of proteoglycan and collagen protein Ⅱ,and promote the repairing of the degenerative intervertebral disc,while the latter has a better therapeutic effect.Therefore,nucleus pulposus mesenchymal stem cells might be further used for the treatment of intervertebral disc degeneration. |
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