华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (6): 651-657.doi: 10.7518/hxkq.2021.06.005

• 基础研究 • 上一篇    下一篇

17β-雌二醇对髁突软骨细胞增殖的影响机制

张帅1(), 王江红1, 田利杰2, 王宝利2, 张娟1()   

  1. 1.天津医科大学口腔医院修复科,天津 300070
    2.天津医科大学朱宪彝纪念医院·内分泌研究所,天津 300134
  • 收稿日期:2020-11-30 修回日期:2021-09-07 出版日期:2021-12-01 发布日期:2021-12-03
  • 通讯作者: 张娟 E-mail:wxwklhhh@163.com;kqzhangjuan@126.com
  • 作者简介:张帅,医师,硕士,E-mail:wxwklhhh@163.com
  • 基金资助:
    天津市自然科学基金重点项目(18JCZDJC3200)

Effect of 17β-estradiol on the proliferation of condylar chondrocytes

Zhang Shuai1(), Wang Jianghong1, Tian Lijie2, Wang Baoli2, Zhang Juan1()   

  1. 1.Dept. of Prosthetics, Stomatological Hospital of Tianjin Medical University, Tianjin 300070, China
    2.Zhu Xianyi Memorial Hospital of Tianjin Medical University & Endocrinology Institute, Tianjin 300134, China
  • Received:2020-11-30 Revised:2021-09-07 Online:2021-12-01 Published:2021-12-03
  • Contact: Zhang Juan E-mail:wxwklhhh@163.com;kqzhangjuan@126.com
  • Supported by:
    Key Project of Tianjin Natural Science Foundation(18JCZDJC3200)

摘要: 目的

研究17β-雌二醇(E2)对髁突软骨细胞增殖调节的影响,并初步探讨磷酸化雷帕霉素哺乳动物靶标(p-mTOR)在该调节过程中发挥的作用。

方法

取6周龄雌性SD大鼠髁突软骨细胞进行原代培养,从第二代分别给予不同浓度的E2和/或雷帕霉素(RAPA);CCK8法检测不同给药条件下,髁突软骨细胞在第24、48、72小时的增殖情况;逆转录聚合酶链式反应(RT-PCR)检测软骨细胞中雌激素受体α(ERα)、雌激素受体β(ERβ)、自噬相关基因6(Beclin-1)、自噬相关基因5(ATG-5)、Ⅱ型胶原(COLⅡ)相关基因的表达;蛋白印迹(Western blot)法检测软骨细胞中ERα、ERβ、Beclin-1、脂质化轻链蛋白3B(LC3-Ⅱ)、p-mTOR相关蛋白的表达及加入雌激素受体拮抗剂后各组细胞p-mTOR的表达。

结果

E2可显著促进体外培养的髁突软骨细胞增殖,并在10-8 mol·L-1 浓度下达到峰值;RAPA可以显著抑制细胞增殖。10-8 mol·L-1 E2上调软骨细胞ERα、COLⅡ基因表达(P<0.01)和ERα、p-mTOR蛋白表达(P<0.05),下调软骨细胞Beclin-1、ATG-5基因表达(P<0.05)和Beclin-1、LC3-Ⅱ蛋白表达(P<0.05);RAPA可以上调细胞Beclin-1和LC3-Ⅱ蛋白水平(P<0.01),下调p-mTOR的表达(P<0.01);ERα拮抗剂可以显著降低细胞中p-mTOR的表达(P<0.01)。

结论

E2在浓度为10-8 mol·L-1时可有效通过ERα-p-mTOR途径激活mTOR的磷酸化,抑制自噬,提高髁突软骨细胞增殖速度。

关键词: 颞下颌关节骨关节病, 17β-雌二醇, 雷帕霉素, 磷酸化雷帕霉素哺乳动物靶标, 髁突软骨细胞

Abstract: Objective

To study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process.

Methods

Condylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR.

Results

E2 could significantly promote the proliferation of chondrocytes cultured in vitro, and maximum promotion was achieved at a concentration of 10-8 mol·L-1. RAPA could significantly inhibit cell proliferation. E2 at aconcentration of 10-8 mol·L-1 could greatly improve the gene expression levels of ERα and COLⅡ (P<0.01) with the protein levels of ERα and p-mTOR (P<0.05), and decrease the gene expression levels of Beclin-1 and ATG-5 (P<0.05) with the protein levels of Beclin-1 and LC3-Ⅱ (P<0.05). RAPA could also enhance the relative protein expression of Beclin-1 and LC3-Ⅱ (P<0.01), and reduce the expression of p-mTOR (P<0.01). Treatment with the ERα antagonist significantly reduced the expression of p-mTOR in cells (P<0.01).

Conclusion

At a concentration of 10-8 mol·L-1, E2 could effectively activate the phosphorylation of mTOR through the ERα-p-mTOR pathway, inhibit cell autophagy, and promote the proliferation of condylar chondrocytes.

Key words: temporomandibular joint osteoarthritis, 17β-estradiol, rapamycin, phosphorylated-mammalian target of rapamycin, condylar chondrocyte

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