基于析因设计探讨腰椎活动范围增大对大鼠黄韧带肥厚的影响

The Influence of Increased Range of Motion of Lumbar on Rat Model of Ligamentum Flavum Hypertrophy with Factorial Design

目的:基于析因设计评价三种腰椎活动范围增大的造模方法,建立一种新的大鼠黄韧带肥厚模型,同时探讨腰椎活动范围的增大所诱发黄韧带肥厚的机制。方法:将72只大鼠随机分为假手术组、肌肉切除组、骨关节切除组和肌肉+骨关节切除组,分别对其L_(5/6)节段进行造模。于造模后的第4,8,12周共3个时间点取材。通过X线片观察屈曲和背伸位时L_(5/6)节段的活动范围和椎间隙高度,通过苏木精-伊红(HE)染色观察L_(5/6)节段黄韧带总体和背侧层的厚度,通过免疫组化法和实时聚合酶链式反应法(Real-Time Polymerase Chain Reaction,RT-PCR)检测黄韧带中白细胞介素1β(Interleukin-1β,IL-1β)、肿瘤坏死因子α(Tumor Necrosis Factor-α,TNF-α)、转化生长因子β1(Transforming Growth Factor-β1,TGF-β1)、Ⅰ型胶原(TypeⅠCollagen,Col1)和Ⅲ型胶原(TypeⅢCollagen,Col3)等因子的表达。结果:肌肉+骨关节切除法可以最大程度增大L_(5/6)节段的屈曲活动范围和黄韧带的厚度,并导致黄韧带中IL-1β、TNF-α、TGF-β1、P-Smad_(2/3)(或Smad2和Smad3)、Col1和Col3的蛋白及mRNA呈高表达,其次为骨关节切除法,最后为肌肉切除法。结论:同时切除棘突、棘间韧带、椎旁肌等结构可以最大程度引起大鼠腰椎屈曲范围的增大和黄韧带的增厚,其机制可能是机械应力导致黄韧带中炎性因子TNF-α和IL-1β的高表达,并诱导TGF-β1/Smads通路致使细胞外基质沉积,进而发生了黄韧带肥厚。

Objective:To establish a new rat model of ligamentum flavum hypertrophy(LFH)by evaluating three modeling methods of increasing lumbar range of motion,and to explore the mechanism of LFH induced by increasing lumbar range of motion.Methods:The 72 rats were randomly divided into sham group(group A),muscle resection group(group B),bone resection group(group C),and the mixture group of B and C(group D),and the L_(5/6)segments were modeled respectively.The specimens were collected at three time points:4th week,8th week,and 12th week after modeling.The motion range of the lumbar and the height of the intervertebral space were observed by X-ray examination.The thickness of LF and its dorsal layer were observed by HE staining.The expression of IL-1β,TNF-α,TGF-β1/Smads pathway,and Col1 and Col3 were detected by immunohistochemistry and real-time-PCR.Results:The model of muscle and bone resection can maximize the flexion range of the L_(5/6)segment and the thickness of LF,and lead to the high expression of mRNA and protein of IL-1β,TNF-α,TGF-β1,P-Smad_(2/3)(or Smad2 and Smad3),Col1 and Col3 in ligamentum flavum followed by the model of bone resection,and finally the model of muscle resection.Conclusion:The simultaneous resection of the spinous process,interspinous ligament,and paravertebral muscle can increase the range of lumbar flexion and thicken the LF in rats.The mechanism may be that mechanical stress leads to inflammatory factors TNF-αand IL-1βwhich induced TGF-β1/Smads pathway,leading to the deposition of extracellular matrix and LFH.