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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (6): 609-616    DOI: 10.3785/j.issn.1008-9292.2019.12.04
原著     
TLR4/MyD88/NF-κB通路基因及相关炎症因子在继发性脊髓损伤患者中的表达
米爽(),吴燕君,洪正华*(),王章富,冯兴兵,郑光彬
浙江省台州医院脊柱外科, 浙江 台州 317000
Expression of TLR4/MyD88/NF-κB pathway genes and its related inflammatory factors in secondary spinal cord injury
MI Shuang(),WU Yanjun,HONG Zhenghua*(),WANG Zhangfu,FENG Xingbing,ZHENG Guangbin
Department of Spinal Surgery, Taizhou Hospital of Zhejiang Province, Taizhou 317000, Zhejiang Province, China
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摘要：

目的: 探讨Toll样受体4（TLR4）/髓样分化因子（MyD88）/NF-κB通路基因及相关炎症因子TNF-α、IL-12、IL-6在继发性脊髓损伤（SSCI）患者中的表达及与预后的相关性。方法: 回顾性分析2017年7月至2018年6月浙江省台州医院收治的105例SSCI患者及40名健康体检者的临床资料。根据Frankel脊髓损伤分级评估结果将SSCI患者分为完全损伤组和不完全损伤组，根据日本矫形外科学会（JOA）患者神经功能改善率将SSCI患者分为预后优良组和预后不良组。对比SSCI患者与健康体检者、完全损伤组与不完全损伤组、预后优良组与预后不良组外周血单个核细胞（PBMC）中TLR4、MyD88、NF-κB mRNA表达及血清TNF-α、IL-12、IL-6水平；采用Logistic回归分析法分析导致SSCI患者预后不良的危险因素，并采用Pearson相关性分析法分析JOA改善率与上述指标的相关性。结果: SSCI患者PBMC中TLR4、MyD88、NF-κB mRNA表达量及血清TNF-α、IL-12、IL-6水平均高于健康体检者（均P < 0.01），完全损伤组上述指标均高于不完全损伤组，且预后不良组高于预后优良组（均P < 0.01）。预后不良组Frankel分级A级、脊髓水肿或出血、脊髓损伤长度超过4 cm患者的比例均高于预后优良组（均P < 0.01），且经Logistic回归分析证实Frankel分级、脊髓水肿或出血、脊髓损伤长度及PBMC中TLR4、MyD88、NF-κB mRNA相对表达量、血清TNF-α、IL-12、IL-6水平均是导致SSCI患者预后不良的危险因素（P < 0.05或P < 0.01）。Pearson相关性分析结果显示，JOA改善率与PBMC中TLR4、MyD88、NF-κB mRNA相对表达量以及血清TNF-α、IL-12、IL-6水平均呈负相关（P < 0.05或P < 0.01）。结论: TLR4/MyD88/NF-κB通路激活及其相关炎症因子TNF-α、IL-12、IL-6表达上调参与SSCI疾病进展且与神经炎症损伤关系密切，可作为评估SSCI患者预后的参考指标。
关键词： Toll样受体4;  核因子κB;  髓样分化因子88;  脊髓损伤;  基因表达;  预后    
Abstract:

Objective: To investigate the expression of Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD88)/nuclear factor-κB (NF-κB) pathway genes and related inflammatory factors tumor necrosis factor-α (TNF-α), interleukin (IL)-12, IL-6 in patients with secondary spinal cord injury (SSCI) and the correlations with prognosis. Methods: The clinical data of 105 SSCI patients and 40 healthy subjects were reviewed. According to Frankel's classification of spinal cord injury, the patients were divided into complete injury group and incomplete injury group, and according to the improvement of Japanese Orthopedic Association (JOA) scores, the patients were divided into good prognosis group and poor prognosis group. The expression of TLR4, MyD88, NF-κB in peripheral blood mononuclear cells (PBMC) and serum TNF-α, IL-12, IL-6 levels were compared between SSCI patients and healthy controls, between patients with complete and incomplete injury, between patients with poor and good prognosis. Logistic regression analysis was used to analyze the risk factors leading to poor prognosis of SSCI, and Pearson's correlation analysis was used to analyze the correlation between JOA score and the above indicators. Results: The expressions of TLR4, MyD88, NF-κB in PBMC and serum TNF-α, IL-12, IL-6 levels in SSCI patients were significantly higher than those in healthy subjects (all P < 0.01), those in complete injury group were higher than those in incomplete injury group, and those in poor prognosis group were higher than those in good prognosis group (all P < 0.01). The proportions of patients with Frankel grade A, spinal cord edema or hemorrhage, spinal cord injury length longer than 4 cm in poor prognosis group was significantly higher than those in good prognosis group (all P < 0.01). Logistic regression analysis showed that Frankel grade, spinal cord edema or hemorrhage, length of spinal cord injury, relative expressions of TLR4, MyD88, NF-κB in PBMC, serum levels of TNF-α, IL-12 and IL-6 were risk factors for poor prognosis in SSCI patients (P < 0.05 or P < 0.01). Pearson's correlation analysis showed that JOA improvement rate was negatively correlated with the relative expressions of TLR4, MyD88, NF-κB mRNA in PBMC and serum TNF-α, IL-12, IL-6 levels (P < 0.05 or P < 0.01). Conclusion: The activation of TLR4/MyD88/NF-κB pathway and the up-regulation of the expression of related inflammatory factors TNF-α, IL-12 and IL-6 are involved in the progression of SSCI, which are closely related to the neuroinflammatory injury, and can be used as reference indexes for evaluating prognosis in SSCI patients.
Key words: Toll-like receptor 4    Nuclear factor kappa B    Myeloid differentiation factor 88    Spinal cord injuries    Gene expression    Prognosis
收稿日期: 2019-08-30 出版日期: 2020-01-19
:  R744  
基金资助: 浙江省中医药科技计划(2018ZB138)
通讯作者: 洪正华     E-mail: scudme@163.com;0001hzh@163.com
作者简介: 米爽(1986—), 男, 硕士, 主治医师, 主要从事脊柱外科学研究; E-mail:scudme@163.com; https://orcid.org/0000-0002-7293-2857
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米爽
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引用本文:

