miR-138-5p在微炎症环境中对人牙髓干细胞成牙本质向分化作用研究

Study on the role of miR-138-5p on odontogenic differentiation of human dental pulp stem cells by microinflammatory state

目的 研究miR-138-5p在微炎症环境对人牙髓干细胞(human dental pulp stem cells,hDPSCs)成牙本质向分化过程中的作用。方法 研究于2021年5月至2022年3月在中国医科大学口腔医学院中心实验室进行。原代培养并分离细胞,通过流式细胞术检测细胞表面间充质干细胞表面标记分子CD29、CD105、CD146及造血干细胞表面标记分子CD14、CD45的细胞阳性率,碱性磷酸酶(alkaline phosphatase,ALP)染色鉴定是否为hDPSCs及其成骨分化能力。根据ALP染色结果确定提高h DPSCs ALP活性的脂多糖(lipopolysaccharide,LPS)有效质量浓度,按照hDPSCs培养条件的不同分为生长培养液(GM)组(仅使用GM培养)、成牙本质向分化诱导培养液(OM)组(仅使用OM培养)、OM+LPS组(使用含有效质量浓度LPS的OM培养)。采用实时荧光定量PCR(qRT-PCR)检测各组hDPSCs白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、成牙本质向分化相关指标[牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、牙本质基质蛋白1(dentin matrix protein 1,DMP1)]及miR-138-5p的表达差异。将上述3组分别分为敲减miRNA阴性对照小干扰RNA(以下简称“NC”)亚组及敲减miR-138-5p小干扰RNA(以下简称“i-138”)亚组,通过qRT-PCR检测各亚组的成牙本质向分化相关指标,以及通过ALP染色检测敲减miR-138-5p后对微炎症环境中hDPSCs成牙本质向分化的影响。结果 分离出的原代细胞中表达间充质干细胞表面标记分子CD29、CD105的细胞阳性率超过95%,表达CD146的细胞阳性率超过30%,而表达造血干细胞表面标记分子CD14、CD45的细胞阳性率低于2%,且OM培养下细胞的ALP染色强于GM。ALP染色结果显示,含有0.01μg/mL质量浓度LPS的OM能有效促进hDPSCs成牙本质向分化。qRTPCR结果显示,OM+LPS组IL-6和TNF-α相对表达量均高于OM组,差异均有统计学意义(均P <0.001)。细胞培养7 d后,OM组DSPP、miR-138-5p相对表达量均显著高于GM组;细胞培养4、7 d后,OM组DMP1相对表达量显著高于GM组;细胞培养1、4、7 d后,OM+LPS组DSPP与DMP1相对表达量均显著高于OM组;细胞培养4、7 d后,OM+LPS组miR-138-5p相对表达量显著高于OM组,差异均有统计学意义(均P <0.001)。敲减miR-138-5p后,i-138 OM组和i-138 OM+LPS组分别与NC OM组和NC OM+LPS组相比,miR-138-5p、DSPP、DMP1相对表达量均减少,差异均有统计学意义(均P <0.001),且ALP染色强度均减弱。结论 微炎症环境可促进hDPSCs的成牙本质向分化,miR-138-5p可能在此过程中对h DPSCs成牙本质向分化起促进作用。

Objective To investigate the role of miR-138-5p in the process of odontogenic differentiation of human dental pulp stem cells(hDPSCs)in the microinflammatory state.Methods The study was conducted in the Central Laboratory,School of Stomatology,China Medical University from May 2021 to March 2022.The positive rates of CD29,CD105,CD146,CD14,CD45 were detected by flow cytometry.ALP staining was used to identify hDPSCs and their osteogenic differentiation ability.According to the results of ALP staining,the effective concentration of lipopolysaccharide(LPS)to improve the ALP activity of hDPSCs was determined.According to the culture conditions of hDPSCs,hDPSCs were divided into growth medium(GM)group(GM culture only),odontogenic differentiation medium(OM)group(OM culture only),and OM+LPS group(OM culture with effective concentration of LPS).The levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),dentin sialophosphoprotein(DSPP),dentin matrix protein 1(DMP1)and miR-138-5p in hDPSCs were detected by real-time fluorescence quantitative PCR(qRT-PCR).The three groups were divided into small interfering RNA(hereinafter referred to as"NC")of knockdown of miRNA negative control subgroup and small interfering RNA(hereinafter referred to as"i-138")of knockdown of miR-138-5p subgroup,and the relevant indicators of odontogenic differentiation in each subgroup were detected by qRT-PCR.ALP staining was used to detect the effect of miR-138-5p knockdown on the differentiation of hDPSCs into odontoblasts in a microinflammatory state.Results More than 95%of the isolated cells expressed CD29 and CD105,and more than 30%expressed CD146.However,less than 2%of the isolated cells expressed CD14 and CD45.The ALP staining of the isolated cells cultured in OM was stronger than that in GM.ALP staining results showed that OM containing 0.01μg/mL LPS could effectively promote the differentiation of hDPSCs into odontoblasts.The results of qRT-PCR showed that the relative expression of IL-6 and TNF-αin OM+LPS group was significantly higher than that in OM group(P<0.001).The relative expression of DSPP and miR-138-5p in OM group was significantly higher than that in GM group after 7 days of cell culture;the relative expression of DMP1 in OM group was significantly higher than that in GM group after 4 and 7 days of cell culture;the relative expression of DSPP and DMP1 in OM+LPS group was significantly higher than that in OM group after 1,4 and 7 days of cell culture;the relative expression of miR-138-5p in OM+LPS group was significantly higher than that in OM group(P<0.001).After knockdown of miR-138-5p,the relative expressions of miR-138-5p,DSPP and DMP1 in the i-138 OM group and i-138 OM+LPS group were significantly lower than those in the NC OM group and NC OM+LPS group,respectively(all P<0.001).And the intensity of ALP staining decreased.Conclusion Microinflammatory state can promote the odontogenic differentiation of hDPSCs,and miR-138-5p may promote the odontogenic differentiation of hDPSCs in this process.