| Objective:Detecting the indexes of cellular immunity and humoral immunity in peripheral venous blood and tissue samples of patients with degenerative scoliosis,comparing with the immunological functional indexes of normal blood from volunteer and tissue samples from patients with spinal trauma.To explore the possible correlation and potential mechanism between immunology and the occurrence and development of degenerative scoliosis.To provide theoretical basis for the etiology of degenerative scoliosis,and provide a new theoretical basis for the diagnosis and treatment of degenerative scoliosis.Methods:1.The data of clinical data,tissue and/or blood samples of 8 patients with degenerative scoliosis,8 patients who underwent surgical treatment due to spinal trauma at the same time,and 8 normal volunteers from November 2019 to February 2020 in the Second Affiliated Hospital of Kunming Medical University were collected.The collection process has been approved by the medical ethics committee of the Second Affiliated Hospital of Kunming Medical University.At the same time,the subjects have signed the informed consent.Subjects with immune related and infection related diseases have been excluded from the study.2.Group:eight patients diagnosed as degenerative scoliosis were included in the observation group,aged 49-63 years(mean age was 56.5±4.50 years old),the Cobb’s angle range of which was 12-16°(mean 13.38 ± 1.51°).Eight normal persons recruited at the same time were included in the control group of normal blood samples,which were 26-51 years old(mean age was 36.25 ± 10.79 years old).Eight patients with spinal trauma were included in the control group,average age of which was 47.0± 7.60 years old.3.Sample acquisition:peripheral venous blood of patients in the observation group was obtained before the operation.During the operation,the intervertebral disc tissue(including nucleus pulposus,fibrous ring and cartilage endplate),ligamentum flavum tissue and paravertebral muscle tissue were obtained from observation group.And the peripheral venous blood of normal people was collected.the normal intervertebral disc,ligamentum flavum tissue and paravertebral muscle tissue were obtained from the control group of tissue sample.(4)Management of samples:the proportion of CD4+T lymphocytes,CD8+T lymphocyte subsets and the ratio of CD4+ T/CD8+T cells were measured by flow cytometry;the levels of IgM,IgG,IgA,complement C3 and C4 were measured by immunoturbidimetry,and the level of IL-6 was measured by electrochemiluminescence.The obtained tissue samples were fixed with 4%paraformaldehyde at room temperature and made into paraffin sections.The pathological changes of the intervertebral disc,ligamentum flavum and paravertebral muscle were observed under the microscope.At the same time,the sections were stained with immunohistochemical staining method to observe the distribution of CD4 positive,CD8 positive T lymphocytes and cytokines IL-6 in different parts of the specimens.The results of immunohistochemistry were analyzed by image and digital pathological system and ImageJ method.All data were analyzed by spss26.0 software.Results:1.the proportion of CD8+T lymphocyte subsets in the observation group was 34.06± 8.45%,which was higher than that in the control group(20.39±2.34%).The ratio of CD4+/CD8+T cells in the observation group was 1.22 ± 0.57,which was lower than that in the control group(2.46±0.75).And the difference between the two groups was significant(P<0.05);The percentage of CD3+ T lymphocyte subsets(76.44±7.09%)and CD4+T lymphocyte subsets(37.77 ± 12.20%)in the observation group were not significantly different from those in the control group[(75.03±0.75%)and(48.78±9.03%)](P>0.05);the content of IgG in the 8 blood samples of the observation group was 7.81±2.50g/l,lower than which in the control group(14.41±1.77 g/L)(P>0.05).The contents of IgM and C3 in the control group were 1.06 ± 0.26 g/L and 0.77±0.15 g/L,lower than which in the control group(1.88±0.63 g/L)and(0.86 ± 0.07g/L)respectively.The difference between the two groups was statistically significant(P<0.05);The difference of serum IgA(1.54± 0.77g/L),complement C4(0.17 ± 0.04g/L)in observation group and serum IgA(2.03±0.56 g/L),complement C4(0.18 ± 0.04g/L)in control group were not statistically significant(P>0.05);2.The results of immunohistochemistry showed that the distribution of CD4 and CD8 positive lymphocytes was found in the intervertebral disc,ligamentum flavum and paravertebral muscle tissues of the observation group.The distribution of CD4 and CD8 positive lymphocytes in the intervertebral disc was mainly in the marginal area of the fibrous ring.The distribution of CD4 and CD8 positive lymphocytes in the ligamentum flavum was mainly in the aggregation area of ossified multicellular types and around the neovascularization.The distribution of CD4 and CD8 positive lymphocytes in paravertebral muscle was mainly around the vascularization.There was no obvious distribution of CD4 and CD8 positive lymphocytes in the control group.In the semi quantitative observation of ImageJ processed scanning image,the CD4,CD8 positive cell rate and immunohistochemical AOD value of the three tissues in the observation group were higher than those in the tissue sample control group(P<0.05).3.In the blood samples of the observation group,the content of serum IL-6 was 5.74±3.23g/l,which was significantly higher than that of the control group(1.01±0.45g/L),the difference between the two groups was statistically significant(P<Q.05);the expression of IL-6 was found in the samples of intervertebral disc,ligamentum flavum and paravertebral muscle of the two groups,but compared with the control group,the positive expression of IL-6 in the three tissues of the observation group was more obvious Obviously.The rate of IL-6 positive cells and the AOD value of immunohistochemistry in the observation group were higher than those in the tissue sample control group after image analysis,digital pathological system and ImageJ software analysis.The difference was statistically significant(P<0.05).Conclusions:1.The systemic immune system function of patients with degenerative scoliosis is different from that of normal people.The cellular immune system and humoral immune system are different from that of normal people.The cellular immune system function is in a relatively "inhibited state" after activation,while the humoral immune function is decreased 2.CD4 and CD8 positive T-lymphocytes were observed in the local lesion tissues of patients with degenerative scoliosis,and there was autoimmune reaction;3.IL-6 participated in the pathological process of degenerative scoliosis,and played a role in the intervertebral disc,ligamentum flavum and paravertebral muscles of degenerative site.2.T lymphocytes with positive expression of CD4 and CD8 were observed in the observation group,indicating the enhancement of local immune activity.3.IL-6 is involved in the pathological process of degenerative scoliosis,and plays a role in the intervertebral disc,ligamentum flavum and paravertebral muscles. |
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