| Part One Construction of rat intervertebral disc injury model by M1 macrophage injectionObjectives:The needle caudal puncture model was used to be a comparation to explore the feasibility of the model of intervertebral disc degeneration caused by M1 macrophage intervertebral disc injection.Methods:1.Using 4-week-old SPF SD rats as the bone marrow extraction animal and then using the medium containing LPS and IFN-? to amplify and induce M1 macrophages derived from rat bone marrow,and perform real-time RT PCR of the obtained M1 macrophages.RT-PCR detects the expression of inflammatory cytokines TNF-? and IL-1b genes.2.The Co6-Co9 tail intervertebral discs of 12-week-old SPF rat were treated with 21 G needle puncture,blank control and M1 macrophage injection.3.At 8 weeks and 12 weeks after treatment,MRI and CT examinations were performed on the intervertebral discs of the tail of the rats,and the degree of disc degeneration and disc height were statistically measured using software.The rats were sacrificed at 12 weeks,and the intervertebral discs of the rats were stained with HE and Safranin and Fast Green and immunohistochemical analysis.Results:1.Observed under a microscope,the bone marrow cells are round floating cells after inoculation,and gradually adhere to the wall with the extension of the culture time.After the 4th day of the primary cells,most of the cells have adhered to the wall(M0 macrophages).After the M1 macrophages are induced to mature,the cell morphology is diverse,with pseudopods and bulges.Real-time RT-PCR detection revealed that IL-1b and TNF-? m RNA were significantly increased compared to the control group,indicating that the M1 macrophages were successfully cultured;2.The signal of the intervertebral disc was significantly reduced at 8 and12 weeks after the injection of M1 macrophages,and the reduction was greater than that of the puncture model.After injection of M1 macrophages,the intervertebral height can be significantly reduced,and the endplate cartilage is invaded and the endplate is destroyed;3.Histological staining showed that the structure of the intervertebral disc in the control group was neat,and the structure of the nucleus pulposus and annulus fibrosus was clear.In the puncture model,the annulus fibrosis of the intervertebral disc is broken,the nucleus pulposus cells are reduced,and the edge of the endplate is irregular.In the M1 macrophage injection group,the contents of the discs further decreased,the structure was obviously disordered,and the endplate was severely irregular.HE staining score control group 4.15±1.15,puncture model 8.73±1.40,M1 macrophage injection group11.52±1.22,Safranin and fast green staining score control group 4.79±1.19,puncture model 11.31±1.38,M1 macrophage injection Group 11.78±0.93;4.Immunohistochemistry results showed that the positive expression rate of proteoglycan in the puncture model was significantly lower than that of the control group,P<0.05,while the positive expression rate of the M1 macrophage injection group was significantly lower than that of the puncture model and the control group,P<0.05;The positive expression rate of collagen type II in the puncture model was significantly lower than that of the control group,P<0.05,and the positive expression rate of M1 macrophage injection group was significantly lower than that of puncture model and control group,P<0.05.Conclusions:1.Both 21 G needle puncture and M1 macrophage injection can create an intervertebral disc injury model,which is jointly manifested by reduced nuclear magnetic signals,reduced intervertebral height and loss of extracellular matrix components;2.The possible mechanism of M1 macrophage injection of intervertebral disc injury model is related to its secreted inflammatory factors.3.The M1 macrophage injection group is more severely injured than the21 G needle puncture group,and it is a severe disc injury model that can be used in the future.Part Two Effect of iPSC on Intervertebral Disc Injury Induced by M1 Macrophage Injection in RatsObjectives:To observe the therapeutic effect of iPSC on the severe disc injury model caused by M1 macrophage injection in rats,and try to explore its mechanism of action.Methods:1.T Using 4-week-old SPF SD rats as the bone marrow extraction animal and then using the medium containing LPS and IFN-? to amplify and induce M1 macrophages derived from rat bone marrow,and inject this M1 macrophages into the Co6-7 and Co8-9 tail discs of the rats to make severe injury models of tail discs.2.8 weeks after M1 macrophage injection,the Co6-7 and Co8-9 tail discs of rats were treated with PBS injection and iPSC injection respectively.3.8 weeks and 12 weeks after iPSC injection treatment,MRI and CT examinations were performed on the intervertebral discs of the tail of the rats,and the degree of disc degeneration and disc height were statistically measured using software.The rats were sacrificed at 12 weeks,and the