椎间盘内注射医用臭氧对椎间盘退变模型自身免疫反应的影响

Experimental study of ozone injection for treating intervertebral disc degeneration with prolapsed type

中文关键词:  腰椎间盘退变  自身免疫应答  臭氧  兔

英文关键词:Medical ozone  Intervertebral disc degeneration  Autoimmunity  Rabbit

中文摘要:

  【摘要】 目的：观察椎间盘内注射医用臭氧对椎间盘退变（intervertebral disc degeneration，IDD）动物模型自身免疫反应的影响，探讨其作用机理。方法：50只新西兰兔随机分为A、B、C、D、E共5组，其中A组为正常组，不作处理；B组为假手术组，仅暴露纤维环而不穿刺；C、D、E组采用后外侧纤维环穿刺的方法建立腰椎间盘退变模型（L6/7），在造模后14d，D组模拟临床手术椎间盘内注射臭氧干预，E组椎间盘内注射医用纯氧，于造模后14d、21d对5组动物进行自由行走疼痛行为学评分，并于21d获取各组动物外周血，L6、L7神经根及L6/7髓核，运用透射比浊法测定各组动物血清中免疫球蛋白IgG和IgM水平，ELISA法测定血清中TNF-α、IL-1、IL-6的含量；免疫组化染色观察髓核中抗原抗体复合物沉积情况，应用图像分析系统记录染色阳性细胞的累积光密度值（integrated optical density，IOD）；BCA蛋白定量法检测腰神经根中PLA2的活性。结果：造模后1周左右（平均7.4d）C、D、E组动物手术侧后肢活动减少，并在爬行前进时明显偏离手术侧，行走疼痛学评分分别为2.81±0.13分、2.69±0.11分、2.64±0.31分，组间比较差异无统计学意义（P>0.05）；D组注射臭氧后上述异常行为逐渐减轻，处死前行走疼痛学评分为0.54±0.12分，低于C组（2.75±0.17分）及E组（2.52±0.96分）（P<0.05）；D组动物血清中IgG、IgM水平（6.05±0.49g/L, 1.98±0.28g/L）低于C组（8.98±0.77g/L，2.40±0.25g/L）及E组（8.52±0.63g/L，2.91±0.31g/L）（P<0.05），与A组（5.43±0.29g/L，1.72±0.15g/L）及B组（4.97±0.35g/L，1.66±0.27g/L）比较差异无统计学意义（P>0.05）；D组炎性细胞因子TNF-α、IL-1、IL-6的含量（98±11pg/ml，247±21pg/ml，149±23.4pg/ml）亦低于C组（256±39pg/ml，404±19pg/ml, 231±41.3pg/ml）及E组（244±45pg/ml， 410±15pg/ml，249±21.7pg/ml）（P<0.05），与A组（70±6pg/ml，223±17pg/ml，141±12.6pg/ml）及B组（79±11pg/ml，238±32pg/ml，138±17.4pg/ml）比较差异无统计学意义（P>0.05）；D组髓核中抗原抗体复合物阳性沉积光密度值（7.40±3.56）小于C组（27.49±12.84）及E组（29.55±10.38）（P<0.05），其相应节段腰神经根PLA2活性（0.0189±0.0072μmol/min·L）较C组（0.1685±0.0103μmol/min·L）及E组（0.1994±0.080μmol/min·L）明显下降（P<0.01），与A组（0.0036±0.0081μmol/min·L和B组（0.0098±0.0043μmoL/min·L）比较差异无统计学意义（P>0.05）。结论：医用臭氧能够在一定程度上抑制腰椎间盘退变模型中异常的自身免疫反应，这可能是臭氧治疗椎间盘退变引起的腰痛的作用机制之一。

英文摘要:

  【Abstract】 Objectives: To observe the influence of ozone injection on the abnormal autoimmunity of intervertebral disc degeneration(IDD) model(L6/7) in animal. Methods: An IDD model was established on rabbits. Then medical ozone was used for treatment while medical purified oxygen was used as the control. The immunoglobulin G and immunoglobulin M in serum were obtained at two weeks after the operation and examined by turbidimetry. The presence and distribution of antigen-antibody complexes in nucleus pulposus were examined by immunohistochemistry. And the levels of cytokine TNF-α, IL-1, IL-6 were detected by enzyme linked immunosorbent assay, and the BCA method was also used to analyze the activity of PLA2 in the nerve root. Results: After the operation, there were abnormal activities in the groups of model, ozone and oxygen, which had the scores of free walk pain showed as 2.81±0.13, 2.69±0.11 and 2.64±0.31 respectively, and improved when the medical ozone was applied. Compared with the levels of the immunoglobulin G and M as (8.98±0.77g/L, 2.40±0.25g/L) and (8.52±0.63g/L， 2.91±0.31g/L) on model group and medical oxygen group, the medical ozone group showed lower levels(6.05±0.49g/L, 1.98±0.28g/L, P<0.05); compared with the model group and medical oxygen group as (256±39pg/ml，404±19pg/ml, 231±41.3pg/ml） and (244±45pg/ml， 410±15pg/ml, 249±21.67pg/ml)(P<0.05) for the leves of cytokine TNF-α, IL-1, IL-6 in the serum, the medical ozone group also showed lower values(98±11pg/L，247±21pg/L， 149±23.4pg/L); compared with the model group and medical oxygen group for the expression of antigen-antibody complexes in the nucleus pulposus(27.49±12.84) and (29.55±10.38), the value of medical ozone group was lower(7.40±3.56)(P<0.05);furthermore, the level of PLA2 in the lumbar nerve root of medical ozone group(0.0189±0.0072μmol/min·L) was also lower than the model group and medical oxygen group as (0.1685±0.0103μmol/(min·L) and (0.1994±0.080μmol/min·L)(P<0.01). Conclusions: Medical ozone injection can an inhibit the abnormal autoimmunity of LDH model to an extent, which can be a main mechanism of therapy for the early stage of IDD.