循环机械压力诱导下兔椎间盘退变器官模型的建立及意义

In vitro organ culture of rabbit degenerative intervertebral disc under cyclic mechanic pressure and its significance

背景:力学因素是导致椎间盘退变(IDD)的重要诱因,建立力学相关性IDD的器官模型能为IDD机制的研究提供理想的模型基础。目的:建立兔椎间盘器官模型,并施以循环机械压力,探究循环机械压力载荷对IDD的影响。方法:6月龄新西兰大白兔随机分为加力组和对照组,静脉给予1.3 ml肝素(5000 U/ml),待肝素体内循环5 min后处死。无菌条件下完整取出带部分椎骨的腰段椎间盘,放入20%胎牛血清的培养液中培养。加力组应用加力器施以0.2 MPa压力值,每日加压1次,每次30 min。经过各个时段的培养后,苏木精-伊红(HE)染色观察椎间盘大体组织形态学变化;氯化硝基四氮唑蓝(NBT)染色及4',6-二眯基-2-苯基吲哚(DAPI)复染检测椎间盘细胞成活率;Realtime RT-PCR和Western-blotting检测蛋白多糖(AGN)、Ⅱ型胶原(COLⅡ)的mRNA和蛋白表达。结果:对照组和加力组培养至第7 d的组织形态学无明显变化,培养至第14 d均表现为组织形态学破坏,且以加力组表现更为明显。对照组培养至第7 d的细胞成活率及AGN、COLⅡ表达与0 d相比无明显变化;对照组培养第14 d与0 d比较,培养第7 d加力组与对照组比较,培养第14 d加力组与对照组比较,均表现为细胞成活率明显下降,AGN、COLⅡ表达下调。结论:成功建立短周期兔椎间盘体外器官模型,并在此模型基础上阐明循环机械压力载荷可直接导致椎间盘退变样改变。

Background：The mechanical factor is the main incentive of intervertebral disc degeneration. The establishment of an in vitro organ culture model can provide a basic condition for studying the mechanism of intervertebral disc degeneration. Objective： To develop an in vitro organ culture model of rabbit intervertebral disc, and to investigate the relationship be-tween cyclic mechanic pressure and intervertebral disc degeneration. Methods：New Zealand white rabbits aged 6 months old were randomly divided into cyclic mechanical pressure group and control group. Heparin （5000 U/ml, 1.3 ml） were intravenously infused, then the animals were killed 5 minutes later. Then intervertebral discs were taken out with part of the vertebrae from lumbar spines by asepsis and cultured in medium with 20%fetal bovine serum supplemented. Cyclic mechanical pressure at 0.2 MPa was applied once every 30 minutes in a day. The change of histomorphology, cell viability and mRNA and protein expression levels of aggrecan, typeⅡcollagen within intervertebral disc tissue were assessed by HE staining, NBT-DAPI, real time RT-PCR and Western-blotting, respectively, at different periods. Results：In the control group, there were no significant defferences in terms of the changes in histomorphology, cell viability and mRNA and protein expression levels of aggrecan and typeⅡcollagen before culture and 7 days after culture. Obvious damage in histomorphology were seen in both control group and cyclic mechanical pressure group 14 days after culture, es-pecially in later one. The cell viability and the expression level of typeⅡcollagen and aggrecan were significantly decreased at 7, 14 days after culture in the cyclic mechanical pressure group and 14 days in the control group. Conclusions：The short-term in vitro organ culture of rabbit intervertebral disc is successfully performed in the study. Cyclic mechanical pressure can induce degeneration of the intervertebral disc in this model.