siRNA沉默Rsk2基因表达抑制人牙周膜细胞的增殖及成骨分化

doi:10.19439/j.sjos.2017.05.007

摘要/Abstract

摘要： 目的研究核糖体S6激酶2（ribosomal S6 kinase,Rsk2）基因对人牙周膜细胞(human periodontal ligament cells,hPDLCs) 增殖及成骨分化的影响及其作用机制。方法收集牙科手术拔除的前磨牙,分离其牙周韧带并进行hPDLCs原代培养,取第4~6代细胞用于实验。采用RT-PCR 和Western 印迹法检测小干扰RNA(siRNA)对hPDLCs内Rsk2的沉默效率,MTT法检测Rsk2 siRNA对细胞增殖的影响,碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测ALP活性变化。p38MAPK信号通路抑制剂SB203580处理转染后的hPDLCs,Western 印迹法检测Rsk2 siRNA对MAPK信号通路p38蛋白磷酸化的影响。RT-PCR和Western 印迹法检测成骨分化标志分子Runt相关转录因子2 (Runt-related transcription factor-2,Runx2)、骨钙蛋白(osteocalcin,OCN) 和成骨蛋白BMP2的表达情况,采用SPSS18.0R软件包对数据进行统计学分析。结果hPDLCs转染Rsk2 siRNA后,Rsk2表达下调,差异显著（P<0.05）。Rsk2 siRNA 显著降低p38蛋白磷酸化（P<0.05）,抑制hPDLCs增殖（P<0.05）,降低ALP活性（P<0.01）,成骨分化标志分子Runx2、OCN和BMP2的表达降低,差异具有显著性。SB203580处理Rsk2 siRNA转染后的hPDLCs发现,细胞增殖、ALP活性、Runx2、OCN和BMP2的表达较Rsk2 siRNA转染组相比,均有显著差异。结论Rsk2 siRNA通过p38MAPK信号通路抑制hPDLCs 增殖和成骨分化。

关键词: Rsk2, 人牙周膜细胞, siRNA, 增殖, 成骨分化

Abstract: PURPOSE: To study the effect of ribosomal S6 kinase (Rsk2) gene on proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) and underlying mechanism. METHODS: Premolar surgically extracted were collected, the periodontal ligament was separated and hPDLCs were primarily cultured. Cells of 4 to 6 passage were used in the experiment. The silencing efficiency of small interfering RNA (siRNA) on Rsk2 in hPDLCs was detected by RT-PCR and Western blot. MTT assay was used to detect the effect of Rsk2 siRNA on cell proliferation. Alkaline phosphatase (ALP) kit was used to detect ALP activity. P38MAPK signal pathway inhibitor SB203580 was used to detect hPDLCs after transfection. Western blot was used to detect the effect of Rsk2 siRNA on MAPK signaling pathway p38 protein phosphorylation. The expressions of Runt-related transcription factor-2 (Runx2), osteocalcin (OCN) and osteogenic protein BMP2 were detected by RT-PCR and Western blot. The data were analyzed using SPSS18.0 software package. RESULTS: The expression of Rsk2 was down-regulated by hPDLCs transfected with Rsk2 siRNA, the difference was statistically significant (P<0.05). Rsk2 siRNA significantly reduced phosphorylation of p38 protein (P<0.05), inhibition of hPDLCs proliferation (P<0.05), decreased ALP activity (P<0.01); the expression of Runx2, OCN and BMP2 was different, and the difference was statistically significant (P<0.05). After SB203580 treatment, hPDLCs transfected with Rsk2 siRNA showed increased cell proliferation, ALP activity, Runx2, OCN and BMP2 expression; compared with Rsk2 siRNA transfected hPDLCs, the difference was statistically significant. CONCLUSIONS: Rsk2 siRNA inhibits hPDLCs proliferation and osteogenic differentiation through p38MAPK signaling pathway.

Key words: Rsk2, Human periodontal ligament cells, siRNA, Proliferation, Osteogenic differentiation

中图分类号:

R781.4

杜莉, 曹伟靖, 王原明. siRNA沉默Rsk2基因表达抑制人牙周膜细胞的增殖及成骨分化[J]. 上海口腔医学, 2017, 26(5): 498-503.

DU Li, CAO Wei-jing, WANG Yuan-ming. SiRNA silencing Rsk2 gene expression inhibits proliferation and osteogenic differentiation of human periodontal ligament cells[J]. Shanghai Journal of Stomatology, 2017, 26(5): 498-503.

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