大鼠尾椎间盘退变不同时期Hippo信号差异性研究

Study on Hippo pathway of different period disc degeneration in rat coccygeal vertebra disc

目的:探讨纤维环穿刺诱导的不同时期大鼠尾椎椎间盘退变过程中Hippo信号通路的表达变化。方法:SD雄性大鼠共60只,随机分为三组:未行尾椎纤维环穿刺的20例作为对照组(A组);40只大鼠进行尾椎纤维环穿刺,构建椎间盘退变动物模型,术后4周(B组,20只)和术后3个月(C组,20只)为实验组,术后4周和3个月行尾椎X线、MRI和病理学HE染色观察,评估上述三组椎间盘退变状况;免疫组化染色检测椎间盘髓核组织内Hippo信号通路下游因子Yes相关蛋白(Yes-associated protein,YAP)、磷酸化Yes相关蛋白(phosphorylat Yes-associated protein,p-YAP)和富半胱氨酸蛋白61 (cysteine-rich angiogenic inducer 61,CYR61)表达情况,分析早期和后期椎间盘退变Hippo信号通路表达变化情况。结果:与A组相比,B和C两组椎间盘纤维环穿刺术后椎间盘高度明显丢失(P<0.05),MRI T2像灰度值增加(P<0.05),表明椎间盘已经发生退变。且与A组(2.44±0.17)相比,B、C两组椎间盘退变病理学分级评分(4.33±0.23、4.22±0.22)显著增加(P<0.05);p-YAP免疫组化和蛋白印迹检测显示B组表达相对减少,在A组中表达稍高,B和C组间p-YAP的表达具有显著性差异(0.33±0.02 vs 0.99±0.11,P<0.05),而B组和A组(0.56±0.05)相比较无明显差异(P>0.05);YAP在B组表达明显增高,而在C组表达明显减少,与B组相比YAP表达具有显著性差异(P<0.05)。但是,与YAP表达不一致的是下游靶基因CYR61在B组中呈低表达,且与C组相比差异无统计学意义(P>0.05)。结论:Hippo信号通路参与不同时期椎间盘退变的发生发展,在维持椎间盘内稳态中起重要作用。

Objectives:To observe the changes in the expression of Hippo signaling pathway during the coc-cygeal vertebra disc degeneration induced by annulus fibrosis puncture in rats.Methods:60 male SD rats,were randomly divided into three groups,20 rats without annulus puncture were used as control group(group A);40 rats performed annulus puncture to construct intervertebral disc degeneration(IDD)model,4 weeks after operation(group B,n=20)and 3 months after operation(group C,n=20)as the experimental groups.The IDD conditions were accessed by X-ray,MRI and HE staining.Yes-associated protein(YAP),phosphorylat Yes-associated protein(p-YAP)and cysteine-rich angiogenic inducer 61(CYR61)were detected by immunohisto-chemical staining to analyze Hippo signal expression in different period IDD.Results:Compared with group A,group B and C were loss disc height obviously after annulus fibrosus puncture and MRI T2 gray-value increased(P<0.05),thus showed that IDD had been made.And when compared with group A(2.44±0.17),group B and C IDD pathological grading score(4.33±0.23,4.22±0.22)increased significantly(P<0.05).Immunohistochemistry and Western blot analysis showed that the p-YAP expression was relatively reduced in group B(0.33±0.02)compare with group A(0.56±0.05).There was a significant difference in p-YAP expression between B and C groups(0.33±0.02 vs 0.99±0.11,P<0.05),but there was no significant difference between group B and A(P>0.05).The expression of YAP in group B was significantly higher,but it decreased significantly(P<0.05)in group C.However,the expression of target gene CYR61 was decreased in group B and there was no statistically difference compared with the group C(P>0.05),which was not consistent with the expression of YAP.Conclusions:Hippo pathway is involved in the development of IDD in different period and plays an important role in the maintenance of intervertebral disc homeostasis.