吴雪 段少宇 梁萍 刘欣

[摘要]目的：探讨miR-34a靶向STAT1基因调控牙周膜细胞增殖及凋亡的分子机制。方法：将PDLCs细胞分成Pm、Py、Pz三组，Pz组（PDLCs细胞不做任何处理），Pm组（转染miR-34a模拟物）、Py组（转染miR-34a抑制物），RT-PCR检测STAT1、miR-34a水平，Westernblot法检测STAT1蛋白表达，流式细胞仪、MTT法分别检测PDLCs细胞凋亡、增殖情况。结果：RT-PCR检测STAT1、miR-34a水平结果显示，与Pz组对比，Py组STAT1、miR-34a水平有显著下降，Pm组STAT1、miR-34a水平最高，Pz组STAT1、miR-34a水平其次，Py组STAT1、miR-34a水平最低（均P<0.05）。Westernblot法检测STAT1蛋白发现，与Pz组对比，Py组STAT1蛋白较Pm组有显著下降，Pm组STAT1蛋白表达最高，Pz组蛋白表达其次，Py组STAT1蛋白表达最低（均P<0.05）。流式细胞术检测发现，与Pz组对比，Pm组PDLCs细胞凋亡明显增加，与Pm组对比，Py组细胞凋亡明显降低（均P<0.05），Pm组细胞凋亡呈三组最高，Pz组其次，Py组最低。MTT检测结果显示：Pz组PDLCs细胞在24h、48h、72h时均缓慢增长，Py组PDLCs细胞24h、48h增殖变化较平稳，72h时增殖速度明显加快，Pm组受miR-34a模拟物影响，PDLCs细胞数量增殖缓慢（均P<0.05）。结论：miR-34a低表达能够抑制PDLCs细胞凋亡，促进其生长，这一作用机制可能与降低STAT1蛋白表达有关。

[关键词]miR-34a;STAT1;牙周膜细胞;牙周炎;增殖;凋亡

[中圖分类号]R329.2+8    [文献标志码]A    [文章编号]1008-6455（2021）04-0115-04

Molecular Mechanisms of MiR-34a Targeting STAT1 Gene Regulating Proliferation and Apoptosis of Periodontal Ligament Cells

WU Xue，DUAN Shao-yu，LIANG Ping，LIU Xin

（Department of Stomatology，Beijing Electric Power Hospital，Beijing 100073，China）

Abstract： Objective  To investigate the molecular mechanism of the proliferation and apoptosis of periodontal ligament cells regulated by STAT1 gene targeting to miR-34a. Methods  Human periodontal ligament cells were isolated， cultured and diluted. According to Lipofectamine 2000 instructions， PDLCs were divided into three groups： Pm group， Py group and Pz group. Pz group （PDLCs were not treated with any treatment）， Pm group （transfected mimic of miR-34a）， Py group （transfected microarray）. miR-34a inhibitor， transfected for about 4 hours， discarded the upper liquid， replaced the medium， cultured for 48 hours. RT-PCR was used to detect the levels of STAT1 and miR-34a. Western blot was used to detect the expression of STAT1. Flow cytometry and MTT were used to detect the apoptosis and proliferation of PDLCs. Results  RT-PCR detection of STAT1 and miR-34a levels showed that compared with Pz group， STAT1 and miR-34a levels in Py group decreased significantly. STAT1 and miR-34a levels in Pm group were the highest， followed by STAT1 and miR-34a levels in Pz group， and STAT1 and miR-34a levels in Py group were the lowest （P<0.05）. Western blot analysis of STAT1 protein showed that compared with Pz group， the expression of STAT1 protein in Py group was significantly lower than that in Pm group. The expression of STAT1 protein in Pm group was the highest， followed by that in Pz group， and the expression of STAT1 protein in Py group was the lowest （P<0.05）. Flow cytometry showed that compared with Pz group， the apoptosis of PDLCs in Pm group increased significantly， and decreased significantly in Py group （P<0.05）. The apoptosis of PDLCs in Pm group was the highest in three groups， followed by Pz group and the lowest in Py group. MTT results showed that PDLCs in Pz group grew slowly at 24h， 48h and 72h. PDLCs  in Py group increased steadily at 24h and 48h， and increased significantly at 72h. The number of PDLCs in Pm group increased slowly under the influence of miR-34a （P<0.05）. Conclusion  The low expression of miR-34a can inhibit PDLCs cell apoptosis and promote its growth. This mechanism may be related to the reduction of STAT1 protein expression.

Key words： miR-34a; STAT1; periodontal ligament cells; periodontitis; proliferation; apoptosis

牙周炎是牙周组织发生慢性炎症病变，患者可能会出现牙龈出血、牙槽骨破坏和牙周袋等症状，随着病情的发展还会发展为牙齿松动和脱落，是现在成年人牙齿缺失的主要原因[1]。牙周炎是常见的口腔疾病，研究表明，牙周膜细胞（PDLCs）生长失衡是导致牙周炎出现的主要原因[2]，PDLCs细胞凋亡会引起其他疾病，严重威胁人们的身体健康[3]。近些年研究发现牙周炎中的致病菌主要对患者体内的炎症反应和免疫反应起作用，PDLCs细胞作为牙周支持组织必不可少的一部分，具有效应性，会受到炎性因子影响增长受限或过剩，牙体出现疾病也与PDLCs细胞有关[4-5]。miR-34a是微小RNA之一，有相关研究证明，miR-34a会激活恶性生物活性和恶性特质，加重疾病进程，PDLCs细胞在维持牙周功能正常运转有重要作用，在牙周炎组织中miR-34a表达水平上升，PDLCs凋亡细胞明显增加，而当miR-34a水平下降时，能有效改善牙周组织的损伤，说明miR-34a与PDLCs凋亡程度呈正相关[6-7]。STAT1信号分布在机体多个组织细胞内，多种因子参与和调控PDLCs细胞分化，抑制STAT1信号表达可以明显减少PDLCs细胞凋亡，说明STAT1信号在牙周炎发展过程中起重要作用[8]。……