李苹苹1 罗俊1 彭志庆2 初颜兵3 王燕4
Li Pingping1, Luo Jun1, Peng Zhiqing2, Chu Yanbing3, Wang Yan4
1. Dept. of Endodontics, The Affiliated Hospital of Sto-matology, Chongqing Medical University, Chongqing 400015, China; 2. Dept. of Stomatology, The Yongchuan Affiliated Hospital, Chongqing Medical University, Chongqing 402160, China; 3. Dept. of Stomatology, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China; 4. Dept. of Preventive and Pediatric Dentistry, The Affiliated Hos-pital of Stomatology, Chongqing Medical University, Chongqing 400015, China
摘要:
目的 研究内质网信号通路Ⅰ型跨膜蛋白α亚型(IRE1α)缺失突变体对人牙周膜成纤维细胞(hPDLFs)细胞周期的影响。方法 在成功构建人IRE1α基因全长重组质粒的基础上,采用重叠聚合酶链反应构建其两个主要功能域Kinase和Rnase的缺失突变体(pD-Kinase、pD-Rnase);然后分别染入hPDLFs细胞,Western blot检测重组基因表达情况,流式细胞仪(FCM)检测转染后hPDLFs细胞的细胞周期变化。结果 酶切及测序结果证实构建的IRE1α缺失突变体重组质粒构建成功;Western blot分析结果显示,3种重组基因均能正确表达;FCM结果分析显示:与衣霉素(TM)组相比,IRE1α实验组hPDLFs细胞S期比例增加而G1期减少(P<0.05);D-Rnase突变体组hPDLFs细胞S期比例减少而G1期增加(P<0.05);D-Kinase突变体组对hPDLFS细胞的增殖和各周期分布影响则无显著差异(P>0.05)。结论 在内质网应激状态下,IRE1α可促进hPDLFs细胞从G1期进入S期,D-Rnase突变体导致hPDLFs细胞生长阻滞于G1期,而D-Kinase则对hPDLFS细胞周期分布无明显影响。