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中华口腔医学研究杂志(电子版) ›› 2018, Vol. 12 ›› Issue (06) : 335 -341. doi: 10.3877/cma.j.issn.1674-1366.2018.06.002

所属专题: 文献

基础研究

不同浓度γ-分泌酶抑制剂对人牙周膜干细胞骨向分化的影响
邱申彩1, 戴晓玮2, 龙晏1, 陈晓燕1, 吴佩玲1,()   
  1. 1. 830063 乌鲁木齐,新疆医科大学第二附属医院口腔科
    2. 830063 乌鲁木齐市口腔医院口腔科
  • 收稿日期:2018-07-23 出版日期:2018-12-01
  • 通信作者: 吴佩玲
  • 基金资助:
    国家自然科学基金(81460103)

Effects of different concentrations of γ-secretase inhibitor on osteogenic differentiation of human periodontal ligament stem cells

Shencai Qiu1, Xiaowei Dai2, Yan Long1, Xiaoyan Chen1, Peiling Wu1,()   

  1. 1. Department of Stomatology, Second Affiliated Hospital of Xinjiang Medical University, Urumqi 830063, China
    2. Department of Stomatology, Urumqi Stomatological Hospital, Urumqi 830063, China
  • Received:2018-07-23 Published:2018-12-01
  • Corresponding author: Peiling Wu
  • About author:
    Corresponding author: Wu Peiling, Email:
引用本文:

邱申彩, 戴晓玮, 龙晏, 陈晓燕, 吴佩玲. 不同浓度γ-分泌酶抑制剂对人牙周膜干细胞骨向分化的影响[J/OL]. 中华口腔医学研究杂志(电子版), 2018, 12(06): 335-341.

Shencai Qiu, Xiaowei Dai, Yan Long, Xiaoyan Chen, Peiling Wu. Effects of different concentrations of γ-secretase inhibitor on osteogenic differentiation of human periodontal ligament stem cells[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(06): 335-341.

目的

研究不同浓度的γ-分泌酶抑制剂(DAPT)对人牙周膜干细胞(hPDLSC)骨向分化的影响。

方法

将体外培养的hPDLSC随机分为5组:(1)2个对照组,即hPDLSC组和二甲基亚砜(DMSO)组(加入0.173 25 μL/L DMSO);(2)3个实验组,分别加入25、50和75 μmol/L的DAPT,即DAPT25组、DAPT50组及DAPT75组。通过细胞计数试剂盒(CCK-8)法检测不同浓度DAPT对hPDLSC的增殖情况的影响;通过茜素红染色、实时荧光定量聚合酶链反应(PCR)以及蛋白免疫印记法(Western blot)检测不同浓度DAPT对hPDLSC的Notch信号抑制能力及成骨能力的影响。使用SPSS 21.0软件对数据进行统计分析,以P<0.05为差异具有统计学意义。

结果

CCK-8结果显示,DMSO浓度为0.173 25 μL/L时对hPDLSC的增殖没有明显影响,同时不同浓度的DAPT均会抑制hPDLSC增殖,且DAPT浓度为50 μmol/L时对细胞的抑制能力最强(P<0.001);茜素红染色结果显示,hPDLSC组及DMSO组染色明显,矿化结节面积大且颜色深,两组间无明显差异,而3个实验组形成的矿化结节均有所减少,其中DAPT浓度为50 μmol/L时较浓度为25及75 μmol/L时形成的矿化结节数量更少且矿化结节面积更小;实时荧光定量PCR结果显示,DAPT浓度为50 μmol/L时,其成骨标志基因牙骨质附着蛋白(CAP,0.574 ± 0.182)、骨钙蛋白(OCN,0.269 ± 0.100)、Runt相关转录因子2(Runx2,0.470 ± 0.080)的表达量均明显减少,Notch信号通路相关分子Notch1Hes-1的表达水平分别为0.467 ± 0.118及0.318 ± 0.015,较其余组也有所减少,且DAPT浓度为50 μmol/L时的相关成骨标志基因表达量最低(P<0.05),Notch信号通路相关分子Notch1Hes-1的表达水平也最低(P<0.05);Western blot结果显示,与对照组相比3个实验组Notch、Hes-1及Runx2的蛋白表达量均有减少,其中50 μmol/L的DAPT组Runx2的蛋白表达量(0.116 ± 0.006)明显比其它组少,差异有统计学意义(P<0.001)。

结论

DMSO浓度为0.173 25 μL/L时对hPDLSC增殖及骨向分化没有明显影响;DAPT可抑制Notch信号通路的传递,从而抑制hPDLSC增殖及骨向分化,同时DAPT浓度为50 μmol/L时抑制能力最强。

Objective

To evaluate the effect of gamma secretase inhibitor (DAPT) on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in different concentrations.

