Prrx1+牙周膜干细胞在牙移动过程中的谱系示踪

Study on Prrx1+periodontal ligament stem cells during orthodontic tooth movement by lineage tracing

目的·通过Cre/loxP重组酶系统研究牙周膜干细胞(periodontal ligament stem cells,PDLSCs)中配对相关同源基因1(paired related homeobox 1,Prrx1)阳性细胞谱系(Prrx1+细胞谱系)在小鼠牙移动过程中的动态分布。方法·利用Cre/loxP重组酶系统,将诱导型Prrx1-Cre^(ERT2)小鼠与R26^(tdTomato)荧光标记小鼠交配繁殖,并通过PCR对其子代小鼠进行基因型鉴定。取8只子代小鼠,向其腹腔注射他莫昔芬(tamoxifen,TA),以标记PDLSCs中的Prrx1+细胞谱系(即tdTomato^(+)细胞)。于小鼠上颌左侧安置加力装置[即正畸牙移动(orthodontics tooth movement,OTM)侧],右侧未加力即为对照侧,以构建小鼠OTM模型。分别于牙移动第3日(OTM 3 d)、第7日(OTM 7 d)时处死小鼠,收集其双侧上颌磨牙及周围牙周组织,经脱钙后行包埋及冰冻切片处理。采用苏木精-伊红染色(hematoxylin-eosin staining,H-E染色)观察张力区和压力区牙周膜的改变,并利用免疫荧光染色观察Prrx1+细胞谱系在张力区及压力区的动态分布。结果·经PCR鉴定,子代小鼠基因型为Prrx1-Cre^(ERT2);R26^(tdTomato)。随着加力装置作用的增加,OTM 7 d小鼠的牙移动距离[(87.44±4.02)μm]较OTM 3 d小鼠[(42.81±5.04)μm]明显增加,提示小鼠OTM模型构建成功。H-E染色结果显示:OTM 3 d时小鼠OTM侧压力区的牙周膜被压缩、间隙变窄,而OTM 7 d时OTM侧牙周膜宽度逐渐恢复;OTM 3 d时OTM侧张力区的牙周膜被拉伸,而OTM 7 d较OTM 3 d时牙周膜排列更规则。免疫荧光染色结果显示:OTM 3 d时OTM侧压力区牙周膜中tdTomato^(+)细胞数量较对照侧减少,OTM 7 d时OTM侧压力区tdTomato^(+)细胞数量较对照侧增加(均P<0.05);OTM 3 d及OTM 7 d时,OTM侧张力区tdTomato^(+)细胞数量较对照侧均有增加(均P<0.05)。结论·成功构建了Prrx1-Cre^(ERT2);R26^(tdTomato)小鼠的OTM模型,并初步证实了Prrx1+细胞谱系参与OTM过程中的牙周改建。

Objective·To investigate the dynamic distribution of paired related homebox 1(Prrx1)positive cell lineage(Prrx1+cell lineage)in periodontal ligament stem cells(PDLSCs)during tooth movement in mice by Cre/loxP recombination system.Methods·By using Cre/loxP recombination system,inducible Prrx1-Cre^(ERT2)mice were mated with R26^(tdTomato)fluorescently labeled mice,and their offspring mice were genotyped by PCR.Eight offspring mice were injected with tamoxifen(TA)intraperitoneally to mark the Prrx1+cell lineage(tdTomato^(+)cells)in PDLSCs.The orthodontics tooth movement(OTM)model was constructed by placing a force-loading device on the left side of maxilla(i.e.,OTM side),and the right side without force was control side(i.e.,Ctrl side).The mice were sacrificed on the third day(OTM 3 d)and the seventh day(OTM 7 d)of tooth movement,and their bilateral maxillary molars and surrounding periodontium were collected,decalcified,embedded and frozen sectioned.The changes of periodontal ligament in the tension area and compression area were observed by hematoxylin-eosin staining(H-E staining),and the dynamic distribution of Prrx1+cell lineage was observed by immunofluorescence staining.Results·The genotype of the offspring mice was identified by PCR as Prrx1-Cre^(ERT2);R26^(tdTomato).With the increased action of the stressing device,the tooth movement distance of OTM 7 d mice[(87.44±4.02)μm]increased significantly compared with that of the OTM 3 d mice[(42.81±5.04)μm],suggesting OTM model was successfully constructed.H-E staining showed that the periodontal ligament and its gap in the compression area of the OTM side was compressed and narrowed on OTM 3 d,and its width was gradually restored on OTM 7 d;the periodontal ligament in the tension area of the OTM side was stretched on OTM 3 d,and the periodontal ligament was more regularly arranged on OTM 7 d than that on OTM 3 d.Immunofluorescence staining showed that the number of tdTomato^(+)cells in the periodontal ligament of the compression area on the OTM side was lower than that of the Ctrl side on OTM 3 d,and the number of tdTomato^(+)cells in compression area on the OTM side was higher than that of the Ctrl side on OTM 7 d(both P<0.05);on both OTM 3 d and OTM 7 d,the number of tdTomato^(+)cells in tension area on the OTM side increased compared with that on the Ctrl side(both P<0.05).Conclusion·The OTM model of Prrx1-Cre^(ERT2);R26^(tdTomato)mice is successfully constructed,and the involvement of Prrx1+cell lineage in periodontal remodeling during OTM is tentatively confirmed.