摘要
该文旨在探讨小檗碱(berberine)对人牙周膜干细胞(hPDLSCs)增殖和成骨分化的影响及潜在的机制.体外培养hPDLSCs,将其分为空白对照(Con)组、PI3K/AKT信号通路抑制剂LY294002(LY)组、小檗碱(Ber)组和小檗碱+PI3K/AKT信号通路抑制剂LY294002(Ber+LY)组,采用细胞计数试剂盒-8(CCK-8)法检测hPDLSCs的增殖活力,流式细胞仪检测细胞周期分布,酶联免疫检测仪检测碱性磷酸酶(ALP)活性,实时荧光定量PCR(qRT-PCR)分析增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)、骨钙蛋白(OCN)、骨膜蛋白(POSTN)和骨桥蛋白(OPN)的表达情况,蛋白免疫印迹法(Western blot)检测PCNA、Cyclin D1、OCN、POSTN、OPN和PI3K/AKT信号通路相关蛋白的表达情况.与Con组相比,Ber组hPDLSCs增殖活力,ALP的活性,S期和G2/M期细胞比例,PCNA、Cyclin D1、OCN、POSTN、OPN、p-PI3K和p-AKT的表达水平均显著升高,G0/G1期细胞比例显著降低(P＜0.05),LY组上述指标呈相反变化(P＜0.05);与Ber组相比,Ber+LY组hPDLSCs增殖活力,ALP的活性,S期和G2/M期细胞比例,PCNA、Cyclin D1、OCN、POSTN、OPN、p-PI3K和p-AKT的表达水平均显著降低,G0/G1期细胞比例显著升高(P＜0.05).小檗碱可通过激活PI3K/AKT信号通路促进hPDLSCs增殖,并诱导hPDLSCs成骨分化.
Abstract
This study aims to investigate the effect and potential mechanism of berberine on the pro-liferation and osteogenic differentiation of hPDLSCs(human periodontal ligament stem cells).hPDLSCs were cultured in vitro,and were divided into blank Con(control)group,LY(PI3K/AKT signaling pathway inhibi-tor LY294002)group,Ber(berberine)group and Ber+LY(berberine+PI3K/AKT signaling pathway inhibitor LY294002)group.CCK-8(cell counting kit-8)method was used to detect the proliferation activity of hPDLSCs.Flow cytometry was used to detect cell cycle distribution.Enzyme-linked immunosorbent assay was used to de-tect ALP(alkaline phosphatase)activity.qRT-PCR(real-time fluorescent quantitative PCR)was used to analysis the expression of PCNA(proliferating cell nuclear antigen),Cyclin D1,OCN(osteocalcin),POSTN(periostin)and OPN(osteopontin).Western blot was used to detect the expression of PCNA,Cyclin D1,OCN,POSTN,OPN and PI3K/AKT signaling pathway-related proteins.Compared with the Con group,the proliferation activ-ity of hPDLSCs in the Ber group,ALP activity,S phase and G2/M phase cell ratio,PCNA,Cyclin D1,OCN,POSTN,OPN,p-PI3K and p-AKT expression levels were significant increased,the proportion of cells in G0/G1 phase was significantly reduced(P＜0.05),while the above indicators in the LY group showed opposite changes(P＜0.05).Compared with the Ber group,the proliferation activity of hPDLSCs in the Ber+LY group,the activity of ALP,the ratio of cells in S phase and G2/M phase,the expression levels of PCNA,Cyclin D1,OCN,POSTN,OPN,p-PI3K and p-AKT were significantly reduced,and the proportion of cells in G0/G1 phase was significantly increased(P＜0.05).Berberine can promote the proliferation of hPDLSCs and induce the osteogenic differentia-tion of hPDLSCs by activating the PI3K/AKT signaling pathway.
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