微重力环境下Smads信号通路对人牙周膜干细胞成骨向分化的影响

目的:了解Smads信号通路在模拟微重力条件下对人牙周膜细胞成骨向分化的影响。方法:自手术拔除的人牙周膜中培养获得牙周膜细胞,利用有限稀释法培养获得人牙周膜细胞(human periodontal ligament cells,hPDLCs)。采用旋转细胞培养系统(rotary cell culture system,RCCS)建立模拟微重力环境,将细胞分为对照组(正常重力组)、模拟微重力组,应用实时定量PCR检测Smads信号分子表达及加入Smads抑制剂后成骨标志物的表达,流式细胞仪检测磷酸化Smad表达。采用SPSS13.0软件包对数据进行统计学处理。结果:与对照组相比,模拟微重力组Smads 2、3、4 mRNA表达量显著增加,呈时间依赖性,在2h达到峰值,随后开始下降。Smads7在2h时开始上升,观测时间内未捕捉到其下降。流式细胞检测发现,p-Smads在30min时开始出现高表达(29.39%),2h时达到峰值,表达量为91.32%。加入Smads抑制剂后,p-Smads表达下降至5.3%,成骨标志物COL1、ALP、OCN表达显著下降(P<0.05)。结论:模拟微重力环境下,Smads信号通路参与了hPDLSCs成骨向分化。

The effect of Smads signal pathway on the osteogenesis of human periodontal ligament stem cells in simulated microgravity

PUPOSE: To investigate the effect of Smads signal pathway on the osteogenesis of human periodontal ligament stem cells(hPDLSCs) in simulated microgravity.METHODS: Human periodontal ligament stem cells were isolated from the ligament of surgically extracted human teeth.Through limiting dilution assay,mono-clone of the cell was obtained,hPDLSCs were isolated from MesenPRO RS medium.Rotary cell culture system(RCCS) was enrolled to set simulated microgravity(SMG).Samples were set to control group(normal gravity group,NG) and simulated microgravity group(SMG).Real-time PCR was used to detect the expression of Smads signals and the expression of markers of osteogenesis before and after SIS3.The amount of phosphated-Smad was assayed by flow cytometry.Statistical analysis of the data was done by ANOVA with SPSS 13.0 software package.RESULTS: Compared with that of the control group,the expression of Smads 2,3,4 was significantly higher in SMG group(P<0.05)in a time-dependent manner.Flow cytometry assay showed increased expression of p-Smads at 30-min,and peak expression was observed at 2h,reaching at 91.32%.The addition of SIS3 led to significant decrease of COL I,ALP,OCN and p-Smads(P<0.05).CONCLUSION: Smads signal pathway was enrolled in the osteogenesis of hPDLSCs in simulated microgravity.