摘要
目的:探究高糖炎性环境下Krüppel样因子4(KLF4)对人牙周膜干细胞(hPDLSCs)线粒体能量代谢和成骨分化的影响。方法:利用免疫荧光和Western blot实验研究高糖炎性环境下KLF4在hPDLSCs的定位和表达情况。通过碱性磷酸酶(ALP)和茜素红染色等检测高糖炎性环境下hPDLSCs成骨能力。通过慢病毒敲低和过表达技术,结合实时荧光定量PCR与Western blot实验检测病毒感染后KLF4的表达,检测KLF4对hPDLSCs成骨分化的影响。采用核酸酶靶向切割和释放(CUT&RUN)技术,分析高糖炎性环境下hPDLSCs中KLF4结合的DNA及相关信号通路变化。使用Seahorse能量代谢仪探索KLF4对hPDLSCs氧化呼吸能力的影响。结果:高糖炎性环境下,hPDLSCs中KLF4表达下调、ALP活性降低、钙结节形成减少(均P<0.001),Runt相关转录因子2(RUNX2)、骨钙素(OCN)表达降低(均P<0.001)。敲低KLF4,导致hPDLSCs的ALP活性降低(P<0.001),钙结节形成减少(P<0.001);过表达KLF4促进hPDLSCs的成骨分化。高糖炎性环境下,KLF4下游调控基因富集于能量代谢、成骨等相关通路。敲低KLF4导致hPDLSCs线粒体耗氧率(OCR)水平下调,而细胞外酸化率(ECAR)水平上调;过表达KLF4使OCR水平上调,而ECAR水平下调。结论:在高糖炎性环境下,hPDLSCs的成骨分化能力受损,KLF4可能通过上调线粒体氧化磷酸化能力来恢复hPDLSCs的成骨分化能力。
Objective:To investigate the effects of Krüppel-like factor 4(KLF4)in a high glucose and inflammatory environment on mitochondrial energy metabolism and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods:The localization and expression of KLF4 in hPDLSCs was investigated under high glucose inflammatory environment using immunofluorescence and Western blot.ALP staining and alizarin red staining were used to study osteogenic differentiation of hPDLSCs in hyperglycemic inflammatory environment.Lentivirus knockdown and overexpression techniques were used to detect the expression of KLF4 after virus infection by qRT-PCR and Western blot.CUT&RUN technique was used to analyze the changes of KLF4 binding DNA and related signaling pathways in hPDLSCs under high glucose inflammatory environment.The impact of KLF4 on oxidative respiratory capacity of hPDLSCs was explored using a Seahorse energy metabolism analyzer.Results:KLF4 expression was downregulated under high glucose inflammatory conditions,accompanied by reduced ALP activity,calcium nodule formation(all P<0.001),and expression of RUNX2 and OCN(P<0.001).Knockdown of KLF4 resulted in a significant decrease in ALP activity(P<0.001)and reduced calcium nodule formation(P<0.001)in hPDLSCs,while overexpression of KLF4 promote osteogenic differentiation.Under high glucose inflammatory environment,the downstream regulatory genes of KLF4 were enriched in energy metabolism,osteogenesis and other related pathways.KLF4 knockdown reduced OCR levels and increased ECAR levels in hPDLSCs,while KLF4 overexpression increased OCR levels and decreased ECAR levels.Conclusions:In a high glucose inflammatory environment,the osteogenic differentiation ability of hPDLSCs was compromised,and KLF4 may enhance the mitochondrial oxidative phosphorylation capacity to restore this ability.
作者
唐婧淇
徐萱雯
周逸
苟惠清
李璐
徐艳
TANG Jingqi;XU Xuanwen;ZHOU Yi;GOU Huiqing;LI Lu;XU Yan(Department of Periodontology,The Affiliated Stomatological Hospital of Nanjing Medical University,Jiangsu Province Key Laboratory of Oral Diseases,Jiangsu Province Engineering Research Center of Stomatological Translational Medicine,Nanjing 210029,China)
出处
《口腔生物医学》
2024年第2期82-90,共9页
Oral Biomedicine
基金
国家自然科学基金(82170962,82370961)
江苏省科教能力提升工程——江苏省研究型医院(YJXYYJSDW4)
江苏省医学创新中心(CXZX202227)。
关键词
人牙周膜干细胞
Krüppel样因子4
牙周炎
糖尿病
线粒体能量代谢
human periodontal ligament stem cells
Krüppel-like factor 4
periodontitis
diabetes mellitus
mitochondrial energy metabolism
