小干扰RNA沉默YAP基因对人牙周膜干细胞增殖凋亡的影响  

Effects of YAP-small interfering RNA on the proliferation and apoptosis of human periodontal ligament stem cells

作　　者：唐翠竹 文勇[1,2] 顾伟亭[3] 张冰[1] 张云鹏[1] 姬雅雯 徐欣[1,2] 

机构地区：[1]山东大学口腔医学院 [2]山东省口腔组织再生重点实验室 [3]山东大学齐鲁医院妇产科,济南250012

出　　处：《华西口腔医学杂志》2015年第6期622-626,共5页West China Journal of Stomatology

基　　金：国家自然科学基金资助项目(81300885);山东省自然科学基金资助项目(ZR2013HQ052;ZR2013HM086);山东省医药卫生发展计划基金资助项目(2013WS0212);山东大学青年学者未来计划基金资助项目(2015WLJH53);山东大学基本科研业务费专项基金资助项目(2015QY003)

摘　　要：目的本研究通过小干扰RNA(siRNA)沉默人牙周膜干细胞(hPDLSCs)YAP基因,观察YAP基因沉默后对细胞增殖凋亡的影响。方法将化学合成siRNA序列以脂质体LipofectamineTM 2000介导瞬时转染h PDLSCs,采用实时荧光定量逆转录聚合酶链反应(RT-PCR)、Western blot检测转染后YAP表达水平,细胞增殖活性检测试剂盒(CCK-8)检测siRNA在体外对h PDLSCs增殖能力的影响,流式细胞仪检测细胞周期及凋亡率的变化。采用SPSS 19.0软件对数据进行处理,设P<0.05为差异有统计学意义。结果在转染48 h后h PDLSCs的YAP mRNA和蛋白水平明显降低(P<0.001),YAP蛋白表达水平在细胞被转染48、72 h后无显著差异,表明si RNA可以持续有效地抑制YAP的表达;CCK-8增殖活性实验结果显示细胞增殖活性受到抑制;沉默YAP基因后细胞早期及晚期凋亡率均增加。细胞周期检测结果显示细胞周期发生改变,G1及S期细胞比例增多(P<0.01),G2期细胞比例明显减少(P<0.05)。结论 si RNA沉默YAP基因后h PDLSCs增殖活性降低,细胞凋亡率明显增加,细胞的周期分布发生改变;YAP基因可以调控h PDLSCs的增殖与凋亡。Objective To investigate the effects of small interfering RNA（si RNA） targeting YAP on the proliferation and apoptosis of human periodontal ligament stem cells（h PDLSCs）. Methods Synthesized sequences of si RNA were transfected into h PDLSCs by Lipofectamine TM 2000. The expression of YAP was identified by using real-time quantitative reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. Proliferation activity was detected by using cell counting kit-8（CCK-8）. Changes in the cell cycle and apoptosis rate were detected by using flow cytometry. Results were analyzed by using SPSS 19.0, and P〈0.05 was considered statistically significant. Results Expression of YAP m RNA and protein were significantly downregulated after 48 h of transfection（P〈0.001）. No obvious difference was found in the expression levels of YAP protein between 48 and 72 h, thus indicating that si RNA could inhibit the expression of YAP persistently and effectively. Proliferation activity was inhibited, and apoptosis rate was increased. Cell cycle was changed as the proportion of G1 and S phases increased（P〈0.01） and G2 phase decreased（P〈0.05）. Conclusion Knocking down YAP gene by si RNA could inhibit proliferation activity, induce apoptosis, and change the cell cycle of h PDLSCs. Thus, YAP could regulate the proliferation and apoptosis of h PDLSCs.

关 键 词：小干扰RNA YAP基因 人牙周膜干细胞 增殖 凋亡 

分 类 号：R78[医药卫生—口腔医学]