摘要
目的建立大鼠膝关节纤维化模型,探讨伴侣素CCT-eta在关节纤维化中的表达及调节作用。方法手术组取8只雄性SD大鼠,通过固定左膝建立关节纤维化模型。对照组为右侧膝关节,未行干预措施。8周后拆除固定测量膝关节活动度,取膝后关节囊以组织块法分离培养成纤维细胞。用qRT-PCR和Western Blot检测成纤维细胞中CCT-eta和α-SMA的mRNA和蛋白质的表达情况。通过成纤维细胞转染siRNA抑制CCT-eta的表达,再用Transwell技术检测成纤维细胞的迁移能力。手术组和对照组关节活动度比较、手术组和对照组、转染组和手术组的qRT-PCR和Transwell细胞迁移实验数据均采用独立样本t检验,P<0.05为有统计学差异。结果手术组固定8周后膝关节伸直角度为(85.7±4.1)°,较对照组(145.7±3.6)°明显降低(t=35.13,P<0.05)。手术组较对照组成纤维细胞CCT-eta的mRNA含量和α-SMA的蛋白质的表达明显升高(t=113.40,P<0.05)。转染组较对照组α-SMA蛋白质的表达受到显著抑制。手术组成纤维细胞较对照组迁移能力明显增强,但转染组中细胞迁移能力受到抑制(t=270.90,P<0.05)。结论 CCT-eta在大鼠膝关节纤维化模型中表达升高,并增强关节囊中成纤维细胞的迁移力,从而促进关节纤维化。
Objective To investigate the expression and regulation of chaperonin containing T- complex polypeptide subunit eta （CCT-eta） in the joint of the rat model of knee arthrofibrosis. Methods Left knees of eight male SD rats were fixed as the experimental group, the right non-immobilized knees of the same rats were set as the control group. After eight weeks immobilization, the internal fixators were removed, then the range of motion of bilateral knees was measured. Fibroblasts were obtained from the posterior joint capsule of the knees. Total mRNA and protein extracted from fibroblasts were subjected to qRT-PCR and Weslern blot assays to determine the relative expression levels of CCT-eta and o^-SMA. CCT- I eta siRNA was employed in fibroblasts from the immobilization group by a transient transfection experiment. The migratory ability of fibroblasts was detected by transwell assay. Two independent samples were compared by independent t-test. Results Compared to the control group （ 145.7 ±3.6 ） °, the full extension of knee in immobilized group （ 85.7 ± 4. 1 ） ° decreased distinctly （ t = 35.13, P 〈 0.05 ）. The mRNA and protein expressions of CCT-eta and α-SMA significantly elevated in the immobilized group compared to the control group （t = 113.40, P 〈 0. 05 ）. The reduction of CCT-eta resulted in a dramatic decrease in both a-SMA mRNA and protein levels. The transwell assay showed that the migration of fibroblasts was higher in immobilized group than in control group, and the migratory ability reduced significantly after transfected by siRNA of CCT-eta （ t = 270. 90, P 〈 0. 05 ）. Conclusion Expression of CCT-eta elevates in the model of knee arthrofibrosis, and it may regulate expression of ct-SMA in fibroblasts, so it may play an important role in arthrofibrosis.
出处
《中华关节外科杂志（电子版）》
CAS
2016年第5期42-47,共6页
Chinese Journal of Joint Surgery(Electronic Edition)
基金
广东省自然科学基金(2016A030313220)
广东省科技计划项目(2016A020215225)
