受体酪氨酸激酶对子宫内膜异位症患者哺乳动物雷帕霉素靶蛋白信号通路的影响

Effect of TAM family receptor tyrosine kinases on signaling pathway of mammalian target of rapamycin in patients with endometriosis

  • 摘要:
    目的 探讨受体酪氨酸激酶(Axl)对子宫内膜异位症(EMS)患者哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的影响。
    方法 收集5例卵巢EMS患者的在位内膜组织和异位内膜组织, 另选择5例正常内膜组织作为对照。分离培养异位子宫内膜间质细胞(HEcESCs)、在位子宫内膜间质细胞(HEuESCs)及正常子宫内膜间质细胞(HEnESCs)并进行免疫组化鉴定,分析在细胞水平靶向上游Axl、mTOR激酶调控PI3K/AKT信号通路影响人类胚胎干细胞(HESCs)凋亡的机制。采用实时荧光定量聚合酶链反应(qRT-PCR)验证HEcESCs、HEuESCs及HEnESCs的AxlmTOR mRNA的表达差异; 在细胞水平选择siRNA转染调控Axl靶点,雷帕霉素调控mTOR靶点,采用蛋白质印迹法、qRT-PCR检测Axl、mTOR、磷酸化mTOR (p-mTOR)及CyclinD1表达差异。
    结果 与HEnESCs比较,HEuESCs及HEcESCs中Axl mRNA、mTOR mRNA表达水平差异有统计学意义(P < 0.05);与空白对照组比较,雷帕霉素处理组、Axl-siRNA处理组Axl mRNA、CyclinD1 mRNA表达水平差异有统计学意义(P < 0.05);与雷帕霉素处理组相比, Axl-siRNA处理组中Axl mRNA表达水平差异有统计学意义(P < 0.05);与阴性对照组比较,雷帕霉素处理组Axl mRNA、CyclinD1 mRNA表达水平差异有统计学意义(P < 0.05);与阴性对照组比较, Axl-siRNA处理组中Axl mRNA、CyclinD1 mRNA表达差异有统计学意义(P < 0.05);与空白对照组比较,雷帕霉素处理组中mTOR mRNA相对表达水平差异有统计学意义(P < 0.05)。4组mTOR蛋白表达差异无统计学意义(P>0.05);与空白对照组比较,雷帕霉素处理组、Axl-siRNA处理组中p-mTOR、CyclinD1蛋白表达差异有统计学意义(P < 0.05), 与阴性对照组的差异也有统计学意义(P < 0.05)。
    结论 调控Axl、mTOR可促进HEcESCs凋亡, Axl通过介导mTOR信号通路参与EMS的发生。

     

    Abstract:
    Objective To explore the effect of TAM family receptor tyrosine kinases (Axl) on signaling pathway of mammalian target of rapamycin (mTOR) in patients with endometriosis (EMS).
    Methods The eutopic and ectopic endometrium tissues of 5 patients with ovarian EMS were collected, and 5 normal endometrium tissues were selected as controls. Human ectopic endometrial stromal cells (HEcESCs), human eutopic endometrial stromal cells (HEuESCs) and human normal endometrial stromal cells (HEnESCs) were isolated and cultured for immunohistochemical identification, and the mechanism of regulating PI3K/AKT signal pathway by targeting upstream Axl and mTOR kinase at cell level to affect apoptosis of human embryonic stem cells (HESCs) was analyzed. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to verify the differences of Axl, mTOR mRNA expressions in HEcESCs, HEuESCs and HEnESCs; at the cell level, siRNA transfection was used to regulate Axl targets, rapamycin was used to regulate mTOR targets, and Western blotting and qRT-PCR were used to detect the differences in the expressions of Axl, mTOR, phosphorylated mTOR (p-mTOR) and CyclinD1.
    Results Compared with HEnESCs, the expression levels of Axl mRNA and mTOR mRNA in HEuESCs and HEcESCs showed significant difference (P < 0.05); compared with the blank control group, the expression levels of Axl mRNA and CyclinD1 mRNA in rapamycin treated group and Axl-siRNA treated group showed significant difference (P < 0.05); compared with the rapamycin treated group, the Axl mRNA expression level in the Axl-siRNA treated group showed significant difference (P < 0.05); compared with the negative control group, the expression levels of Axl mRNA and CyclinD1 mRNA in the rapamycin treated group showed significant difference (P < 0.05); compared with the negative control group, the expressions of Axl mRNA and CyclinD1 mRNA in the Axl-siRNA treated group showed significant difference (P < 0.05); compared with the blank control group, the relative expression level of mTOR mRNA in the rapamycin treated group showed significant difference (P < 0.05). There was no significant difference in mTOR protein expression among the four groups (P>0.05); compared with the blank control group, the expressions of p-mTOR and CyclinD1 protein in the rapamycin treated group and Axl-siRNA treated group showed significant difference (P < 0.05), and the difference compared with the negative control group was also statistically significant (P < 0.05).
    Conclusion Regulation of Axl and mTOR can promote apoptosis of HEcESCs, and Axl participates in the occurrence of EMS by mediating mTOR signal pathway.

     

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