【作者】 班碧秀；

【导师】 罗佐杰；

【作者基本信息】 广西医科大学 ， 内分泌（专业学位）， 2022， 博士

【摘要】 目的和意义:甲状腺未分化癌(ATC),是致命的恶性肿瘤之一。手术、放化疗等常规治疗方法不能有效阻止ATC疾病进程。目前,寻找ATC的有效治疗依然是临床治疗过程中需要面临的巨大问题。硒-甲基硒代半胱氨酸(MSC)是有机硒的一种,在乳腺癌、前列腺癌等癌症的临床前实验和临床研究中都显示了良好的抗癌特性,但在ATC中的抗癌机制尚未阐明。本研究我们以ATC细胞8305C为对象,研究MSC对其增殖、凋亡、侵袭迁移和糖酵解的影响,并探讨MMPs、ERK1/2信号通路和糖酵解在其中的作用和联系。方法:体外培养8305C细胞,使用不同浓度的MSC干预。相差显镜观察各浓度MSC处理组8305C细胞形态及密度的变化。MSC对8305C细胞增殖抑制率通过CCK-8实验检测。Hoechst33258染色后观察MSC对8305C细胞核染色体形态的改变。JC-1探针检测细胞内线粒体膜电位,了解MSC对8305C细胞线粒体膜电位的影响。细胞划痕(伤口愈合)和Transwell迁移实验检测MSC影响下细胞迁移能力,Transwell基质胶侵袭实验检测MSC作用下细胞侵袭的能力。DCFH-DA检测细胞内ROS的累积,联合抗氧化剂NAC验证ROS在MSC抑制癌细胞增殖中的作用。通过WB检测ERK1/2信号通路的ERK1/2和p-PERK1/2蛋白,转移相关因子MMP-2和MMP-9蛋白,并分别用通路激动剂EGF和通道阻滞剂U0126联用验证ERK/2信号通路在MSC抗肿瘤中的作用。体外培养8305C细胞和人甲状腺正常细胞株Nthy-ori 3-1,使用试剂盒检测养基中乳酸的含量和葡萄糖消耗量。WB方法检测糖酵解关键酶GLUT1、HK2、LDHA及HIF-1α表达水平。糖酵解抑制剂2-DG联合MSC干预后观察8305C细胞增殖及细胞内ROS的改变。结果:(1)CCK-8结果显示:MSC组8305C细胞的生长活性明显下降,表明MSC对8305C细胞具有生长抑制作用(p<0.05)。(2)与对照组相比,MSC干预后8305C细胞相差显微镜下表现出明显凋亡特征,细胞皱缩,数目显著减少。(3)JC-1探针检测结果显示MSC引起8305C细胞线粒体膜电位剂量依赖性的降低(P<0.05),Hoechst33258染色提示MSC对8305C有诱导凋亡的作用。(4)DCFH-DA染色表明MSC剂量依赖性地诱导8305C细胞内ROS的积累(P<0.05)。(5)划痕(伤口愈合实验)、transwell迁移和侵袭实验发现MSC可有效地抑制了8305C细胞的迁移和侵袭(P<0.05)。(6)经过不同浓度MSC干预后,8305C细胞的p-ERK1/2水平较对照组降低(P<0.05);联合ERK激动剂EGF,可减弱MSC对细胞增殖、迁移、侵袭的抑制作用(P<0.05);而联合通路抑制剂U0126则进一步增强MSC对8305C细胞的增殖、迁移、侵袭的抑制作用(P<0.05)。(7)MSC干预可降低8305C细胞MMP-2、MMP-9蛋白表达(P<0.05)。(8)与人Nthy-ori 3-1细胞比较,8305C细胞的葡萄糖消耗量和乳酸生成量明显增加(P<0.05),糖酵解途径的关键酶GLUT1、HK2、LDHA蛋白表达水平升高(P<0.05)。(9)MSC干预后,8305C细胞的葡萄糖消耗量和乳酸生成量降低(P<0.05),GLUT1、HK2、LDHA、HIF-1α蛋白表达水平下降,具有统计意义(P<0.05)。联合糖酵解抑制剂2-DG干预可以增强MSC对8305C细胞的抑制作用,进一步升高细胞内ROS(P<0.05)。结论:MSC作用于8305C细胞后,抑制ERK1/2信号通路,从而影响8305C细胞的增殖、凋亡、迁移、侵袭等恶性生物学行为,其中ROS发挥分子作用机制。与Nthy-ori 3-1细胞比较,8305C细胞具有更高的糖酵解水平。MSC作用于8305C细胞后,下调细胞的有氧糖酵解水平,降低HIF-1α水平,从而影响8305C细胞的恶性生物学行为。MSC联合2-DG产生协同氧化,增强MSC的抗肿瘤作用。更多还原

