本文選題：自噬 + UCH-L1��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究自噬相關(guān)基因LC3Ⅱ、P62及泛素蛋白酶體家族成員UCH-L1在甲狀腺癌病人甲狀腺組織和PBMC中的表達情況;研究二甲雙胍、雷帕霉素對甲狀腺癌BCPAP細胞生長增殖的影響;比較分析二甲雙胍、雷帕霉素處理BCPAP細胞后LC3Ⅱ、P62和UCH-L1基因和蛋白水平上的表達差異,為甲狀腺癌的治療提供新靶點和新思路。方法:1.CCK-8檢測不同濃度的(0.5、1、2.5、5、10m M)二甲雙胍及不同濃度的(1、5、10、15、20μM)雷帕霉素對甲狀腺癌BCPAP細胞生長增殖的影響。2.應(yīng)用Real time PCR法和Western blot法分別檢測對照組、二甲雙胍組、雷帕霉素組、二甲雙胍+3-MA組及雷帕霉素+3-MA組處理后,LC3Ⅱ、P62和UCH-L1mRNA與蛋白的表達。3.透射電鏡觀察對照組、二甲雙胍組、雷帕霉素組,二甲雙胍+3-MA組及雷帕霉素+3-MA組處理后BCPAP細胞內(nèi)自噬小體的改變。4.應(yīng)用Real time PCR法檢測60例甲狀腺癌患者PBMC中LC3Ⅱ、P62和UCH-L1 mRNA表達水平,并與30例健康對照者相比較;檢測10例甲狀腺癌患者癌組織和癌旁組織中LC3Ⅱ、P62和UCH-L1 mRNA表達水平。結(jié)果:1.與各自對照組相比,1μM的雷帕霉素處理組、0.5 m M的二甲雙胍處理組細胞存活率稍有下降,但差異無統(tǒng)計學(xué)意義(p0.05),5、10、15、20μM的雷帕霉素處理組、1、2.5、5、10m M的二甲雙胍處理組細胞存活率明顯下降,差異均有統(tǒng)計學(xué)意義(p0.01)。隨著雷帕霉素或二甲雙胍濃度不斷增加,細胞存活率也不斷降低,且具有濃度依賴關(guān)系。2.與對照組相比,雷帕霉素處理組LC3ⅡmRNA相對表達量上調(diào)(p0.01)、P62和UCH-L1 mRNA相對表達量下調(diào)(p0.01);與雷帕霉素組相比,雷帕霉素+3-MA組LC3ⅡmRNA相對表達量下調(diào)(p0.05)、P62和UCH-L1 mRNA相對表達量上調(diào)(p0.01、p0.05)。與對照組相比,二甲雙胍組LC3ⅡmRNA相對表達量上調(diào)(p0.01)、P62和UCH-L1 mRNA相對表達量下調(diào)(p0.01);與二甲雙胍組相比,二甲雙胍+3-MA組LC3ⅡmRNA相對表達量下調(diào)(p0.01)、P62和UCH-L1 mRNA相對表達量上調(diào)(p0.01)。3.與對照組相比,雷帕霉素組LC3Ⅱ在蛋白水平表達上調(diào)(p0.01)、P62和UCH-L1在蛋白水平表達下調(diào)(p0.01、p0.05);與雷帕霉素組相比,雷帕霉素+3-MA組LC3Ⅱ在蛋白水平表達下調(diào)(p0.05)、P62和UCH-L1在蛋白水平表達上調(diào)(p0.01、p0.05)。與對照組相比,二甲雙胍組LC3Ⅱ在蛋白水平表達上調(diào)(p0.01)、P62和UCH-L1在蛋白水平表達下調(diào)(p0.01);與二甲雙胍組相比,二甲雙胍+3-MA組LC3Ⅱ在蛋白水平表達下調(diào)(p0.05)、P62和UCH-L1在蛋白水平表達上調(diào)(p0.01、p0.05)。4.通過透射電鏡觀可以察到,對照組中BCPAP細胞內(nèi)細胞核完整,線粒體豐富并且完整。與對照組相比,雷帕霉素組、二甲雙胍組BCPAP細胞內(nèi)自噬體雙層膜結(jié)構(gòu)明顯增多;分別與雷帕霉素組、二甲雙胍組相比,雷帕霉素+3-MA組、二甲雙胍+3-MA組細胞內(nèi)自噬小體明顯減少。5.在PBMC中,與健康對照組相比,無淋巴轉(zhuǎn)移組、淋巴轉(zhuǎn)移組LC3ⅡmRNA相對表達量均下調(diào),差異有統(tǒng)計學(xué)意義(p0.01)。與健康對照組相比,淋巴轉(zhuǎn)移組P62 mRNA相對表達量上調(diào),差異有統(tǒng)計學(xué)意義(p0.05);而P62的表達在健康對照組與無淋巴轉(zhuǎn)移組之間、無淋巴轉(zhuǎn)移組與淋巴轉(zhuǎn)移組之間差異均無統(tǒng)計學(xué)意義。UCH-L1 mRNA相對表達量在健康對照組、無淋巴轉(zhuǎn)移組和淋巴轉(zhuǎn)移組之間差異均無統(tǒng)計學(xué)意義。在甲狀腺組織中,相較于癌旁組織,癌組織中LC3ⅡmRNA相對表達量下調(diào),而P62和UCH-L1 mRNA相對表達量上調(diào),差異均有統(tǒng)計學(xué)意義(p0.01)。結(jié)論:1.二甲雙胍和雷帕霉素均可有效抑制甲狀腺癌BCPAP細胞增殖,該增殖抑制作用與自噬有關(guān)。二甲雙胍、雷帕霉素誘導(dǎo)自噬并造成增殖抑制作用可以為治療甲狀腺癌提供新思路和新途徑。2.甲狀腺癌的發(fā)生可能與自噬水平有關(guān)。3.UCH-L1可能作為促癌因子與甲狀腺癌的形成有關(guān),且UCH-L1可能參與到甲狀腺癌細胞的自噬過程。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R736.1

【參考文獻】

相關(guān)期刊論文 前1條

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本文編號：1828730