siRNA沉默CPNE7基因表达抑制人牙周膜细胞的增殖及骨向分化

Effect of CPNE7 siRNA on cell proliferation and osteogenic differentiation in human periodontal ligament cells

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.