摘要目的 建立兔椎间盘器官培养模型,在此基础上施以循环机械压力(CMP),观察兔椎间盘器官细胞成活率、组织形态学的变化及ANK蛋白的表达情况,并观察外源性转化生长因子(TGF)-β1对ANK蛋白表达的调控作用.方法 6月龄新西兰大白兔随机分为加力组和对照组,无菌条件下完整取出带部分椎骨的腰段椎间盘,放入含有20％胎牛血清的培养液里进行培养.加力组椎间盘应用加力器对其施以0.2 MPa压力值,每日1次,每次30 min.2组器官模型培养选取0、7、14天3个时间点,HE染色观察大体组织形态学变化;氯化硝基四氮唑蓝(NBT)染色及4',6-二脒基-2-苯基吲哚(DAH)复染检测细胞成活率;Western blot检测ANK蛋白的表达.另外,在加力组培养至第7天时,培养液中加入外源性TGF-β1再培养4h,Western blot检测ANK蛋白的表达.结果 对照组培养至第7天时,其组织形态学、细胞成活率及ANK蛋白的表达与0天相比差异无统计学意义(P＞0.05);培养至第14天时,组织形态学破坏,细胞成活率下降、ANK蛋白的表达下调,差异有统计学意义(P值均＜0.05).加力组培养至第7天时,组织形态学无明显变化,但细胞成活率下降、ANK蛋白的表达下调(P值均＜0.05);培养至第14天时,组织形态学破坏、细胞成活率下降及ANK蛋白的表达下调均更明显(P值均＜0.05).另外,加力组培养至第7天时加入外源性TGF-β1后,ANK蛋白的表达上调(P＜0.05).结论 CMP载荷可明显增加兔椎间盘器官模型细胞和组织学结构的破坏,使ANK蛋白的表达明显下调,可能与椎间盘退变的发生、发展密切相关.而外源性TGF-β1对ANK蛋白的表达具有正向调控作用,可能对CMP作用导致的退变椎间盘具有一定的修复作用.

abstractsObjective To develop an in vitro organ culture model of rabbit intervertebral disc,and cyclic mechanic pressure was applied based on this model,and to investigate the change of ANK.To explore the regulatory effect of exogenous TGF-β1 on the expression of ANK.Methods The New Zealand white rabbits of 6 months old were randomly divided into cyclic mechanical pressure group and control group.Rabbits intervertebral disc were taken out completely with part of the vertebrae from lumbar spines by asepsis.The whole rabbit intervertebral discs were cultured in medium with 20％ fetal bovine serum supplemented.And cyclic mechanical pressure at 0.2 MPa was applied for cyclic mechanical pressure group different periods,once every 30 minutes in a day.The change of histomorphology,the cell viability and the protein expression levels of progressive ankylosis gene within intervertebral disc tissue were assessed by HE staining,NBT-DAPI,Western blotting at 0 day,7 days and 14 days.The protein expression levels of progressive ankylosis gene within intervertebral disc were cultured at cyclic mechanical pressure for 7 days after exogenous TGF-β1 stimulation assessed by Western blotting.Results Compared with 0 day group,there were minimal changes in histomorphology,cell viability and the protein expression levels of progressive ankylosis gene at control group for 7 days (P ＞ 0.05).There were minimal changes in histomorphology at cyclic mechanical pressure for 7 days.There were obvious change of damage in histomorphology both control group and cyclic mechanical pressure group for 14 days(P ＜0.05).The cell viability was decreased at 7,14 days of cyclic mechanical pressure group and 14 days of control group(P ＜0.05).The expression levels of progressive ankylosis gene were significantly decreased at 7,14 days of cyclic mechanical pressure group and 14 days of control group (P ＜ 0.05).On the other hand,after exogenous TGF-β1 stimulation,the expression level of progressive ankylosis gene was significantly increased at 7 days of cyclic mechanical pressure group (P ＜ 0.05).Conclusions Based on the model of rabbit intervertebral disc,cyclic mechanical pressure could induce significant decrease of the expression level of progressive ankylosis gene.It may be closely related with the occurrence and development of intervertebral disc degeneration.And it confirms that exogenous TGF-β1 has a positive regulatory role for the expression level of progressive ankylosis gene to protect intervertebral disc from degeneration.