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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。
Objective: To investigate the effect of CPNE7 si RNA on the proliferation and differentiation of human periodontal ligament cells (h PDLs) and its possible mechanism. Methods: h PDLs were isolated and cultured in vitro. The plasmid p SUPER-CPNE7 silenced CPNE7 was transfected into h PDLs by lipofection. The expression of CPNE7 was detected by RT-PCR and Western blotting, and the effect of CPNE7 si RNA on NF-κB activity was detected by ELISA. H PDLs were pretreated with 10 μmol / L NF-κB inhibitor pyrrolidinedithiocarbamic acid ammonium salt (PDTC) for 30 min. CCK8 was used to detect the effect of CPNE7 on cell proliferation. ELISA was performed to detect alkaline phosphatase phosphatase (ALP) activity). The expressions of RUNX2, OSX and OCN were detected by RT-PCR and Western blot. SPSS11.0 software package for statistical analysis of the data. Results: h PDLs transfected with p SUPER-CPNE7, CPNE7 expression was significantly decreased (P <0.05). CPNE7 si RNA significantly enhances NF-κB activity. Compared with the control group (CON), CPNE7 si RNA significantly inhibited the proliferation of h PDLs, down-regulated the activity of ALP and the expression of RUNX2, OSX and OCN, PDTC promoted the proliferation of h PDLs, increased ALP activity and up-regulated RUNX2, OSX and OCN expression. CONCLUSION: CPNE7 si RNA inhibits the proliferation and differentiation of h PDLs through NF-κB pathway.