Ⅰ型跨膜蛋白α亚型缺失突变体对人牙周膜成纤维细胞周期影响.docVIP

Ⅰ型跨膜蛋白α亚型缺失突变体对人牙周膜成纤维细胞周期影响 [文章编号] 1000-1182(2014)03-0221-04 　　[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）。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。 　　[关键词] Ⅰ型跨膜蛋白α亚型；缺失体；牙周膜成纤维细胞；细胞周期 　　[中图分类号]Q 781 [文献标志码] A [doi] 10.7518/hxkq.2014.03.003 　　 　　Effect of type#8198;Ⅰ#8198;transmembrane protein deletions on the cell cycle of human periodontal ligament fibroblasts cells 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) 　　[Abstract] Objective To determine the effect of type Ⅰ transmembrane protein (IRE1α) deletions on the cell cycle of human periodontal ligament fibroblasts (hPDLFs) cells. Methods Based on the IRE1α deletions, a full-length model was successfully constructed. Moreover, overlapping polymerase chain reaction mutagenesis facilitated the establishment of two deletion mutants of IRE1α (pD-Kinase, pD-Rnase). The full-length model and two mutant eukaryotic expression vectors were transfected into hPDLFs cells. Western blot analysis was performed to identify the expression in the cells. The changes in the cell cycle of hPDLFS cells were detected by flow cytometry (FCM). Results The two deletion mutants of