DU Li, Cao Wei-Jing, Wang Yuan-Ming
Department of Stomatology, Affiliated Hospital of Yan'an University. Yan'an 716000, Shaanxi Province, China. E-mail:
Shanghai Kou Qiang Yi Xue. 2017 Oct;26(5):498-503.
To study the effect of ribosomal S6 kinase (Rsk2) gene on proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) and underlying mechanism.
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.
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.
Rsk2 siRNA inhibits hPDLCs proliferation and osteogenic differentiation through p38MAPK signaling pathway.
研究核糖体S6激酶（Rsk2）基因对人牙周膜细胞（hPDLCs）增殖和成骨分化的影响及其潜在机制。
收集外科拔除的前磨牙，分离牙周膜并原代培养hPDLCs。实验采用4至6代细胞。通过RT-PCR和蛋白质免疫印迹法检测小干扰RNA（siRNA）对hPDLCs中Rsk2的沉默效率。采用MTT法检测Rsk2 siRNA对细胞增殖的影响。使用碱性磷酸酶（ALP）试剂盒检测ALP活性。转染后用P38丝裂原活化蛋白激酶（MAPK）信号通路抑制剂SB203580处理hPDLCs。采用蛋白质免疫印迹法检测Rsk2 siRNA对MAPK信号通路p38蛋白磷酸化的影响。通过RT-PCR和蛋白质免疫印迹法检测与 runt 相关的转录因子2（Runx2）、骨钙素（OCN）和成骨蛋白BMP2的表达。使用SPSS18.0软件包分析数据。
转染Rsk2 siRNA的hPDLCs使Rsk2表达下调，差异具有统计学意义（P<0.05）。Rsk2 siRNA显著降低p38蛋白磷酸化水平（P<0.05），抑制hPDLCs增殖（P<0.05），降低ALP活性（P<0.01）；Runx2、OCN和BMP2的表达也有差异，且差异具有统计学意义（P<0.05）。SB203580处理后，转染Rsk2 siRNA的hPDLCs细胞增殖、ALP活性、Runx2、OCN和BMP2表达增加；与转染Rsk2 siRNA的hPDLCs相比，差异具有统计学意义。
Rsk2 siRNA通过p38MAPK信号通路抑制hPDLCs增殖和成骨分化。
