摘要
目的研究Lin28过表达通过哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对人牙髓干细胞(hDPSCs)增殖和成骨分化的影响。方法通过慢病毒介导稳定转染Lin28基因到hDPSCs中,qRT-PCR检测Lin28相对表达量。CCK-8法检测Lin28过表达对细胞增殖活性的影响;qRT-PCR法检测成骨相关基因碱性磷酸酶(ALP)、骨桥素(OPN)和骨钙素(OCN)的mRNA相对表达水平;Western blot法检测ALP和OPN的蛋白表达水平;茜素红染色检测细胞矿化能力。结果与对照组比较,转染组增殖能力增强(P<0.05);转染组ALP、OPN、OCN mRNA及蛋白表达水平显著低于对照组(P<0.05);茜素红染色结果显示转染组形成的矿化结节大小与数目均显著低于空载体组(P<0.05)。加入mTOR抑制剂雷帕霉素(rapamycin)后,ALP和OPN mRNA表达水平降低(P<0.05)。过表达Lin28的同时加入rapamycin,相比对照组LV-Lin28组,OPN和OCN的表达增强(P<0.05)。结论Lin28过表达可能部分通过mTOR信号通路抑制成骨分化。
Objective To investigate the effects of Lin28 overexpression on the proliferation and osteogenic differentiation of human dental pulp stem cells(hDPSCs)through mTOR signaling pathway.Methods After transfecting lentiviral vectors of Lin28 gene in hDPSCs,the relative expression of Lin28 was detected by Real-time PCR.CCK-8 assay was applied to detect the effect on cell proliferation.qRT-PCR was used to research the expression levels of alkaline phosphatase(ALP),osteopontin(OPN)and osteocalcin(OCN).Western blot assay was processed to investigate the effects on the relative expression levels of ALP and OPN proteins.Alizarin red staining was utilized to detect the mineralized nodules.Results Compared with the control group,the cell proliferation of transfection group was promoted(P<0.05);The mRNA and protein expression levels of ALP,OPN and OCN in transfection group were significantly lower than those in control group(P<0.05),the expression level of ALP apparently decreased after the addition of mTOR inhibitor rapamycin(P<0.05);Alizarin red staining showed that the size and number of mineralized nodules formed in transfection group were markedly declined compared with empty carrier group(P<0.05).Conclusion Overexpression of Lin28 can inhibit the osteogenic differentiation of hDPSCs through suppress mTOR signaling pathway.
作者
邹雨希
孙晶
孙玉
索溪
周文凯
杨建光
刘岩
Zou Yuxi;Sun Jing;Sun Yu;Suo Xi;Zhou Wenkai;Yang Jianguang;Liu Yan(Xuzhou Medical University,Xuzhou 221004;School and Hospital of Stomatology,Cheeloo College of Medicine,Shandong University,Jinan 250014;Affiliated Stomatological Hospital of Xuzhou Medical University,Xuzhou 221002)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第9期1507-1513,共7页
Acta Universitatis Medicinalis Anhui
基金
江苏省高等学校基础科学研究面上省资助项目(编号:22KJB320024)
徐州市重点研发计划项目(编号:KC22218)。