【Abstract】 Objective(s)Anaplastic thyroid carcinoma(ATC),is one of the fatal malignant tumors.Conventional treatments such as surgery,radiotherapy and chemotherapy cannot effectively stop the progression of ATC disease.At present,finding an effective treatment for ATC is still a huge problem to be faced in the clinical treatment process.Se-Methylselenocysteine(MSC),a kind of organic selenium,has been shown to have good anticancer properties in preclinical experiments and clinical studies,but the anticancer mechanism of ATC has not been clarified.In this study,we took ATC cell 8305C as the target to study the effects of MSC on its proliferation,apoptosis,invasion,migration and glycolysis,and to explore the roles and associations of MMPs,ERK1/2signaling pathways and glycolysis.Method(s)ATC cell 8305C were cultured in vitro and treated with different concentrations of MSC.After 8305C was treated by MSC,the changes of cell morphology and density were observed under phase contrast microscope.The proliferation inhibition rate of MSC on 8305C cells was detected by CCK-8assay.After Hoechst33258 staining,the changes of MSC on nuclear chromosome morphology of 8305C were observed.Intracellular mitochondrial membrane potential was detected by JC-1 probe to understand the effect of MSC on mitochondrial membrane potential of 8305C cells.Scratches(wound healing)and migration assays were used to detect the cell migration under the influence of MSC,and in vitro invasion assays were used to detect the cell invasion under the influence of MSC.DCFH-DA was used to detect the accumulation of intracellular ROS and combined with antioxidant NAC to verify the role of ROS in MSC in inhibiting the proliferation of cancer cells.The ERK1/2 and P-ERK1/2 proteins of ERK1/2 signaling pathway were detected by WB,and the transfer-related factors MMP-2 and MMP-9 proteins were detected.The role of ERK1/2 signaling pathway in the anti-tumor effect of MSC was verified by the combination of pathway agonist EGF and channel blocker U0126 respectively.ATC cell 8305C and human thyroid normal cell line Nthy-ori 3-1 were cultured in vitro.The content of lactate production and glucose consumption in the culture medium were detected using the kit.The expression levels of GLUT1,HK2,LDHA and HIF-1αwere detected by WB.The cell proliferation and intracellular ROS changes of 8305C cells were observed after the intervention of glycolysis inhibitor 2-DG combined with MSC.Result(s)(1)CCK-8 results showed that compared with normal cells,the cell activity of the administration group was significantly decreased,indicating that MSC had a growth inhibition effect on 8305C cell(P<0.05).(2)Compared with the control group,8305C cells in MSC administration group showed obvious apoptotic characteristics,cell shrinkage and significantly reduced number under phase contrast microscope.(3)Jc-1 probe detection showed that MSC caused the dose-dependent decrease of mitochondrial membrane potential of 8305C cells(P<0.05),and Hoenchst33258 staining suggested that MSC could induce apoptosis of 8305C cells.(4)Staining showed that MSC dose-dependant induced ROS accumulation in 8305C cells(P<0.05).(5)Scratch(wound healing test)and Transwell migration and invasion test showed that MSC could effectively inhibit migration and invasion of 8305C cells(P<0.05).(6)After MSC intervention,the level of p-ERK1/2 in ATC cells 8305C was lower than that in control group(P<0.05).Combined with EGF,the inhibition of MSC on cell proliferation,migration and invasion was weakened(P<0.05).The combined pathway inhibitor U0126 further inhibited the proliferation,migration and invasion of 8305C cells(P<0.05).(7)MSC intervention could decrease the expression of MMP-2 and MMP-9 protein in 8305C cells(P<0.05).(8)Compared with Nthy-ori 3-1 cell,glucose consumption and lactate production of 8305C were significantly increased(P<0.05),and the protein expression levels of GLUT1,HK2 and LDHA,the key enzymes of glycolysis pathway,were increased(P<0.05).(9)After MSC intervention,glucose consumption and lactate production of 8305C cells were decreased(P<0.05),GLUT1,HK2 and LDHA protein expression levels of key enzymes in glycolysis pathway also increased(P<0.05).Combined with glycolysis inhibitor 2-DG,the inhibition of MSC on 8305C cells was enhanced and intracellular ROS was further increased(P<0.05).Conclusion(s)After MSC was treated with 8305C cells,ERK1/2signaling pathway was inhibited,thus affecting the malignant biological behaviors of 8305C cells such as proliferation,apoptosis,migration and invasion,among which ROS played a molecular mechanism.Compared with Nthy-ori I3-1 cells,human undifferentiated thyroid cancer cell line 8305C had higher glycolysis level.After MSC was treated with 8305C cells,the level of aerobic glycolysis was down-regulated by lowering HIF-1α,thus affecting the malignant biological behavior of 8305C cells.The synergistic oxidation of MSC combined with 2-DG enhanced the anti-tumor effect of MSC.更多还原