Objective: To explore the distribution characteristics of glioma-associated oncogene homolog 1 (Gli1) positive cells during orthodontic tooth movement process and conduct a proteomic analysis of these cells. Methods: Forty Gli1-LacZ transgenic mice were used to establish an in vivo orthodontic tooth movement (OTM) model for labeling Gli1 positive cells in Gli1-LacZ transgenic mice (OTM group) and an unforced control group, with tooth movement distance measured using micro-CT. The spatial relationship and distribution characteristics of Gli1 positive cells and H-type vessels of CD31 and endomucin (EMCN) in periodontal tissues were detected by immunofluorescence staining. Twenty Gli1-membrane-targeted tandem dimer Tomato (mT)/membrane-targeted green fluorescent protein (mG) double-genotype mice were bred and Gli1 positive cells were sorted for proteomic sequencing after tamoxifen induction. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases were used for enrichment analysis. Results: The micro-CT three-dimensional reconstruction results showed that the mesial movement of the maxillary first molar in mice after 7 days of force application was (69±15) μm, indicating the successful establishment of the Gli1-LacZ transgenic mouse OTM model. Immunofluorescence staining showed that the blood vessels in periodontal tissue were mostly H-type vessels of CD31 and EMCN. The blood vessels in the periodontal tissues are predominantly H-type vessels positive for both CD31 and EMCN. The percentage of Gli1 positive cells in the OTM group, expressed as (54.5±13.2)%, and the relative fluorescence intensity, expressed as 2.6±0.9, were both significantly greater than those in the control group, which had a Gli1 positive cell percentage of (36.3±9.1)% (P<0.01) and a relative fluorescence intensity of 1.0±0.3 (P<0.001). In contrast to the control group where only a small number of Gli1 positive cells were consistent with the distribution of H-type vessels, in the OTM group the number of Gli1 positive cells increased on the tension side were closely associated with the spatial distribution of H-type vessels. GO enrichment analysis of biological processes found that a large number of proteins in Gli1 positive cells were enriched in pathways such as angiogenesis and tissue remodeling. KEGG enrichment analysis found that related proteins were mainly enriched in pathways related to angiogenesis and Gli1, such as hypoxia-inducing factor 1 signaling pathway, vascular endothelial growth factor signaling pathway and hedgehog signaling pathway. Conclusions: The number of Gli1 positive cells increased on tension side and were closely related to H-type blood vessels in response to mechanical force during orthodontic tooth movement. This may be related to profile of inducing blood vessel formation and tissue remodeling.
目的： 探讨小鼠正畸牙移动过程中锌指蛋白（Gli1）阳性细胞的分布特征并对其蛋白质组学进行分析。 方法： 选择40只Gli1-β-半乳糖苷酶（Gli1-LacZ）转基因小鼠，构建体内标记Gli1阳性细胞的正畸牙移动组（正畸组）及未加力的对照组（每组20只），使用显微CT（micro-CT）检测牙移动距离。免疫荧光染色检测牙周组织中Gli1阳性细胞与CD31和内皮黏蛋白（EMCN）双阳性H型血管的空间位置关系和分布特征。繁育20只Gli1-膜靶向的串联番茄红荧光蛋白/膜靶向的绿色荧光蛋白（Gli1-mT/mG）双基因型小鼠，他莫昔芬诱导后获取长骨组织，分选Gli1阳性细胞进行蛋白质组学测序，通过基因本体功能注释（GO）和京都基因和基因组数据库（KEGG）进行富集分析。 结果： micro-CT三维重建结果显示，正畸组加力7 d后小鼠上颌第一磨牙近中移动（69±15）μm，表明成功构建Gli1-LacZ转基因小鼠正畸牙移动模型。免疫荧光染色结果显示，牙周组织中的血管多为CD31和EMCN双阳性H型血管，正畸组牙根张力侧Gli1阳性细胞数量百分比［（54.5±13.2）%］和相对荧光强度（2.6±0.9）均显著大于对照组Gli1阳性细胞百分比［（36.3±9.1）%］（P<0.01）和相对荧光强度（1.0±0.3）（P<0.001）。对照组仅少量Gli1阳性细胞与H型血管分布一致，正畸组张力侧Gli1阳性细胞数量增加且与H型血管空间分布紧密。GO富集分析显示，Gli1阳性细胞中大量蛋白质富集于促进血管形成和组织改建等生物学过程的通路，KEGG富集分析显示，相关蛋白主要富集于血管形成和GLi1相关通路，如缺氧诱导因子1信号通路、血管内皮生长因子信号通路以及刺猬信号通路。 结论： 正畸牙移动过程中Gli1阳性细胞响应机械力，张力侧数量上升且与H型血管空间分布紧密，可能与其自身高表达血管形成和组织改建相关蛋白的特性有关。.