17β-雌二醇对髁突软骨细胞增殖的影响机制

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

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。

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(pmTOR)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 secondgeneration 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 typeⅡ(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 10-8mol·L^(-1).RAPA could significantly inhibit cell proliferation.E2 at aconcentration of 10-8mol·L^(-1) could greatly improve the gene expression levels of ERαand COLⅡ(P<0.01)with the protein levels of ERαand 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 ERαantagonist significantly reduced the expression of pmTOR in cells(P<0.01).Conclusion At a concentration of 10-8mol·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.