米爽,吴燕君,洪正华,王章富,冯兴兵,郑光彬. TLR4/MyD88/NF-κB通路基因及相关炎症因子在继发性脊髓损伤患者中的表达[J]. 浙江大学学报（医学版）, 2019, 48(6): 609-616.

MI Shuang,WU Yanjun,HONG Zhenghua,WANG Zhangfu,FENG Xingbing,ZHENG Guangbin. Expression of TLR4/MyD88/NF-κB pathway genes and its related inflammatory factors in secondary spinal cord injury. J Zhejiang Univ (Med Sci), 2019, 48(6): 609-616.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.12.04    或    http://www.zjujournals.com/med/CN/Y2019/V48/I6/609

引物 引物序列
TLR4 上游：5′-TAGCGAGTAGCTGATGCT-3′
下游：5′-ATCGTAGCTGTAGCTGAC-3′
MyD88 上游：5′-CTAGCTAGCTGTAGCTGC-3′
下游：5′-ATCGTATCGTATCTATCGT-3′
NF-κB 上游：5′-ATCGTAGCTAGCTGTAG-3′
下游：5′-TGCTAGCTAGCTAGCTC-3′
β-actin 上游：5′-TAGCTAGCTAGCTAGCT-3′
下游：5′-CGTATGCGTACGTGATC-3′
表 1  引物序列
变量名称 变量 赋值说明
TLR4：Toll样受体4；MyD88：髓样分化因子；NF-κB：核因子-κB；TNF-α：肿瘤坏死因子-α；IL-12：白细胞介素-12；IL-6：白细胞介素-6.
性别 X1 女=0、男=1
年龄 X2 不超过60岁=0、超过60岁=1
致伤原因 X3 车祸伤=0、坠落伤=1、砸伤=2、摔伤=3
受伤至就诊时间 X4 不超过8 h=0、超过8 h=1
损伤部位 X5 骶尾段=0、胸腰段=1、颈段=2
Frankel分级 X6 A级=0、B~D级=1
脊髓水肿或出血 X7 无变化=0、脊髓水肿或出血=1
脊髓损伤长度 X8 不超过4 cm=0、超过4 cm=1
治疗方式 X9 手术=0、非手术=1
大剂量甲强龙冲击治疗 X10 是=0、否=1
PBMC中TLR4 mRNA X11 连续变量
PBMC中MyD88 mRNA X12 连续变量
PBMC中NF-κB mRNA X13 连续变量
血清TNF-α X14 连续变量
血清IL-12 X15 连续变量
血清IL-6 X16 连续变量
表 2  影响因素赋值说明
组别 n TLR4 MyD88 NF-κB TNF-α(pg/mL) IL-12(pg/mL) IL-6(pg/mL)
“—”：无相关数据.SSCI：继发性脊髓损伤；TLR4：Toll样受体4；MyD88：髓样分化因子；NF-κB：核因子-κB；TNF-α：肿瘤坏死因子-α；IL-12：白细胞介素-12；IL-6：白细胞介素-6.
SSCI患者 105 2.14±0.35 3.18±0.45 2.52±0.37 38.50±4.22 22.13±3.50 3.58±0.64
健康体检者 45 0.95±0.13 1.03±0.16 1.07±0.15 7.61±0.91 5.15±0.84 2.13±0.37
t值 — 22.127 31.166 25.371 48.534 32.093 23.990
P值 — ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01
表 3  SSCI患者与健康体检者TLR4/MyD88/NF-κB基因及相关炎症因子表达比较
组别 n TLR4 MyD88 NF-κB TNF-α(pg/mL) IL-12(pg/mL) IL-6(pg/mL)
“—”：无相关数据.SSCI：继发性脊髓损伤；TLR4：Toll样受体4；MyD88：髓样分化因子；NF-κB：核因子-κB；TNF-α：肿瘤坏死因子-α；IL-12：白细胞介素-12；IL-6：白细胞介素-6.
完全损伤组 33 3.62±0.52 5.71±0.80 4.13±0.61 52.01±7.25 30.00±4.59 5.84±0.72
不完全损伤组 72 1.46±0.27 2.02±0.34 1.78±0.30 32.31±5.80 17.15±3.14 2.71±0.40