Methods

hPDLSCs cultured in vitro were randomly divided into, (1) two control groups: hPDLSCs group (without DAPT) and 0.173 25 μL/L dimethyl sulfoxide (DMSO) group, and (2) three experimental groups: 25, 50 and 75 μL/L DAPT groups. The effects of different concentrations of DAPT on the proliferation of hPDLSCs were evaluated by cell counting kit (CCK-8) in control group and three experimental groups (25, 50 and 75 μmol/L DAPT) . Notch letters of different concentrations of DAPT on hPDLSCs were detected by alizarin red staining, real-time fluorescence quantitative polymerase chain reaction (PCR) and western blot. SPSS 21.0 was used for statistical description and analysis, when α= 0.05 was set as the statistical significance level.

Results

CCK-8 results showed that DMSO concentration of 0.17325 ul/L had no significant effect on the proliferation of periodontal ligament stem cells, and DAPT at different concentrations inhibited the proliferation of hPDLSCs, and DAPT concentration of 50 μmol/L had the strongest inhibitory effect on cells (P<0.001) ; Alizarin red staining results showed that the mineralized nodules in SC and DMSO groups were large and dark. There was no significant difference between the two groups. In contrast, the mineralized nodules formed in the three experimental groups were less. Specifically, the mineralized nodules formed in the group of 50 μmol/L DAPT were less and smaller than that in the groups of 25 and 75 μmol/L. The results of real-time PCR showed that the expressions of CAP, OCN and Runt-related transcription factor 2 (Runx2) were 0.574 ± 0.182, 0.269 ± 0.100 and 0.470 ± 0.080, respectively, in the group of 50 μmol/L DAPT, less than that in other groups (P<0.05) . The expression levels of Hes-1 and Notch signaling pathway related molecules Notch1 and Hes-1 were 0.467 ± 0.118 (P<0.001) and 0.318 ± 0.015 (P<0.024) , respectively, the lowest among the groups. The expression of Notch protein, Hes-1 and Runx2 in the control group was higher than that of the three experimental groups, and the expression of Runx2 protein in the DAPT group at 50 μmol/L was 0.116 ± 0.006, which was significantly lower than that in the other groups (P<0.001) .

Conclusions

DMSO concentration of 0.173 25 μL/L had no significant effect on the proliferation and bone differentiation of periodontal ligament stem cells. DAPT may inhibit the proliferation and bone differentiation of hPDLSCs by blocking Notch signaling pathway. Such an inhibitory effect was found to be strongest in the group of 50 μmol/L DAPT.

表1 实时荧光定量聚合酶链反应目的基因的引物序列
图1 二甲基亚砜(DMSO)对人牙周膜干细胞(hPDLSC)生长曲线的影响
图2 不同浓度γ-分泌酶抑制剂(DAPT)对人牙周膜干细胞(hPDLSC)生长曲线的影响
表2 细胞计数试剂盒(CCK-8)法测定二甲基亚砜(DMSO)对人牙周膜干细胞(hPDLSC)增殖影响的吸光度(A)值( ± s
表3 细胞计数试剂盒(CCK-8)法测定γ-分泌酶抑制剂(DAPT)对人牙周膜干细胞(hPDLSC)增殖影响的吸光度(A)值( ± s
图3 不同浓度γ-分泌酶抑制剂(DAPT)对人牙周膜干细胞(hPDLSC)成骨分化的影响(茜素红染色)
图4 二甲基亚砜(DMSO)对人牙周膜干细胞(hPDLSC)成骨基因表达的影响
图5 不同处理组人牙周膜干细胞(hPDLSC)成骨基因的表达
表4 实时荧光定量聚合酶链反应检测二甲基亚砜(DMSO)对人牙周膜干细胞(hPDLSC)成骨基因表达量的影响( ± s
表5 实时荧光定量聚合酶链反应检测不同浓度γ-分泌酶抑制剂(DAPT)对人牙周膜干细胞(hPDLSC)成骨基因表达量的影响( ± s
图6 人牙周膜干细胞(hPDLSC)组与不同浓度γ-分泌酶抑制剂(DAPT)实验组成骨相关蛋白的蛋白免疫印迹检测结果
表6 不同浓度γ-分泌酶抑制剂(DAPT)对人牙周膜干细胞(hPDLSC)成骨相关蛋白表达的影响
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