Hur Junseok W, Bae Taegeun, Ye Sunghyeok, Kim Joo-Hyun, Lee Sunhye, Kim Kyoungmi, Lee Seung-Hwan, Kim Jin-Soo, Lee Jang-Bo, Cho Tai-Hyoung, Park Jung-Yul, Hur Junho K
Department of Neurosurgery, College of Medicine, Korea University, Seoul, Korea.
Center for Genome Engineering, Institute for Basic Science, Seoul, Republic of Korea.
Eur Spine J. 2017 Aug;26(8):2021-2030. doi: 10.1007/s00586-017-4981-2. Epub 2017 Feb 8.
Majority of the previous studies compared lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) patients for analyses of LFH. However, the separation of normal/hypertrophied LF has often been ambiguous and the severity of hypertrophic activity differed. Here, we present a novel analysis scheme for LFH in which myofibroblast is proposed as a major etiological factor for LFH study.
Seventy-one LF patient tissue samples were used for this study. Initially, mRNA levels of the samples were assessed by qRT-PCR: angiopoietin-like protein-2 (ANGPTL2), transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin-6, collagen-1, 3, 4, 5, and 11, and elastin. Myofibroblasts were detected by immune stain using α-smooth muscle actin (αSMA) as a marker. To study the myofibroblast in TGF-β pathway, LF tissues were analyzed for protein levels of αSMA/TGF-β1 by Western blot. In addition, from LF cells cultured with exogenous TGF-β1 conditioned medium, expression of αSMA/collagen-1 was assessed and the cell morphology was identified.
The comparative analysis of mRNA expression levels (LSS vs LDH) failed to show significant differences in TGF-β1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-β1, and collagen-1 and 3, which represent common trends in hypertrophic activity (p < 0.05). We detected myofibroblast in the patient samples by αSMA staining, and the protein levels of αSMA were positively correlated with TGF-β1. In LF cell culture, exogenous TGF-β1 upregulated αSMA/collagen-1 mRNA levels and facilitated trans-differentiation to myofibroblast.
We conclude that the transition of fibroblast to myofibroblasts via TGF-β pathway is a key linker between inflammation and fibrosis in LFH mechanism.
以往大多数研究比较腰椎管狭窄症（LSS）和腰椎间盘突出症（LDH）患者以分析腰椎肥厚（LFH）。然而，正常/肥厚的LF分离往往不明确，且肥厚活动的严重程度不同。在此，我们提出一种新的LFH分析方案，其中提出肌成纤维细胞作为LFH研究的主要病因因素。
本研究使用了71份LF患者组织样本。首先，通过qRT-PCR评估样本的mRNA水平：血管生成素样蛋白2（ANGPTL2）、转化生长因子-β1（TGF-β1）、血管内皮生长因子（VEGF）、白细胞介素-6、胶原蛋白-1、3、4、5和11以及弹性蛋白。使用α-平滑肌肌动蛋白（αSMA）作为标志物通过免疫染色检测肌成纤维细胞。为了研究TGF-β途径中的肌成纤维细胞，通过蛋白质印迹分析LF组织中αSMA/TGF-β1的蛋白质水平。此外，在用外源性TGF-β1条件培养基培养的LF细胞中，评估αSMA/胶原蛋白-1的表达并鉴定细胞形态。
mRNA表达水平的比较分析（LSS与LDH）未显示TGF-β1有显著差异（p = 0.08）；然而，我们发现ANGPTL2、VEGF、TGF-β1以及胶原蛋白-1和3之间存在显著正相关，这代表了肥厚活动的共同趋势（p < 0.05）。我们通过αSMA染色在患者样本中检测到肌成纤维细胞，且αSMA的蛋白质水平与TGF-β1呈正相关。在LF细胞培养中，外源性TGF-β1上调了αSMA/胶原蛋白-1的mRNA水平并促进向肌成纤维细胞的转分化。
我们得出结论，成纤维细胞通过TGF-β途径向肌成纤维细胞转变是LFH机制中炎症与纤维化之间的关键连接点。
