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论文摘要

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的影响

Effect of type Ⅰ transmembrane protein deletions on the cell cycle of human periodontal ligament fibroblasts cells

作者:李苹苹 罗俊 彭志庆 初颜兵 王燕

Author:Li Pingping, Luo Jun, Peng Zhiqing, Chu Yanbing, Wang Yan

收稿日期:          年卷(期)页码:2014,32(3):221-221-224

期刊名称:华西口腔医学杂志

Journal Name:West China Journal of Stomatology

关键字:Ⅰ型跨膜蛋白&alpha,亚型,缺失体,牙周膜成纤维细胞,细胞周期,

Key words:type Ⅰ transmembrane protein,deletion,periodontal ligament fibroblasts,cell cycle,

基金项目:

中文摘要

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(P0.05)。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。

英文摘要

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 IRE1α with eukaryotic expression vectors were successfully constructed and correctly expressed in hPDLFs cells based on Western blot analysis. Under stress conditions, the FCM assay showed that cell percentage of S phases increased, whereas that of G1 phases decreased in the IRE1α group (P0.05).Conclusion Under stress conditions, IRE1α can improve the cell cycle of hPDLFs cells from the G1 to the S phase. The deletion mutant D-Rnase cause hPDLFs cell growth arrest at the G1 phase, whereas deletion mutant D-Kinase has no significant effect.

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