药物阻断mGlu3受体通路对脑胶质瘤干细胞增殖与分化的影响

Effect of drug blockade of type-3 metabotropic glutamate receptor on proliferation and differentiation in glioma stem cells

目的探讨应用亲代谢性谷氨酸受体亚型3(mGlu3)特异性拮抗剂LY341495阻断受体通路后对脑胶质瘤干细胞(glioma stem cells,GSCs)生物学特性的作用及相关机制。方法将培养人脑胶质瘤干细胞以及裸鼠移植瘤动物模型分为对照组、激动剂组,拮抗剂组,采用CCK-8法测定细胞增殖率,流式细胞术观察细胞周期变化,免疫组织化学染色激光共聚焦显微镜观察细胞CD133表达情况,RT-PCR和Western blot分别检测β-catenin、c-myc等mRNA和蛋白表达,观察裸鼠移植瘤生长变化。结果与对照组比较,拮抗剂组GSCs增殖率和进入S期的比例均显著降低(P<0.05),且呈时间依赖性;在分化培养基条件下,拮抗剂组GSCs向星形胶质细胞分化明显,Nestin阳性细胞减少而GFAP阳性细胞增多(P<0.05);mGlu3拮抗剂干预后,诱导β-catenin、c-myc等mRNA和蛋白表达明显下调(P<0.05);与对照组比较,拮抗剂组裸鼠移植瘤生长速度及肿瘤体积明显降低(P<0.05)。结论药物阻断mGlu3受体通路可显著抑制体外培养的GSCs以及裸鼠体内移植瘤的生长,并促使GSCs由去分化状态转向分化状态,此过程可能经由Wnt/β-catenin通路发挥作用。

Objective To determine the effect of type-3 metabotropic glutamate receptor（mGluR3） antagonist LY341495 on the biological behavior of cultured human glioma stem cells（GSCs）.Methods Cultured human GSCs and nude mouse xenograft models were divided into three groups including a control group,an agonist group and an antagonist group.Cell proliferation rate was assessed by CCK-8 assay.Cell cycle was measured by flow cytometry.Expression of CD133 was assessed by immunohistochemistry and laser scanning confocal microscopy.The mRNA and protein expressions of β-catenin and c-myc were measured by real-time RT-PCR and Western blotting.Results As compared with the control group,the cell proliferation rate and the proportion of GSCs at S stage of cell cycle in the antagonist group significantly decreased in a time-dependent manner（P0.05）.The number of GSCs differentiating into astrocytes significantly increased in the antagonist group.The number of the nestin-positive cells decreased,but the number of the GFAP-positive cells increased（P0.05）.LY341495 significantly decreased the mRNA and protein expressions of β-catenin and c-myc（P0.05）.LY341495 significantly decreased the growth and volume of transplanted tumors in nude mouse xenograft models（P0.05）.Conclusion In the biological behavior of human GSCs,mGluR3 plays an important role.The drug blockade of mGluR3 can inhibit the growth of cultured GSCs and the transplanted tumors in nude mouse xenograft models via Wnt/β-catenin pathway.