siRNA沉默CPNE7基因表达抑制人牙周膜细胞的增殖及骨向分化

摘要/Abstract

摘要： 目的探讨沉默Copine Ⅶ（CPNE7 siRNA）对人牙周膜细胞（human periodontal ligament cells, hPDLs）增殖及骨向分化的作用及其可能机制。方法体外分离培养hPDLs,采用脂质体转染法将沉默CPNE7的质粒pSUPER-CPNE7转染至hPDLs。采用RT-PCR和Western印迹法检测CPNE7的表达,ELISA检测CPNE7 siRNA对NF-κB活性的作用。给予10 μmol/L NF-κB抑制剂吡咯烷二硫代甲酸铵盐（pyrrolidinedithiocarbamic acid ammonium salt,PDTC）预处理hPDLs 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hPDLs经转染pSUPER-CPNE7,CPNE7表达下调,有显著性差异（P<0.05）。CPNE7 siRNA显著增强NF-κB的活性。与对照组（CON）相比,CPNE7 siRNA显著抑制hPDLs增殖,下调ALP活性及RUNX2、OSX和OCN的表达,给予PDTC促进hPDLs增殖,增强ALP活性并上调RUNX2、OSX和OCN的表达。结论CPNE7 siRNA通过NF-κB通路抑制hPDLs增殖和骨向分化。

关键词: 人牙周膜细胞, Copine Ⅶ, 碱性磷酸酶, 骨向分化, NF-κ, B

Abstract: 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.

Key words: Human periodontal ligament cells, Copine Ⅶ, Alkaline phosphatase, Osteogenic differentiation, NF-κB

刘彩宏, 呼海燕. siRNA沉默CPNE7基因表达抑制人牙周膜细胞的增殖及骨向分化[J]. 上海口腔医学, 2016, 25(4): 420-425.

LIU Cai-hong, HU Hai-yan. Effect of CPNE7 siRNA on cell proliferation and osteogenic differentiation in human periodontal ligament cells[J]. Shanghai Journal of Stomatology, 2016, 25(4): 420-425.

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