Su Ying, Wang Feng, Qi Hu, Zhao Shi Guang, Li Xue, Cui Hao
Department of Ophthalmology, First Clinic College of Harbin Medical University, Harbin, China.
Mol Vis. 2011 Jan 22;17:247-56.
Fibroblast cell proliferation is major reason for recurrence of pterygia. In the present study, we investigated if small interfering RNA (siRNA)-mediated gene silencing of S phase-kinase-interacting protein 2 (Skp2) can be employed to inhibit protein 27 kinase inhibition protein 1 (p27(kip1)) down-regulation in pterygium fibroblast cells (PFC) in vitro and in vivo.
A plasmid containing transgenes encoding Skp2 siRNA was used to decreasing the high constitutive levels of Skp2 protein in PFC and normal fibrboblast cells (NFC) in vitro and in vivo which can lead to consequent degradation of p27(kip1). Cell proliferation and viability were investigated using cell counts, 59-bromodeoxyuridine incorporation (BrdU assay) and tetrazolium reduction (MTT assay).
Infection of PFC and NFC with Skp2 siRNA resulted in significant inhibition of cell proliferation and metabolic activity in vitro. Immunoflurescence showed decreased levels of Skp2 and increased levels of p27(kip1) in pSkp2 siRNA infected cells, but not in plasmid and uninfected cells.
Skp2 siRNA inhibited the cell proliferation of PFC in vitro and in vivo.
成纤维细胞增殖是翼状胬肉复发的主要原因。在本研究中，我们调查了小干扰RNA（siRNA）介导的S期激酶相关蛋白2（Skp2）基因沉默是否可用于在体外和体内抑制翼状胬肉成纤维细胞（PFC）中蛋白27激酶抑制蛋白1（p27(kip1)）的下调。
使用含有编码Skp2 siRNA转基因的质粒，在体外和体内降低PFC和正常成纤维细胞（NFC）中Skp2蛋白的高组成水平，这可能导致随后p27(kip1)的降解。使用细胞计数、5-溴脱氧尿苷掺入（BrdU测定）和四唑盐还原（MTT测定）研究细胞增殖和活力。
用Skp2 siRNA感染PFC和NFC导致体外细胞增殖和代谢活性受到显著抑制。免疫荧光显示，在感染pSkp2 siRNA的细胞中Skp2水平降低，p27(kip1)水平升高，但在质粒转染细胞和未感染细胞中未出现此现象。
Skp2 siRNA在体外和体内均抑制了PFC的细胞增殖。
