标题：细胞因子bFGF和NF-κB在腰椎黄韧带肥厚中的表达及意义

      作者：林伟文，凌华军，钟思龙，罗鹏刚，吴增志，夏雄超，赖茂松，熊浩    广东医科大学附属高明医院骨科，广东 佛山 528500

      卷次： 2019年30卷7期

      【摘要】 目的 了解成纤维细胞生长因子(bFGF)和核转录因子kappaB (NF-κB)的表达水平，并探讨其在腰椎黄韧带肥厚中的作用机制，为临床治疗提供理论基础。方法 选取佛山市高明区人民医院骨科 2017年 1月至2018年6月收治的15例影像学显示黄韧带厚度符合相应分组的临床标本，其中对照组(下腰椎骨折需行后路椎板及黄韧带切除术或反复盘源性腰痛行固定术黄韧带切除患者) 5例，突出组(单纯腰椎间盘突出症患者行黄韧带切除术患者) 5例，腰椎管狭窄组(黄韧带肥厚型腰椎管狭窄行黄韧带切除患者) 5例，对其组织进行培养，分离出成纤维细胞通过CCK8检测其增殖活性。使用ELISA试剂盒检测培养基上清中细胞因子浓度。将培养基上清加入巨噬细胞中通过实时荧光定量PCR (PT-qPCR)及免疫荧光检测巨噬细胞活化情况。结果 对照组、突出组、腰椎管狭窄组患者黄韧带厚度分别为(2.26±0.31) mm、(3.52±0.45) mm、(4.56±0.51) mm，腰椎管狭窄组患者黄韧带较对照组及突出组肥厚，差异均有统计学意义(P<0.05)；3 d和7 d成纤维细胞密度及成纤维细胞增殖CCK8结果显示，椎管狭窄组的细胞增殖速度较其他两组均明显增快，差异均有统计学意义(P<0.05)；对照组、突出组、腰椎管狭窄组成纤维细胞上清中bFGF细胞因子浓度分别为(0.72±0.04) ng/mL、(0.81±0.06) ng/mL、(1.16±0.15) ng/mL，培养基上清中NF-κB浓度分别为(0.81±0.07) ng/mL、(1.09±0.14 ) ng/mL、(1.27±0.13) ng/mL，腰椎管狭窄组3 d后的细胞培养基中bFGF和NF-κB浓度较对照组及突出组均明显升高，差异均有统计学意义(P<0.05)；通过RT-qPCR及免疫荧光检测表明，加入腰椎管狭窄组成纤维细胞上清的巨噬细胞系(RAW2647)细胞高表达促炎指标 iNOS、低表达抗炎指标Arg，分别与对照组及突出组比较，差异均有统计学意义(P<0.05)。结论 细胞因子bFGF和NF-κB可能通过刺激成纤维细胞增生、促进巨噬细胞活化而促使黄韧带肥厚的发生与发展。
      【关键词】 成纤维细胞生长因子；核转录因子kappaB；黄韧带肥厚；腰椎；免疫荧光
      【中图分类号】 R686 【文献标识码】 A 【文章编号】 1003—6350（2019）07—0824—05

Expression and significance of cytokines fibroblast growth factor and nuclear factor-kappaB in lumbarhypertrophy of ligamentum flavum.
LIN Wei-wen, LING Hua-jun, ZHONG Si-long, LUO Peng-gang, WU Zeng-zhi,XIA Xiong-chao, LAI Mao-song, XIONG Hao. Department of Orthopedics, Gaoming Hospital Affiliated to Guangdong MedicalUniversity, Foshan 528500, Guangdong, CHINA
【Abstract】 Objective To understand the expression levels of fibroblast growth factor (bFGF) and nuclear fac-tor-kappaB (NF-κB), and to explore its mechanism of action in lumbar ligamentum flavum hypertrophy, providing a the-oretical basis for clinical treatment. Methods The clinical specimens of 15 patients admitted to the Department of Or-thopaedics at Gaoming Hospital Affiliated to Guangdong Medical University from January 2017 to June 2018 were se-lected, including 5 cases in the control group (patients with lower lumbar vertebrae fractures requiring posterior laminaand ligamentum flavum resection or repeated discogenic low back pain requiring fixation and ligamentum flavum resec-tion), 5 cases in the disc herniation group (patients with simple lumbar disc herniation requiring ligamentum flavum re-section), and 5 cases in the lumbar spinal stenosis group (patients with ligamentum flavum stenosis requiring ligamen-tum flavum resection). The resected ligamentum flavum tissue was cultured, and the fibroblasts were isolated and testedfor their proliferative activity by CCK8. ELISA kit was used to detect cytokine concentrations in culture supernatants.The supernatant of the culture medium was added to macrophages, and the activation of macrophages was detected by re-al-time fluorescent quantitative PCR (RT-qPCR) and immunofluorescence. Results The thickness of the ligamentumflavum in the control group, the disc herniation group, and the lumbar spinal stenosis group was (2.26±0.31) mm, (3.52±0.45) mm, and (4.56±0.51) mm, respectively. The ligamentum flavum in the lumbar spinal stenosis group was signifi-cantly thicker than that in the control group and the disc herniation group (P<0.05). CCK8 results of fibroblast densityand fibroblast proliferation cultured for 3 d and 7 d showed that the cell proliferation rate in the spinal stenosis groupwas significantly faster than that in the other two groups (P<0.05). In the control group, the disc herniation group and thelumbar spinal stenosis group, the concentration of bFGF cytokines in fibroblast supernatants was respectively (0.72 ±0.04) ng/mL, (0.81±0.06) ng/mL, and (1.16±0.15) ng/mL; the concentrations of NF-κB in the supernatants was respec-tively (0.81±0.07) ng/mL, (1.09±0.14) ng/mL, and (1.27±0.13) ng/mL. The concentration of bFGF and NF-κB in the cul-ture medium of the lumbar spinal stenosis group was significantly higher than that of the control group and the disc her-niation group (P<0.05). The results of RT-qPCR and immunofluorescence showed that the cells of macrophage cell line(RAW2647) cultu