华西口腔医学杂志
華西口腔醫學雜誌
화서구강의학잡지
WEST CHINA JOURNAL OF STOMATOLOGY
2014年
3期
221-224
,共4页
李苹苹%罗俊%彭志庆%初颜兵%王燕
李蘋蘋%囉俊%彭誌慶%初顏兵%王燕
리평평%라준%팽지경%초안병%왕연
Ⅰ型跨膜蛋白α亚型%缺失体%牙周膜成纤维细胞%细胞周期
Ⅰ型跨膜蛋白α亞型%缺失體%牙週膜成纖維細胞%細胞週期
Ⅰ형과막단백α아형%결실체%아주막성섬유세포%세포주기
type Ⅰ transmembrane protein%deletion%periodontal ligament fibroblasts%cell cycle
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，Westernblot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；Westernblot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。
目的：研究內質網信號通路Ⅰ型跨膜蛋白α亞型（IRE1α）缺失突變體對人牙週膜成纖維細胞（hPDLFs）細胞週期的影響。方法在成功構建人IRE1α基因全長重組質粒的基礎上，採用重疊聚閤酶鏈反應構建其兩箇主要功能域Kinase和Rnase的缺失突變體（pD-Kinase、pD-Rnase）；然後分彆染入hPDLFs細胞，Westernblot檢測重組基因錶達情況，流式細胞儀（FCM）檢測轉染後hPDLFs細胞的細胞週期變化。結果酶切及測序結果證實構建的IRE1α缺失突變體重組質粒構建成功；Westernblot分析結果顯示，3種重組基因均能正確錶達；FCM結果分析顯示：與衣黴素（TM）組相比，IRE1α實驗組hPDLFs細胞S期比例增加而G1期減少（P<0.05）；D-Rnase突變體組hPDLFs細胞S期比例減少而G1期增加（P<0.05）；D-Kinase突變體組對hPDLFS細胞的增殖和各週期分佈影響則無顯著差異（P>0.05）。結論在內質網應激狀態下，IRE1α可促進hPDLFs細胞從G1期進入S期，D-Rnase突變體導緻hPDLFs細胞生長阻滯于G1期，而D-Kinase則對hPDLFS細胞週期分佈無明顯影響。
목적：연구내질망신호통로Ⅰ형과막단백α아형（IRE1α）결실돌변체대인아주막성섬유세포（hPDLFs）세포주기적영향。방법재성공구건인IRE1α기인전장중조질립적기출상，채용중첩취합매련반응구건기량개주요공능역Kinase화Rnase적결실돌변체（pD-Kinase、pD-Rnase）；연후분별염입hPDLFs세포，Westernblot검측중조기인표체정황，류식세포의（FCM）검측전염후hPDLFs세포적세포주기변화。결과매절급측서결과증실구건적IRE1α결실돌변체중조질립구건성공；Westernblot분석결과현시，3충중조기인균능정학표체；FCM결과분석현시：여의매소（TM）조상비，IRE1α실험조hPDLFs세포S기비례증가이G1기감소（P<0.05）；D-Rnase돌변체조hPDLFs세포S기비례감소이G1기증가（P<0.05）；D-Kinase돌변체조대hPDLFS세포적증식화각주기분포영향칙무현저차이（P>0.05）。결론재내질망응격상태하，IRE1α가촉진hPDLFs세포종G1기진입S기，D-Rnase돌변체도치hPDLFs세포생장조체우G1기，이D-Kinase칙대hPDLFS세포주기분포무명현영향。
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 (P<0.05) compared with the control group of tunicamycin (TM) treatment. Moreover, the cell percentage of the S phases decreased, whereas that of the G1 phases increased in the D-Rnase group (P<0.05) compared with the control. The deletion mutant D-Kinase had no influence on hPDLFS cell proliferation and cycle (P>0.05). 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.
