mTOR抑制剂对甲状腺癌BCPAP细胞增殖的影响及其机制研究

发布时间：2018-05-01 09:23

本文选题：自噬 + UCH-L1　；参考：《大连医科大学》2017年硕士论文

【摘要】：目的:研究自噬相关基因LC3Ⅱ、P62及泛素蛋白酶体家族成员UCH-L1在甲状腺癌病人甲状腺组织和PBMC中的表达情况;研究二甲双胍、雷帕霉素对甲状腺癌BCPAP细胞生长增殖的影响;比较分析二甲双胍、雷帕霉素处理BCPAP细胞后LC3Ⅱ、P62和UCH-L1基因和蛋白水平上的表达差异,为甲状腺癌的治疗提供新靶点和新思路。方法:1.CCK-8检测不同浓度的(0.5、1、2.5、5、10m M)二甲双胍及不同浓度的(1、5、10、15、20μM)雷帕霉素对甲状腺癌BCPAP细胞生长增殖的影响。2.应用Real time PCR法和Western blot法分别检测对照组、二甲双胍组、雷帕霉素组、二甲双胍+3-MA组及雷帕霉素+3-MA组处理后,LC3Ⅱ、P62和UCH-L1mRNA与蛋白的表达。3.透射电镜观察对照组、二甲双胍组、雷帕霉素组,二甲双胍+3-MA组及雷帕霉素+3-MA组处理后BCPAP细胞内自噬小体的改变。4.应用Real time PCR法检测60例甲状腺癌患者PBMC中LC3Ⅱ、P62和UCH-L1 mRNA表达水平,并与30例健康对照者相比较;检测10例甲状腺癌患者癌组织和癌旁组织中LC3Ⅱ、P62和UCH-L1 mRNA表达水平。结果:1.与各自对照组相比,1μM的雷帕霉素处理组、0.5 m M的二甲双胍处理组细胞存活率稍有下降,但差异无统计学意义(p0.05),5、10、15、20μM的雷帕霉素处理组、1、2.5、5、10m M的二甲双胍处理组细胞存活率明显下降,差异均有统计学意义(p0.01)。随着雷帕霉素或二甲双胍浓度不断增加,细胞存活率也不断降低,且具有浓度依赖关系。2.与对照组相比,雷帕霉素处理组LC3ⅡmRNA相对表达量上调(p0.01)、P62和UCH-L1 mRNA相对表达量下调(p0.01);与雷帕霉素组相比,雷帕霉素+3-MA组LC3ⅡmRNA相对表达量下调(p0.05)、P62和UCH-L1 mRNA相对表达量上调(p0.01、p0.05)。与对照组相比,二甲双胍组LC3ⅡmRNA相对表达量上调(p0.01)、P62和UCH-L1 mRNA相对表达量下调(p0.01);与二甲双胍组相比,二甲双胍+3-MA组LC3ⅡmRNA相对表达量下调(p0.01)、P62和UCH-L1 mRNA相对表达量上调(p0.01)。3.与对照组相比,雷帕霉素组LC3Ⅱ在蛋白水平表达上调(p0.01)、P62和UCH-L1在蛋白水平表达下调(p0.01、p0.05);与雷帕霉素组相比,雷帕霉素+3-MA组LC3Ⅱ在蛋白水平表达下调(p0.05)、P62和UCH-L1在蛋白水平表达上调(p0.01、p0.05)。与对照组相比,二甲双胍组LC3Ⅱ在蛋白水平表达上调(p0.01)、P62和UCH-L1在蛋白水平表达下调(p0.01);与二甲双胍组相比,二甲双胍+3-MA组LC3Ⅱ在蛋白水平表达下调(p0.05)、P62和UCH-L1在蛋白水平表达上调(p0.01、p0.05)。4.通过透射电镜观可以察到,对照组中BCPAP细胞内细胞核完整,线粒体丰富并且完整。与对照组相比,雷帕霉素组、二甲双胍组BCPAP细胞内自噬体双层膜结构明显增多;分别与雷帕霉素组、二甲双胍组相比,雷帕霉素+3-MA组、二甲双胍+3-MA组细胞内自噬小体明显减少。5.在PBMC中,与健康对照组相比,无淋巴转移组、淋巴转移组LC3ⅡmRNA相对表达量均下调,差异有统计学意义(p0.01)。与健康对照组相比,淋巴转移组P62 mRNA相对表达量上调,差异有统计学意义(p0.05);而P62的表达在健康对照组与无淋巴转移组之间、无淋巴转移组与淋巴转移组之间差异均无统计学意义。UCH-L1 mRNA相对表达量在健康对照组、无淋巴转移组和淋巴转移组之间差异均无统计学意义。在甲状腺组织中,相较于癌旁组织,癌组织中LC3ⅡmRNA相对表达量下调,而P62和UCH-L1 mRNA相对表达量上调,差异均有统计学意义(p0.01)。结论:1.二甲双胍和雷帕霉素均可有效抑制甲状腺癌BCPAP细胞增殖,该增殖抑制作用与自噬有关。二甲双胍、雷帕霉素诱导自噬并造成增殖抑制作用可以为治疗甲状腺癌提供新思路和新途径。2.甲状腺癌的发生可能与自噬水平有关。3.UCH-L1可能作为促癌因子与甲状腺癌的形成有关,且UCH-L1可能参与到甲状腺癌细胞的自噬过程。
[Abstract]:Objective: To study the expression of autophagy related gene LC3 II, P62 and the members of the ubiquitin proteasome family in thyroid tissue and PBMC of thyroid cancer patients; to study the effect of metformin and rapamycin on the growth and proliferation of BCPAP cells in thyroid carcinoma, and to compare and analyze LC3 II, P62 and UCH-L1 after BCPAP cells treated by rapamycin. Differential expression on the level of gene and protein to provide new targets and new ideas for the treatment of thyroid cancer. Methods: 1.CCK-8 detection of different concentrations (0.5,1,2.5,5,10m M) metformin and different concentrations (1,5,10,15,20 M) effect of rapamycin on the growth and proliferation of thyroid carcinoma BCPAP cells.2. application Real time PCR method and Western blot method Do not detect the control group, the metformin group, the rapamycin group, the metformin +3-MA group and the group +3-MA of rapamycin, the expression of LC3 II, P62 and UCH-L1mRNA and the protein expression.3. transmission electron microscope observation group, the metformin group, the rapamycin group, the metformin +3-MA group and the ramamycin +3-MA group after treatment, the change.4. of the autophagic corpuscle in the BCPAP cell.4. The expression level of LC3 II, P62 and UCH-L1 mRNA in 60 patients with thyroid cancer was detected by Real time PCR method and compared with 30 healthy controls. The expression level of LC3 II, P62 and UCH-L1 mRNA in the cancer tissues and adjacent tissues of 10 thyroid cancer patients was detected. Results: 1. compared with each group, the 1 micron rapamycin treatment group was 0.5. The cell survival rate of metformin treatment group decreased slightly, but the difference was not statistically significant (P0.05). The cell survival rate