臺灣博碩士論文加值系統

黃韌帶肥厚而造成神經根的壓迫和腰椎椎體間結構的改變，是導致椎管狹窄的主要因素之一。黃韌帶受到物理性的刺激所造成的發炎反應，與肥厚之間有互相關聯，也許我們抑制黃韌帶發炎可以用來治療椎管狹窄症。在正常情況下黃韌帶中彈性蛋白與膠原蛋白含量的比例為 2:1 ，但隨著年齡增長，彈性蛋白與膠原蛋白的比例會逐漸下降。有研究指出肌腱和韌帶上有雌激素的受器，此外雌激素在細胞外基質扮演調控者的角色，與彈性蛋白、膠原蛋白和蛋白多糖體的調節相關。黃韌帶的發炎已經被認為是造成黃韌帶肥厚的原因之一，有研究指出藉由力學刺激誘導黃韌帶發炎。已經有研究顯示雌激素與力學刺激能調控成骨細胞的增生與骨形成；另外也有研究指出在黃韌帶鈣化中雌激素受體的表現量增加。目前雌激素與黃韌帶發炎的機制尚未清楚。
在本篇研究我們所使用的黃韌帶細胞來自接受脊椎手術病患，透過離心力的刺激誘導黃韌帶細胞發炎，然後再加入10-7 ~10-9 M的雌激素做刺激。所以本篇研究的是探討雌激素對力學刺激誘發人類黃細胞發炎的影響。
本研究結果顯示雖然雌激素對黃韌帶細胞並無顯著增生的影響，但有雌激素的刺激有上升的趨勢，在濃度 10-9 M 下有較好的表現。加入力學刺激後對細胞的增生有下降的趨勢，但力學刺激前給予雌激素刺激對照單獨力學刺激組有上升的趨勢。mRNA 表現中單獨雌激素刺激組IL-1α, IL-1β, IL-6, TNF-α 這些發炎前趨因子都有顯著的下降表現；單獨力學刺激下IL-1α, IL-6, TNF-α 有顯著上升表現；力學刺激前給予雌激素中IL-1α, IL-6, TNF-α 與單獨力學刺激做比較有明顯現降的表現。此外黃韌帶細胞分泌發炎前趨蛋白中IL-6 在力學刺激前給予雌激素表現量有顯著的下降，雖然 IL-1β, TNF-α 沒有顯著差異但有下降的趨勢。最後在MAPK 細胞內訊號傳遞實驗這部分實驗結顯示出透過p-ERK來調控發炎反應。因此我們初步推論雌激素確實有抑制力學刺激所誘導黃韌帶細胞的發炎。

Ligamentum flavum hypertrophy leads to the formation of spinal canal with subsequent neural compression. Previous findings suggest that mechanical stresses induce inflammatory reactions and scar formation play an important role in the progression of ligamentum flavum hypertrophy. The normal ligamentum flavum is composed of 70% elastin protein and 30% collagen protein, but the composition ratio of elastin protein and collgen protein change in the elderly. Previous studies indicated the presence of estrogen receptors in tendons and ligament. Therefore, estrogen play an important role in the regulation of lastin protein and collgen protein. Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. Mechanical force induces human ligamentum flavum fibroblasts inflammation. It also demonstrated estrogen and mechanical force are positive regulators for osteoblasts proliferation and bone formation. In addition, the expression of estrogen receptor was enhanced in calcium crystal deposit ligamentum flavum.
In this study, we used human ligamentum flavum fibroblasts were obtained from twenty-one patients undergoing lumbar spine surgery. Centrifugal force induces inflammation of human ligamentum flavum fibroblasts, and then enter 10-9 M of estrogen. Hence, the aim of the present study was to investigate the effects of estrogen on mechanical stress-induced inflammatory response in ligamentum flavum cells.
The investigation demonstrated that cell viability of ligamentum flavum fibroblast was no significant differences but rise trend and the cell viability was better in 10-9 M 17β-estradiol stimulation. Besides, The cell viability were the same reduction trend on mechanical or 17β-estradiol combined with mechanical stress stimulation groups when ligamentum flavum fibroblast treated centrifugal stimulation but had 17β-estradiol stimulation were better than mechanical stress stimulation. The IL-1α, IL-6 and TNF-α mRNA pro-inflammatory cytokines mRNA expression were indeed reduction by mechanical force stimulation but pretreated 17β-estradiol stimulation were inhibited expression. Finally, the intracellular MAPK signaling transduction pathways results were shown pro-inflammatory cytokines regulate through the p-ERK.
This initial study suggested that estrogen inhibite on mechanical stress-induced inflammatory response in ligamentum flavum cell.

