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中华口腔医学研究杂志(电子版) ›› 2016, Vol. 10 ›› Issue (02) : 104 -111. doi: 10.3877/cma.j.issn.1674-1366.2016.02.005

所属专题: 文献

基础研究

雌激素对人牙周膜干细胞干性维持的影响
戴旭彬1, 王萧萧1, 杨凡巧1, 欧乾民1, 姚斯琦1, 王彦1, 林雪峰1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2015-01-21 出版日期:2016-04-01
  • 通信作者: 林雪峰
  • 基金资助:
    国家自然科学基金(81171575)

Effects of estrogen on the stemness maintenance of human periodontal ligament stem cells

Xubin Dai1, Xiaoxiao Wang1, Fanqiao Yang1, Qianmin Ou1, Siqi Yao1, Yan Wang1, Xuefeng Lin1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2015-01-21 Published:2016-04-01
  • Corresponding author: Xuefeng Lin
  • About author:
    Corresponding author: Lin Xuefeng, Email:
引用本文:

戴旭彬, 王萧萧, 杨凡巧, 欧乾民, 姚斯琦, 王彦, 林雪峰. 雌激素对人牙周膜干细胞干性维持的影响[J]. 中华口腔医学研究杂志(电子版), 2016, 10(02): 104-111.

Xubin Dai, Xiaoxiao Wang, Fanqiao Yang, Qianmin Ou, Siqi Yao, Yan Wang, Xuefeng Lin. Effects of estrogen on the stemness maintenance of human periodontal ligament stem cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2016, 10(02): 104-111.

目的

研究雌激素对人牙周膜干细胞(hPDLSC)干性维持的影响。

方法

分离、培养原代hPDLSC并完成其干细胞鉴定;雌激素处理48 h,采用流式细胞术检测雌激素对hPDLSC细胞周期的影响;定量聚合酶链反应(q-PCR)检测人端粒酶逆转录酶(hTERT)基因、干性相关基因Oct4、Sox2、c-Myc以及衰老相关基因p16和p53的变化;雌激素长期处理后观察细胞克隆形成能力及骨向分化能力的改变。应用SPSS 17.0软件,利用配对样本t检验对组间均数进行统计学分析,P<0.05为差异具有统计学意义。

结果

实验获得的hPDLSC符合间充质干细胞鉴定标准。流式细胞术结果显示,雌激素处理48 h后hPDLSC增殖能力(29.730 ± 1.845)较对照组(23.587 ± 2.905)明显提高,差异有统计学意义(t= 9.913,P= 0.010)。q-PCR结果显示雌激素处理48 h后,hTERT表达量(1.958 ± 0.338)较对照组(1.000 ± 0.018)明显提高(t= 7.00,P= 0.001);Oct4表达量(2.539 ± 0.493)较对照组(1.000 ± 0.011)明显提高(t= 6.26,P= 0.001);Sox2表达量(2.234 ± 0.255)较对照组(1.016 ± 0.221)明显提高(t= 6.26,P= 0.003);c-Myc表达量(1.328 ± 0.091)较对照组(1.003 ± 0.088)明显提高(t= 5.67,P= 0.002);而衰老相关基因p16表达量(0.460 ± 0.085)较对照组(1.009 ± 0.163)明显降低(t= 12.24,P= 0.007);p53表达量(0.301 ± 0.041)较对照组(1.004 ± 0.115)明显降低(t= 7.77,P= 0.016)。雌激素长期处理后,雌激素处理组的克隆形成率(0.058 ± 0.008)显著高于对照组(0.013 ± 0.008),差异有统计学意义(t= 5.60,P= 0.028),成骨能力显著强于对照组(A对照组= 1.238 ± 0.084;AE2=2.460 ± 0.182,t= 16.41,P<0.001)。

结论

雌激素能提高hTERT和Oct4、Sox2、c-Myc的基因表达量,维持细胞增殖,抑制细胞衰老。雌激素长期处理能维持体外扩增时hPDLSC的干性。

Objective

To investigate the biological effect of estrogen (E2) on the stemness maintenance of human periodontal ligament stem cells (hPDLSCs) .

Methods

Primary hPDLSCs were isolated and characterized. After treatment with estrogen for 48 h, flow cytometry was used to evaluate cell cycle. The changes in human telomerase reverse transcriptase (hTERT) genes, stemness related genes Oct4, Sox2, c-Myc, aging related genes p16 and p53 were detected by q-PCR. After long-term culture with estrogen, colony formation ability and osteogenic differentiation potential were evaluated. The mean values between groups were analyzed by Paired-Samples T Test in SPSS 17.0. A significance level was set at 0.05.

Results

We isolated hPDLSCs and confirmed their capacity as mesenchymal stem cells. Proliferation ability of hPDLSCs was increased under 48 hours estrogen treatment (t= 9.913, P= 0.010) . q-PCR results showed that the expression level of hTERT (1.958 ± 0.338) was higher than the control group (1.000 ± 0.018) , there was a statistically significant difference between the groups (t= 7.00, P= 0.001) ; the expression level of Oct4 (2.539 ± 0.493) was higher than the control group (1.000 ± 0.011) , there was a statistically significant difference between the groups (t= 6.26, P= 0.001) ; the expression level of Sox2 (2.234 ± 0.255) was higher than the control group (1.016 ± 0.221) , there was a statistically significant difference between the groups (t= 6.26, P= 0.003) ; the expression level of c-Myc (1.328 ± 0.091) was higher than the control group (1.003 ± 0.088) , there was a statistically significant difference between the groups (t= 5.67, P= 0.002) , while senescence related genes that the expression level of p16 (0.460 ± 0.085) was lower than the control group (1.009 ± 0.163) , there was a statistically significant difference between the groups (t= 12.24, P= 0.007) and the expression level of p53 (0.301 ± 0.041) was lower than the control group (1.004 ± 0.115) , there was a statistically significant difference between the groups (t= 7.77, P= 0.016) . After long-term culture, the ability of clone formation (0.058 ± 0.008) was stronger than that of the NC group (0.013 ± 0.008) , there was a statistically significant difference between the groups (t= 5.60, P= 0.028) . And the osteogenic ability (ANC= 1.238 ± 0.084; AE2= 2.460 ± 0.182, t= 16.41, P<0.001) of hPDLSCs with estrogen treatment was significantly stronger than that of the NC group.

Conclusions

Estrogen can increase the expression of hTERT and Oct4, Sox2, c-Myc genes, maintain cell proliferation and inhibit cell senescence. Estrogen also can maintain stemness of hPDLSCs in long-term culture in vitro.

表1 q-PCR引物的碱基序列
图1 人牙周膜干细胞生长形态及克隆形成能力鉴定
图2 人牙周膜干细胞表面标志物鉴定
图3 人牙周膜干细胞多向分化能力鉴定
图4 雌激素对人牙周膜干细胞基因表达的影响(aP<0.05)
图5 雌激素对人牙周膜干细胞周期的影响
图6 雌激素长期处理对人牙周膜干细胞克隆形成能力的影响
图7 雌激素长期处理对人牙周膜干细胞成骨能力的影响
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