of 1,2.5,5,10m M treated group of rapamycin was significantly decreased, the difference was statistically significant (P0.01). With the increasing concentration of rapamycin or metformin, the cell survival rate was also continuous. Compared with the control group, the relative expression of LC3 II mRNA in the rapamycin treatment group was up up (P0.01) and the relative expression of P62 and UCH-L1 mRNA was down (P0.01), and the relative expression of LC3 II mRNA in the rapamycin group was higher than that of the rapamycin group, compared with the control group, and the relative expression of the LC3 II mRNA in the rapamycin group was up (P0.05), and the relative expression of mRNA was up. Compared with the control group, the relative expression of LC3 II mRNA in the metformin group was up (P0.01), and the relative expression of P62 and UCH-L1 mRNA was down (P0.01). Compared with the metformin group, the relative expression of LC3 II mRNA in the group of metformin +3-MA was down down (P0.01), and the relative expression was up to the control group compared with the control group. The expression of white level was up-regulated (P0.01), and the expression of P62 and UCH-L1 was down regulated (P0.01, P0.05). Compared with the rapamycin group, the expression of LC3 II in the rapamycin group +3-MA LC3 II was down regulated (P0.05), P62 and UCH-L1 increased in protein level (P0.01, P0.05). The expression of UCH-L1 was down regulated at protein level (P0.01); compared with metformin group, the expression of LC3 II in metformin group +3-MA was down regulated (P0.05), P62 and UCH-L1 were up-regulated in protein level (P0.01, P0.05).4. through transmission electron microscopy, and the nuclei of the BCPAP cells in the control group were intact, and the mitochondria were rich and complete. Compared with the group of rapamycin and metformin group, the autophagic double layer membrane structure increased significantly in the BCPAP cells. Compared with the rapamycin group and the metformin group, the autophagic body in the group +3-MA of rapamycin and the metformin +3-MA group decreased the.5. in PBMC significantly. Compared with the healthy group, there was no lymphatic metastasis group and LC3 II mRNA phase in the lymph node group. The difference was statistically significant (P0.01). Compared with the healthy control group, the relative expression of P62 mRNA in the lymph node group was up, and the difference was statistically significant (P0.05), while the expression of P62 was not statistically significant between the healthy control group and the lymph node free group, and there was no significant difference between the lymphatic metastasis group and the lymphatic metastasis group.UCH-L1 mRNA. There was no significant difference in the relative expression between the non lymphatic and lymph node groups in the healthy control group. In the thyroid tissue, the relative expression of LC3 II mRNA in the carcinoma tissue was down, while the relative expression of P62 and UCH-L1 mRNA was up, and the difference was statistically significant (P0.01). Conclusion: 1. metformin and Rapa Mycophencin can effectively inhibit the proliferation of thyroid cancer BCPAP cells. The proliferation inhibition is related to autophagy. Metformin, rapamycin induced autophagy and proliferation inhibition can provide new ideas and new pathways for the treatment of thyroid cancer. The occurrence of.2. thyroid cancer may be related to autophagic water level related to.3.UCH-L1 as a cancer promoting factor and a UCH-L1 may be involved in the autophagy process of thyroid cancer cells.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R736.1

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