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

中文关键词:  Hippo  Yes相关蛋白  椎间盘退变  动物模型  生物修复

英文关键词:Hippo  Yes-associated protein  Intervertebral disc degeneration  Animal model  Bioremediation

中文摘要:

  【摘要】 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。

英文摘要:

  【Abstract】 Objectives: To observe the changes in the expression of Hippo signaling pathway during the coccygeal 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 immunohistochemical 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.