t值 — 28.042 33.260 26.523 14.907 16.735 28.674
P值 — ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01
表 4  SSCI完全损伤组与不完全损伤组TLR4/MyD88/NF-κB基因及相关炎症因子表达比较
组别 n TLR4 MyD88 NF-κB TNF-α(pg/mL) IL-12(pg/mL) IL-6(pg/mL)
“—”：无相关数据.SSCI：继发性脊髓损伤；TLR4：Toll样受体4；MyD88：髓样分化因子；NF-κB：核因子-κB；TNF-α：肿瘤坏死因子-α；IL-12：白细胞介素-12；IL-6：白细胞介素-6.
预后不良组 37 3.48±0.54 5.44±0.72 3.92±0.53 50.43±6.23 31.37±4.59 5.29±0.75
预后优良组 68 1.41±0.21 1.95±0.26 1.76±0.31 32.01±4.51 17.10±2.78 2.65±0.39
t值 — 28.038 36.003 26.377 17.418 19.844 23.771
P值 — ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01
表 5  SSCI预后不良组与预后优良组TLR4/MyD88/NF-κB基因及相关炎症因子表达比较
可能的影响因素 预后不良组(n=37) 预后优良组(n=68) χ2/t值 P值
SSCI：继发性脊髓损伤.
性别
  男 23 39 0.229 ＞0.05
  女 14 29
年龄(岁)
  ＞60 16 24 0.642 ＞0.05
  ≤60 21 44
致伤原因
  车祸伤 15 32 0.157 ＞0.05
  坠落伤 10 16
  砸伤 5 8
  摔伤 7 12
受伤至就诊时间(h)
  ＞8 h 10 15 0.326 ＞0.05
  ≤8 h 27 53
Frankel分级
  A 18 15 7.861 ＜0.01
  B~D 19 53
损伤部位
  颈段 10 12 1.231 ＞0.05
  胸腰段 21 46
  骶尾段 6 10
脊髓水肿或出血
  无 16 12 8.028 ＜0.01
  有 21 56
脊髓损伤长度(cm)
  ＞4 6 2 5.999 ＜0.05
  ≤4 31 66
治疗方式
  手术 12 25 0.197 ＞0.05
  非手术 25 43
大剂量甲泼尼龙
  冲击治疗
  是 9 20 0.310 ＞0.05
  否 28 48
表 6  其他可能影响SSCI患者预后的因素比较
变量 β值 SE Wald χ2值 P值 OR值 95%CI
“—”：无相关数据；SSCI：继发性脊髓损伤；PBMC：外周血单个核细胞；TLR4：Toll样受体4；MyD88：髓样分化因子；NF-κB：核因子-κB；TNF-α：肿瘤坏死因子-α；IL-12：白细胞介素-12；IL-6：白细胞介素-6.
Frankel分级 0.553 0.250 4.893 ＜0.05 1.738 1.214~2.263
脊髓水肿或出血 0.629 0.252 6.230 ＜0.01 1.876 1.220~2.531
脊髓损伤长度 0.714 0.262 7.427 ＜0.01 2.042 1.317~2.767
PBMC中TLR4 mRNA 0.850 0.301 7.975 ＜0.01 2.340 1.464~3.215
PBMC中MyD88 mRNA 0.932 0.328 8.074 ＜0.01 2.540 1.532~3.547
PBMC中NF-κB mRNA 0.994 0.334 8.857 ＜0.01 2.702 1.540~3.864
血清TNF-α 1.136 0.351 10.475 ＜0.01 3.114 1.975~4.252
血清IL-12 1.247 0.366 11.608 ＜0.01 3.480 2.061~4.899
血清IL-6 1.334 0.368 13.141 ＜0.01 3.796 2.100~2.492
常数项 -31.017 0.013 6.782 ＜0.01 0.000 —
表 7  SSCI患者预后不良危险因素的多因素Logistic回归分析
图 1  JOA改善率与SSCI患者TLR4、MyD88、NF-κB mRNA表达量及血清TNF-α、IL-12、IL-6水平的Pearson相关性分析散点图(n=105)
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