周期性牵张应力作用下KLF5对人牙周膜细胞增殖和成骨分化的影响

KLF5 modulates proliferation and osteogenic differentiation of human periodontal ligament cells subjected to cyclic tensile stress

目的 :探讨周期性牵张应力(cyclic tensile stress,CTS)作用下Kruppel样转录因子5(Kruppel-like factor 5,KLF5)在人牙周膜细胞(human periodontal ligament cells,h PDLCs)中的表达及其对h PDLCs增殖和成骨分化的影响。方法:酶解组织块法体外培养h PDLCs,对其施加形变率10%,频率0.5 Hz的周期性牵张应力1、4、8、12、24 h,采用RT-PCR和Western印迹法检测细胞中KLF5 m RNA和蛋白的表达。转染si RNA(si-KLF5)至h PDLCs沉默KLF5表达,RT-PCR检测KLF5 m RNA的表达。同时,将过表达碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF、FGF2)的pc DNA3.1-FGF2转染至稳定转染si-KLF5的h PDLCs,加力8 h后,以CCK8法检测各组细胞的增殖活性,碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测ALP活性,RT-PCR检测成骨分化因子Runx2和Osterix的m RNA表达,Western印迹法检测Runx2、Osterix、FGF2、GSK-3β、P-GSK-3β(ser 9)、β-catenin蛋白的表达。采用SPSS22.0软件包对数据进行统计学分析。结果 :10%CTS刺激呈时间依赖性地诱导h PDLCs中KLF5 m RNA和蛋白表达。si-KLF5转染可显著抑制10%CTS诱导的h PDLCs的增殖,降低其ALP活性,减少Runx2和Osterix的m RNA和蛋白表达,并抑制FGF2-GSK-3β/β-catenin信号通路的激活;而过表达FGF2可以部分逆转沉默KLF5对h PDLCs增殖和成骨分化的抑制效应。结论 :周期性牵张应力作用下,KLF5通过FGF2-GSK-3β//β-catenin信号通路影响人牙周膜细胞的增殖和成骨分化。

PURPOSE： To investigate the expression of Kruppel-like factor 5（KLF5） in human periodontal ligament cells（h PDLCs） under cyclic tensile stress（CTS） and its effect on proliferation and osteogenic differentiation of h PDLCs.METHODS： h PDLCs were primarily cultured in vitro by tissue explant and enzyme digestion method. RT-PCR and Western blot were used to detect m RNA and protein expression of KLF5 in h PDLCs stimulated by 10% CTS at a frequency of 0.5 Hz for 1, 4, 8, 12 and 24 h, respectively.KLF5 was then knocked-down in h PDLCs using si RNA（siKLF5） and its m RNA expression was determined by RT-PCR. Meanwhile, basic fibroblast growth factor（FGF2） was overexpressed in KLF-5 silenced h PDLCs by using pc DNA3.1-FGF2. After stimulation with CTS for 8 h, the proliferation of h PDLCs was assayed by CCK-8 kit, alkaline phosphatase（ALP） activity was assayed by ALP kit, the m RNA expression of Runx2 and Osterix was examined by RT-PCR, and the protein expression of Runx2, Osterix, FGF2, GSK-3β, P-GSK-3β（ser 9）, β-catenin was determined by Western blot. The data were analyzed using SPSS 22.0 software package.RESULTS： Stimulation with 10% CTS time-dependently induced m RNA and protein expression of KLF5 in h PDLCs.Transfection of si-KLF5 significantly reduced the proliferation of h PDLCs, decreased ALP activity and expression of Runx2 and Osterix in h PDLCs stimulated by 10% CTS. Simultaneously, CTS-induced activation of FGF2-GSK-3β/β-catenin signaling pathway was also inhibited by si-KLF5 transfection. However, overexpression of FGF2 can partly neutralized the inhibitiory effect of si-KLF5 on proliferation and osteogenic differentiation of h PDLCs subjected to 10%CTS. CONCLUSIONS： KLF5 promotes proliferation and osteogenesis differentiation of h PDLCs subjected to cyclic tensile stress through FGF2-GSK-3β/β-catenin signaling pathway.