摘要
目的 :探讨沉默CopineⅦ(CPNE7 si RNA)对人牙周膜细胞(human periodontal ligament cells,h PDLs)增殖及骨向分化的作用及其可能机制。方法:体外分离培养h PDLs,采用脂质体转染法将沉默CPNE7的质粒p SUPER-CPNE7转染至h PDLs。采用RT-PCR和Western印迹法检测CPNE7的表达,ELISA检测CPNE7 si RNA对NF-κB活性的作用。给予10μmol/L NF-κB抑制剂吡咯烷二硫代甲酸铵盐(pyrrolidinedithiocarbamic acid ammonium salt,PDTC)预处理h PDLs 30 min,采用CCK8检测CPNE7对细胞增殖的影响,ELISA检测碱性磷酸酶(alkaline phosphatase,ALP)活性,RT-PCR和Western印迹法检测RUNX2、OSX和OCN的表达。采用SPSS11.0软件包对数据进行统计学分析。结果:h PDLs经转染p SUPER-CPNE7,CPNE7表达下调,有显著性差异(P<0.05)。CPNE7 si RNA显著增强NF-κB的活性。与对照组(CON)相比,CPNE7 si RNA显著抑制h PDLs增殖,下调ALP活性及RUNX2、OSX和OCN的表达,给予PDTC促进h PDLs增殖,增强ALP活性并上调RUNX2、OSX和OCN的表达。结论 :CPNE7 si RNA通过NF-κB通路抑制h PDLs增殖和骨向分化。
PURPOSE: To investigate the effect and mechanism of CPNE7 siRNA on proliferation and osteogenic differentiation in human periodontal ligament cells (hPDLs). METHODS: hPDLs were isolated by enzyme digestion, transfected with pSUPER-CPNE7 in order to knock down CPNE7. The expression of CPNE7 mRNA and protein was detected by Western blot and RT-PCR; ELISA was carried out for the activity of NF-κB; hPDLs were pretreated with PDTC (10 μmol/L) for 30 min, CCK8 was used to evaluate the proliferation; ALP activity was assayed with ELISA; The expression of RUNX2, OSX and OCN was measured with RT-PCR and Western blot. The data were analyzed with SPSS11.0 software package. RESULTS: After transfection with pSUPER-CPNE7, CPNE7 expression was significantly decreased (P〈0.05). ELISA assay indicated that CPNE7 siRNA enhanced NF-κB activity. CCK8 and RT-PCR assay showed that CPNE7 siRNA inhibited cell proliferation, ALP activity and decreased the expression of RUNX2, OSX and OCN in hPDLs; however, these effects were abolished by PDTC. CONCLUSIONS: CPNE7 siRNA inhibits the proliferation and osteogenic differentiation of hPDLs through NF-κB pathway.
出处
《上海口腔医学》
CAS
CSCD
2016年第4期420-425,共6页
Shanghai Journal of Stomatology