MIP-1α对人牙周膜干细胞骨向分化的影响及其机制  

Effect and mechanism of MIP-1αon osteo-differentiation of human periodontal ligament stem cells

作　　者：王钊鑫 李淑慧[1] 代慧娟 齐鲁[1] 刘紫薇 尼加提·吐尔逊[1] Wang Zhao-Xin;Li Shu-Hui;Dai Hui-Juan;Qi Lu;Liu Zi-Wei;Negati Tursun(Department of Stomatology,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang 830011,China;Graduate School of Xinjiang Medical University,Urumqi,Xinjiang 830011,China)

机构地区：[1]新疆医科大学第二附属医院口腔科,新疆乌鲁木齐830011 [2]新疆医科大学研究生院,新疆乌鲁木齐830011

出　　处：《解放军医学杂志》2023年第3期283-291,共9页Medical Journal of Chinese People's Liberation Army

基　　金：新疆医科大学研究生科研创新项目(CXCY2022023);新疆维吾尔自治区自然科学基金(2019D01C231)。

摘　　要：目的 探讨巨噬细胞炎性蛋白-1α(MIP-1α)对人牙周膜干细胞(hPDLSCs)增殖、迁移、骨向分化的影响及其可能的机制。方法 收集2020年11月-2021年10月在新疆医科大学第二附属医院口腔科门诊就诊的12~25岁患者拔除的健康牙(正畸减数前磨牙或阻生的第三磨牙)共16颗,组织块法联合酶消化法培养原代干细胞,并采用流式细胞术鉴定细胞表型。(1)细胞生物学特性实验:第3代hPDLSCs分为0(对照组)、1、10μg/ml MIP-1α组,各组再分别加入含体积分数为10%胎牛血清、100 U/ml青链霉素、2 mmol/L谷氨酰胺的α-MEM培养基,干预24、48、72 h后采用CCK-8法检测各组细胞增殖能力,干预24 h后采用划痕实验检测各组细胞横向迁移能力。(2)对骨向分化的影响及可能的机制:第3代hPDLSCs分为0(对照组)、1、10μg/ml MIP-1α组,分别加入成骨诱导液,干预7 d后采用碱性磷酸酶(ALP)染色及半定量分析、干预14 d后采用茜素红染色及半定量分析检测细胞成骨能力;干预7 d后采用qRT-PCR和Western blotting检测成骨相关基因Runt相关转录因子2(Runx2)、骨桥蛋白(OPN)、转录因子SP7(Osterix)及Notch信号通路相关分子Notch1受体、Jagged1配体及下游因子Hey1的mRNA及蛋白表达情况。结果 流式细胞术检测结果显示,hPDLSCs STRO-1、CD146呈阳性表达,CD34呈阴性表达。(1)细胞生物学特性实验中,CCK-8法结果显示,与对照组比较,24、48 h时1、10μg/mlMIP-1α组hPDLSCsOD值差异无统计学意义(P>0.05);72 h时,与对照组比较,1μg/mlMIP-1α组hPDLSCs OD值差异无统计学意义(P>0.05),而10μg/mlMIP-1α组hPDLSCsOD值明显增高(P<0.05)。划痕实验结果显示,与对照组比较,1μg/ml MIP-1α组细胞划痕愈合率差异无统计学意义(P>0.05),而10μg/ml MIP-1α组划痕愈合率明显增高(P<0.05)。(2)对骨向分化的影响及可能的机制实验中,ALP染色及半定量结果显示,1、10μg/mlMIP-1α组ALP活性均明显低于对照组(P<0.05)。茜素�Objective To investigate the effect of macrophage inflammatory protein-1α(MIP-1α)on proliferation,migration and osteo-differentiation of human periodontal stem cells(hPDLSCs)and its possible mechanism.Methods A total of 16 healthy teeth(orthodontic minus premolar or blocked third molar)extracted from patients aged 12 to 25 years attending the outpatient clinic of the Department of Stomatology,the Second Affiliated Hospital of Xinjiang Medical University from November 2020 to October 2021 were collected and primary stem cells were cultured by tissue block method combined with enzymatic digestion,and the cell phenotype was identified by flow cytometry.(1)Cell biological characteristics experiment:the 3rd generation hPDLSCs were divided into 0(control group),1 and 10μg/ml MIP-1αgroups,and each group was then added withα-MEM medium containing volume fraction of 10%fetal bovine serum,100 U/ml penicillin,and 2 mmol/L glutamine,respectively,and the proliferation ability of each group was detected by CCK-8 method after 24,48 and 72 h of intervention.The lateral migration ability of cells in each group was detected by scratch assay after 24 h of intervention.(2)Effect and possible mechanism of osteo-differentiation:3rd generation hPDLSCs were divided into 0(control group)1 and 10μg/ml MIP-1αgroups,and osteogenic induction solution was added to each group,and osteogenic ability of cells was detected by alkaline phosphatase(ALP)staining and semi-quantitative analysis 7 d after intervention and by alizarin red staining and semi-quantitative analysis 14 d after intervention;osteogenic ability of cells was detected by qRT-PCR and Western blotting.The mRNA and protein expression of osteogenic genes Runt-related transcription factor 2(Runx2),bone bridge protein(OPN)and transcription factor SP7(Osterix)and Notch1 receptor,Jagged 1 ligand and downstream factor Hey1 were detected by qRT-PCR and Western blotting 7 d after intervention.Results Flow cytometry results showed that hPDLSCs STRO-1 and CD146 showed positive expressi

关 键 词：人牙周膜干细胞 NOTCH信号通路 巨噬细胞炎性蛋白-1Α 成骨分化 

分 类 号：R781.4[医药卫生—口腔医学]