Liu Xiao, Wang Deguo, Zhang Zhengzheng, Zhu Fenghui, Yao Aiming, Tian Jiwei, Miao Dengshun
Department of Orthopedics, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, P.R. China.
Emergency Center, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.
Exp Ther Med. 2017 Aug;14(2):1141-1145. doi: 10.3892/etm.2017.4584. Epub 2017 Jun 12.
This study aims to investigate the function and related mechanism of gene in intervertebral disc (IVD) degeneration of mice. X-ray, immunohistochemical staining, and alkaline phosphatase (ALP) histochemical staining were used to analyze the phenotypic difference of the intervertebral discs of 4-week-old mice with P27 gene knockout (P27) and wild-type (WT) mice in the same brood. Protein in the intervertebral disc was extracted and western blot analysis was employed to detect the changes in the expression of related molecules in the Shh-signal pathways, including Shh, Patched, Smoothened and Gli2. As a result, the ALP histochemical staining revealed that the ALP-positive area of mice in the P27 group was obviously increased compared to the 4-week-old mice of the same brood in the WT group. In addition, the Col-I immunohistochemical staining showed that the Col-I-positive area of mice in the P27 group was significantly increased compared to mice in the WT group. Furthermore, Smo-positive cell rate of mice in the P27 group was apparently increased compared to mice in the WT group. Western blot analysis revealed that in terms of changes of protein expression levels of Shh, Patched, Smoothened and Gli2 in the intervertebral disc, protein expression levels of Shh, Patched, Smoothened and Gli2 of mice in the P27 group were significantly increased compared to those of mice in the WT group. The results show that P27 deficiency activates the expression of Shh-signal pathway and promotes the proliferation of osteoblast, thus, playing a role in promoting IVD degeneration, which provides a scientific and reliable experimental basis for the treatment of the IVD degeneration-related diseases in clinical practice.
本研究旨在探讨基因在小鼠椎间盘(IVD)退变中的作用及相关机制。采用X射线、免疫组织化学染色和碱性磷酸酶(ALP)组织化学染色,分析同窝4周龄P27基因敲除(P27)小鼠和野生型(WT)小鼠椎间盘的表型差异。提取椎间盘内蛋白质,采用蛋白质印迹分析检测Shh信号通路中相关分子(包括Shh、Patched、Smoothened和Gli2)表达的变化。结果显示,ALP组织化学染色表明,与WT组同窝4周龄小鼠相比,P27组小鼠的ALP阳性面积明显增加。此外,Col-I免疫组织化学染色显示,与WT组小鼠相比,P27组小鼠的Col-I阳性面积显著增加。而且,与WT组小鼠相比,P27组小鼠的Smo阳性细胞率明显增加。蛋白质印迹分析显示,就椎间盘内Shh、Patched、Smoothened和Gli2的蛋白质表达水平变化而言,P27组小鼠的Shh、Patched、Smoothened和Gli2的蛋白质表达水平与WT组小鼠相比显著增加。结果表明,P27缺乏激活了Shh信号通路的表达并促进成骨细胞增殖,从而在促进IVD退变中发挥作用,这为临床治疗IVD退变相关疾病提供了科学可靠的实验依据。