中文摘要 I
ENGLISH ABSTRACT III
CONTENTS V
FIGURES VIII
TABLES X
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Objective of this study 3
CHAPTER 2 PAPER REVIEW 4
2.1 Lumbar Spinal Stenosis 4
2.2 Ligamentum Flavum 5
2.3 Estrogen 6
2.3.1 Introduction 6
2.3.2 Boiosynthesis of Estrogen 6
2.3.3 Estradiol 7
2.3.4 Estrogen Receptor 7
2.4 Cytokines 9
2.4.1 Introduction 9
2.4.2 Interleukin-1 10
2.4.3 Interleukin-6 10
2.4.4 Tumor necrosis factor-α 11
CHAPTER 3 MATERIALS AND METHODS 12
3.1 Material 12
3.1.1 Reagents 12
3.1.2 Drug 13
3.1.3 Antibodies 14
3.2 Methods 16
3.2.1 Cell Culture 16
3.2.2 Application of Mechanical Stress 17
3.2.3 Cell Proliferation Reagent WST-1 Assay 18
3.2.4 Ribonucleic Acid (RNA) Isolation and Real-time Polymerase
Chain Reaction (PCR) 19
3.2.5 Western Blotting Analysis 22
3.2.6 Enzyme Immunoassay (EIA) 22
3.2.7 Statistical Analysis 23
CHAPTER 4 RESULTS 24
4.1 Differential Effect of Estrogen Concentration on Cell Viability 24
4.2 Differential Effect of Estrogen Concentration and Mechanical
Stress on Cell Viability 25
4.3 Effect of Estrogen and Mechanical Stress on IL-1α Mrna
Expression 27
4.4 Effect of Estrogen and Mechanical Stress on IL-1β mRNA
Exression 28
4.5 Effect of Estrogen and Mechanical Stress on IL-6 mRNA
Exression 29
4.6 Effect of Estrogen and Mechanical Stress on TNF-α mRNA
Exression 30
4.7 Effect of Estrogen and Mechanical Stress on Ligamentum
Flavum Firbroblast IL-1β Protein Release 31
4.8 Effect of Estrogen and Mechanical Stress on Ligamentum
Flavum Firbroblast IL-6 Protein Release 32
4.9 Effect of Estrogen and Mechanical Stress on Ligamentum
Flavum Firbroblast TNF-α Protein Release 33
4.10 Time Course of Mechanical Stress Induced Signaling
Transduction pathways 34
4.11 Effect of Estrogen on Mechanical Stress Induced Phosphorylation of p38 35
4.12 Effect of Estrogen on Mechanical Stress Induced
Phosphorylation of ERK 36
4.13 Effect of Estrogen on Mechanical Stress Induced
Phosphorylation of JNK 37
CHAPTER 5 DISCUSSION 38
CHAPTER 6 CONCLUSION 42
REFERENCE 43

Fig 4.1 Estrogen stimulated the expression of WST-1 assay for cell
viability 24
Fig 4.2 Estrogen and Mechanical stress stimulated the expression of
WST-1 assay for cell viability 25
Fig 4.3 Effect of estrogen and mechanical stress on IL-1α mRNA
expression after 24 hours stimulation 27
Fig 4.4 Effect of estrogen and mechanical stress on IL-1β mRNA
expression after 24 hours stimulation 28
Fig 4.5 Effect of estrogen and mechanical stress on IL-6 mRNA
expression after 24 hours stimulation 29
Fig 4.6 Effect of estrogen and mechanical stress on TNF-αmRNA
expression after 24 hours stimulation 30
Fig 4.7 Effect of estrogen and mechanical stress on ligamentum flavum
fibroblast IL-1β protein release 31
Fig 4.8 Effect of estrogen and mechanical stress on ligamentum flavum
fibroblast IL-6 protein release 32
Fig 4.9 Effect of estrogen and mechanical stress on ligamentum flavum
fibroblast TNF-α protein release 33
Fig 4.10 Time course of mechanical stress induced signaling transduction
pathways 34
Fig 4.11 Effect of estrogen on mechanical stress induced phosphorylation
of p38 35
Fig 4.12 Effect of estrogen on mechanical stress induced phosphorylation
of ERK 36
Fig 4.13 Effect of estrogen on mechanical stress induced phosphorylation 
of JNK 37


Table 3.2 Forward and reverse primer and probes 21

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