层粘连蛋白α3在膝关节纤维化发生发展中的作用研究

Study on laminin α3 in the development of knee fibrosis

目的:观察层粘连蛋白α3(LAMA3)在膝关节纤维化发生发展中的作用并探讨其作用机制。方法:制备兔膝关节纤维化动物模型,HE染色和Masson染色观察术区标本纤维化发展情况,免疫组化检测标本中LAMA3的表达。体外培养成纤维细胞,进行慢病毒转染,构建LAMA3过表达稳定细胞株。EdU染色观察LAMA3对成纤维细胞增殖的影响,Western blot检测LAMA3对细胞增殖相关蛋白的作用;Tunel染色观察LAMA3对成纤维细胞凋亡的影响,Western blot检测LAMA3对细胞凋亡相关蛋白的作用;Western blot检测成纤维细胞过表达LAMA3基因后,Wnt/β-catenin信号通路蛋白Wnt3a、p-GSK、β-catenin的表达;EdU染色观察Wnt/β-catenin信号通路抑制剂XAV-939处理后细胞增殖能力的变化。结果:动物实验结果显示,随着术后时间的延长,膝关节纤维化程度加重,LAMA3表达随之增加。细胞实验结果显示,成纤维细胞过表达LAMA3后,细胞增殖能力增强,增殖相关蛋白PCNA和Cyclin D1表达增加。Tunel染色显示,成纤维细胞过表达LAMA3后细胞凋亡数量减少,且凋亡相关蛋白cleaved-PARP、Bax表达下调,而凋亡抑制蛋白Bcl-2表达上调。成纤维细胞过表达LAMA3后,Wnt3a、p-GSK、β-catenin蛋白表达上调,Wnt/β-catenin信号通路抑制剂XAV-939处理后,LAMA3促进成纤维细胞增殖的作用明显减弱。结论:LAMA3可以促进成纤维细胞增殖,抑制其凋亡,从而促进膝关节纤维化,Wnt3a/β-catenin信号通路参与成纤维细胞增殖调控过程。

Objective: To observe the role of laminin α3 in the development of knee joint fibrosis and explore its possible mechanism. Methods: The animal model of knee joint fibrosis was prepared, and the development of fibrosis and LAMA3 expression in animal specimens were observed by HE and Masson. Fibroblasts were cultured in vitro and transfected with lentivirus to construct LAMA3 overexpressed stable cells. EdU staining and western-blot were used to detect proliferation-related protenins to observe the effect of LAMA3 on the proliferation of fibroblasts. Tunel staining and western-blot were used to detect apoptosis-related proteins to observe the effect of LAMA3 on apoptosis of fibroblasts. Finally,the protein expression of wnt/β-catenin signaling pathway after LAMA3 overexpression was detected, and wnt/β-catenin signaling pathway inhibitor XAV-939 was added to observe the changes of cell proliferation ability. Results: Animal experimental results showed that with the prolongation of postoperative time, the degree of fibrosis after knee joint surgery became worse, and LAMA3 expression also increased. Cytological experiment results showed that overexpression of LAMA3enhanced the DNA synthesis ability of fibroblasts, and increased the expression of proliferation-related proteins PCNA and Cyclin D1. Tunel staining results showed that the number of apoptotic cells decreased, and western-blot results also showed that apoptosis related protein cleaved-PAPR and Bax expression decreased, while Bcl-2 expression increased. After overexpression of LAMA3, the expression of wnt/β-catenin signaling pathway proteins Wnt3a, P-GSK and β-catenin increased. After addition of wnt/β-catenin signaling pathway inhibitor XAV-939, EdU staining showed that the promotion of DNA synthesis of fibroblasts by overexpression of LAMA3 was partially reversed. Conclusion: LAMA3 can promote the proliferation of fibroblasts and inhibit their apoptosis to improve knee fibrosis, and wnt3a/β-catenin signaling pathway is involved in the regulation of fibroblast proliferation.