基于Integrin/FAK信号通路的电针对颈椎病模型软骨细胞凋亡影响及机制研究

Basic pathological changes of cervical spondylosis are disc degeneration whose key factor is chondrocyte apoptosis. Preliminary studies (see research base) proved that EA was effective to treat cervical spondylosis and slow down disc degeneration. Based on this, the rat models of cervical spondylosis are replicated, EA on acupoints Fengchi, Tianzhu, neck Jiaji, Houxi, the TUNEL staining, the ANNEXIN Ⅴ/PI double staining and electron microscopy to detect chondrocyte apoptosis, immunohistochemical observation on Bax, Bcl-2, Fas and FasL, ELISA assay of caspases-3, 8, 9 and RT-PCR and Western Blot are detected on ECM, P- integrin, integrin, P- FAK, FAK, p130cas, of Paxillin, of Grb2, Src, Ras, Crk, PI3K, STAT3 mRNA and protein expression levels to observe effect on EA of cartilage cell apoptosis and the apoptosis pathway and its intracellular Integrin/FAK signaling pathways, to clarify the mechanism of EA delaying chondrocyte degeneration via the signaling pathway, and to provide a reliable experimental basis for the prevention and treatment of cervical degenerative disc diseases.

颈椎病基本病理变化是椎间盘退变，其启动因素为软骨细胞凋亡，课题组研究（见研究基础）证明电针能有效治疗颈椎病并延缓椎间盘退变，基于此，本课题在复制大鼠颈椎病模型基础上，通过电针风池、天柱、颈夹脊、后溪等穴位，以TUNEL法、ANNEXINⅤ/PI法及电镜检测软骨细胞凋亡，免疫组化观察Bax、Bcl-2、Fas、FasL，ELISA检测Caspases3、8、9的表达，RT-PCR、Western Blot检测ECM、integrin(蛋白含磷酸化)、FAK（蛋白含磷酸化）、p130cas、Grb2、Src、Ras、Crk、PI3K、STAT3 mRNA和蛋白表达水平，观察电针对软骨细胞凋亡影响和凋亡通路确定及其对胞内Integrin/FAK信号通路影响，阐明电针介导该信号通路在延缓软骨细胞退变过程中的作用机制，为防治颈椎间盘退变性疾病提供可靠实验依据。

目的：观察电针对颈椎病模型大鼠椎间盘细胞凋亡及Integrin/FAK 信号通路的影响。方法：SPF级SD大鼠40只，采用完全随机化区组设计方案，将40只SD大鼠分成空白对照组（假手术组）、模型对照组（模型组）、模型电针治疗组（模针组）、模型美洛昔康片对照治疗组（美洛昔康组），每组10只大鼠。建立动静力失衡性颈椎病模型，手术造模后饲养两个月。模针组取手三里与颈夹脊穴进行电针针刺治疗，每次留针25 min，1次/天，14天为1个疗程，两疗程之间间隔2 d，共治疗2个疗程。美洛昔康组:给予美洛昔康片灌胃治疗两个疗程。空白组、模型组均只作固定而不做其它任何处理。采用HE染色和Toluidine blue染色观察颈椎椎间盘形态学的变化， TUNEL法检测椎间盘细胞阳性表达，PCR检测ECM、integrin、FAK、ERK2、Grb2、Ras、Raf、Crk、PI3K、STAT3等基因的表达，western blot检测ECM、integrin、FAK、ERK2、Grb2、Ras、Raf、Crk、PI3K、STAT3等蛋白的表达情况。结果：电针能降低椎间盘凋亡细胞的阳性表达，与模型组相比有显著差异。电针能显著提高ECM、integrin、FAK、ERK2、Grb2、Ras、Raf、Crk、PI3K、STAT3mRNA和蛋白的表达水平。结论：电针能抑制颈椎病模型大鼠的凋亡细胞，其取效途径与电针激活Integrin/FAK信号通路有密切关系。