Impact of tissue inhibitor of matrixmetalloprotease-1 (TIMP-1) protein on the proliferation and gene expression of rabbit Achilles tenocytes and rabbit adipose-derived stem cells in vitro

In order to know the effect of TIMP-1 on in vitro cell culture of rabbit Achilles tenocytes and rabbit adipose-derived stem cells, we assessed their proliferation with Alamar Blue® assay and furthermore report the effect on gene expression of Col1A1 (extracellular matrix marker collagen I), MKI67 (proliferation marker ki67), TNMD (tenomodulin) and ALP (alkaline phosphatase, a marker for early osteogenesis), respectively, the latter because TIMP-1 has been reported to affect stem cell fate towards osteogenic commitment. As reference gene, 18S was utilized. These experiments have been performed at different concentrations of TIMP-1 supplementation; i.e. at 1, 10, and 100 ng/mL TIMP-1. The same experiment without TIMP-1 served as control.为探究基质金属蛋白酶组织抑制剂-1（TIMP-1）对兔跟腱肌腱细胞与兔脂肪来源干细胞体外培养的影响，本研究采用Alamar Blue®分析法检测两类细胞的增殖水平，并进一步分别分析两类细胞中Col1A1（细胞外基质标志物Ⅰ型胶原，collagen I）、MKI67（增殖标志物Ki67）、TNMD（腱调蛋白，tenomodulin）以及ALP（碱性磷酸酶，alkaline phosphatase，早期成骨标志物）的基因表达变化；其中选择ALP作为检测靶点的原因在于，已有研究报道TIMP-1可调控干细胞向成骨细胞分化。本研究以18S作为内参基因。 本研究设置了1、10与100 ng/mL三个TIMP-1添加浓度梯度，以未添加TIMP-1的平行实验体系作为空白对照组。