模拟炎症微环境下紫草素对人牙周膜干细胞增殖和成骨分化的影响  

Effect of Shikonin on proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)under simulated inflammatory microenvironment

作　　者：陈意磊 于倩[1,2] 张文杰 赵今 CHEN Yilei;YU Qian;ZHANG Wen Jie;ZHAO Jin(Department of Cariology and Endodontics,The First Affiliated Hospital of Xinjiang Medical University/Affiliated Stomatology Hospital;Institute of Stomatology of Xinjiang Uygur Autonomous Region,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院(附属口腔医院)牙体牙髓病科 [2]新疆维吾尔自治区口腔医学研究所,乌鲁木齐830054

出　　处：《新疆医科大学学报》2024年第4期480-487,493,共9页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区“天山英才”科技领军创新人才项目(2023TSYCLJ0032);新疆医科大学第一附属医院“青年科研起航”专项基金(2022YFY-QKQN-39)。

摘　　要：目的探究紫草素在牙龈卟啉单胞菌脂多糖诱导的炎症微环境下对人牙周膜干细胞增殖和成骨分化的影响。方法从健康人牙周膜组织中分离培养人牙周膜干细胞,采用流式细胞术鉴定细胞表面标志物CD29、CD105、CD34、CD45。CCK-8法检测不同浓度紫草素对正常及模拟炎症微环境下人牙周膜干细胞增殖活性的影响:首先筛选出紫草素促进增殖人牙周膜干细胞作用的适宜浓度范围,再取牙周膜干细胞进行后续实验分组:空白组(单纯培养基)、紫草素组(实验药物浓度紫草素)、LPS组(牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,P.g-LPS,10μg/mL)、紫草素+LPS组(实验药物浓度紫草素+P.g-LPS)。3 d时采用酶联免疫吸附试验检测细胞上清液中IL-6、IL-18水平;7 d时进行碱性磷酸酶染色及碱性磷酸酶活性测试、21 d时进行茜素红染色检测成骨的分化能力;168 h时进行RT-PCR检测骨钙素、RUNT相关转录因子2、碱性磷酸酶等成骨基因的表达。结果分离培养的人牙周膜干细胞主要呈束状长梭形,流式鉴定结果表面标志物CD29(99.91%)、CD105(92.34%)、CD34(0.76%)、CD45(0.32%),证实分离细胞符合干细胞特性。CCK-8检测结果显示:正常状态下,与空白组相比,紫草素0.0625、0.125、0.25、0.5μmol/L组人牙周膜干细胞相对细胞活力升高(P<0.05);模拟炎症环境下,紫草素0.5μmol/L组人牙周膜干细胞活性最好(P<0.05)。同时与LPS组相比,0.5μmol/L浓度的紫草素可促进炎症状态下细胞迁移,降低炎症状态下IL-6、IL-18的水平(P<0.05),对炎症状态下人牙周膜干细胞成骨分化作用明显,可以增强炎症状态下骨钙素、RUNT相关转录因子2、碱性磷酸酶等成骨基因的表达(P<0.05)。结论模拟炎症微环境下,适当浓度的紫草素可降低炎症环境下IL-6、IL-18水平,同时具有一定促进人牙周膜干细胞增殖、迁移和成骨分化的作用,可作为慢性牙周炎防治�Objective To investigate the effects of Shikonin on the proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)in a lipopolysaccharide-induced inflammatory microenvironment.Methods hPDLSCs were isolated and cultured from healthy human periodontal tissues,andthe cell surface markers CD29,CD105,CD34and CD45 were identified by flow cytometry.CCK-8 method was used to detect the effects of different concentrations of Shikonin on the proliferative activity of hPDLSCs in normal and simulated inflammatory microenvironments.Firstly,the suitable concentration range of Shikonin to promote the proliferation of human periodontal stem cells were screened out.And then periodontal stem cells were taken for subsequent experiments:blank group(simple medium),Shikonin group(experimental drug concentration Shikonin),LPS group(Porphyromonas gingivalis lipopolysaccharide,P.g-LPS,10μg/mL)and Shikonin+LPS group(experimental drug concentration Shikonin+P.g-LPS).The levels of IL-6 and IL-18 in the cell supernatant were detected by enzyme-linked immunosorbent assay at 3 d;alkaline phosphatase staining and alkaline phosphatase activity test was performed at 7 d;and alizarin red staining was performed to detect the differentiation ability of osteogenic bone at 21 d;and RT-PCR was performed to detect the expression of osteogenic genes,such as osteocalcin,RUNT-associated transcription factor 2,and alkaline phosphatase,at 168 h.Results Isolated cultured hPDLSCs were mainly bundled long shuttle-shaped.Flow cytometry identified the results of surface markers CD29(99.91%),CD105(92.34%),CD34(0.76%)and CD45(0.32%).The results confirmed that the isolated cells conformed to stem cell characteristics.The results of CCK-8 assay showed that the relative cell viability of hPDLSCs was elevated in the 0.0625,0.125,0.25 and 0.5μmol/L groups of Shikonin compared with blank group under normal state(P<0.05);and that the activity of hPDLSCs in the 0.5μmol/L group of Shikonin was the best under the simulated inflammatory e

关 键 词：紫草素 慢性牙周炎 牙周膜干细胞 成骨分化 

分 类 号：R781.4[医药卫生—口腔医学]