肌腱是体内主要的承重组织之一;承受巨大的峰值应力,因此容易受伤。肌腱损伤的细胞反应很复杂,涉及炎症和修复成分,后者同时雇用居民和招募的外源细胞群。基因表达分析是研究肌腱损伤的有价值的工具,允许评估修复过程和病理反应,如纤维化,并允许评估治疗性药理学干预措施。定量聚合酶链反应(qPCR)是此类研究的常用方法,但是通过这种方法获得的数据必须标准化为参考基因:已知在所研究的实验条件之间稳定表达的基因。因此,建立合适的肌腱损伤参考基因至关重要。因此,我们研究了肌腱损伤大鼠模型中mRNA表达的稳定性,比较受伤和未受伤的肌腱,以及雷帕霉素治疗的效果,在受伤后1周和3周。我们使用了11个候选基因(18S,ACTB,AP3D1,B2M,CSNK2A2,GAPDH,HPRT1,PAK1IP1,RPL13a,SDHA,UBC),并通过四种互补算法(Bestkeeper,deltaCt,geNorm,Normfinder)。我们的结果表明,ACTB,CSNK2A2、HPRT1和PAK1IP1均在肌腱中稳定表达,无论损伤或药物治疗:这些中的任何三个将作为普遍适用的参考基因组,用于在大鼠肌腱损伤模型中标准化qPCR表达数据。我们还揭示了18S,UBC,GAPDH,和SDHA一直是得分较差的候选人(后两者表现出与雷帕霉素和损伤相关的变化,分别):应避免这些基因。
Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.