intervertebral discs of the rats were stained with HE and Safranin?Fast Green and immunohistochemical analysis.Results:1.8 weeks and 12 weeks after iPSC injection,the intervertebral disc signal of Co6-7(degeneration group)was significantly lower than that of Co7-8(blank group),the height of the intervertebral disc was significantly decreased,and the endplate cartilage was invaded and the endplate was damaged;Compared with the degeneration group,the Co8-9(puncture + iPSC group)showed a significant increase in the intervertebral height,and the intervertebral disc signal was significantly enhanced,which has a certain therapeutic effect on the degeneration of the intervertebral disc.2.Histological staining showed that the structure of the intervertebral disc in the control group was neat,and the structure of the nucleus pulposus and annulus fibrosus was clear.The annulus fibrosus of the degenerative model intervertebral disc is broken,the nucleus pulposus and the annulus fibrosus borders are interrupted,the nucleus pulposus cells are reduced,the extracellular matrix is coagulated,and the endplate edges are irregular.Compared with the degeneration group,the structure of the intervertebral disc in the iPSC treatment group was increased in nucleus pulposus cells,and the boundaries of the annulus fibrosus and nucleus pulposus were clear,which improved significantly compared with the degeneration group.HE staining score blank group 4.55±0.89,degeneration group 7.18±0.98,iPSC group5.17±0.70,iPSC group score was significantly lower than that of degeneration group,P<0.05;Safranin and green staining score blank group 4.4±1.17,degeneration Group 11.60±1.38,iPSC group 9.02±1.13,iPSC group score was significantly lower than degeneration group,P<0.05.3.The results of immunohistochemistry showed that the positive expression rate of proteoglycan in the degeneration group was significantly lower than that in the control group,P<0.05,while the positive expression rate in the iPSC group was lower than that in the control group but significantly higher than the degeneration group,P<0.05;The expression rate of histone type II collagen was significantly lower than that of the control group,P<0.05,while the positive expression rate of the iPSC group was lower than that of the control group but significantly higher than the degeneration group,P<0.05;the expression rate of TGF-? in the degeneration group was significantly lower than the iPSC group,P<0.05,the expression rate of TGF-? in the iPSC group was lower than the control group,P<0.05.Conclusions:1.iPSC has a certain therapeutic effect on intervertebral disc degeneration caused by M1 macrophages,can increase the signal intensity of the intervertebral disc and increase the height of the intervertebral disc,and has the effect of promoting the synthesis of proteoglycan and type II collagen;2.The mechanism of iPSC in the treatment of intervertebral disc degeneration caused by M1 macrophages may be related to the increase in the amount of intervertebral disc cells,the promotion of the synthesis of extracellular matrix and the regulation of the TGF-? pathway related to intervertebral disc degeneration.Part Three Predictors of poor outcome in cervical spondylotic myelopa-thy patients underwent anterior hybrid approachObjectives:This study was a retrospective multivariable analysis for risk factors of poor outcome in patients who underwent anterior hybrid approach,and discussed the causes of worsening of postoperative local alignment.Methods:A total of 86 patients with progressive spinal cord compression and local kyphosis underwent an anterior hybrid approach(ACDF+ACCF),between June 2011 and June 2017.We evaluated clinical outcome by the Japanese Orthopaedic Association(JOA)score and recovery rate.Patients were divided into two groups according to the worsening and improving of postoperative local alignment.Multivariate logistic regression analysis was applied to the evaluation of risk factors.Mann-Whitney U test,independent t test,and chisquared test were performed for the comparison of local kyphosis between postoperative and last follow-up.Results:There were twenty patients who had a recovery rate of less than 50%.Advance age,longer duration of symptoms,bigger T1 slope angle,and lower change of local kyphosis angle were significantly associated with a poor clinical outcome by multivariate logistic regression analysis.The cause of worsening of postoperative local alignment had T1 slope,C2–7 sagittal vertical axis(SVA),adjacent segment degeneration(ASD),and implant subsidence.Conclusions:1.Age at operation,duration of symptoms,T1 slope angle,and change of local kyphosis were the risk factors of clinical outcome in patients with CSM and cervical kyphosis after the hybrid approach.2.Postoperative ASD,implant subsidence,T1 slope,and C2–7 Cobb were associated with recurrence of postoperative cervical kyphosis. |
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