本文選題：子宮內膜癌　切入點：上皮間質轉化　出處：《華中科技大學》2016年博士論文　論文類型：學位論文

【摘要】：[目的]肌層浸潤及遠處侵襲轉移是影響子宮內膜癌患者預后的主要因素。近年來越來越多的研究表明,EMT在子宮內膜癌的原位侵襲及遠處轉移中發(fā)揮著重要的作用,嚴重影響患者的預后。研究還發(fā)現(xiàn),缺氧可以誘導EMT過程,同時鈣敏感受體CaSR在其中可能發(fā)揮著關鍵作用。因此,我們探討缺氧與子宮內膜癌EMT之間的聯(lián)系,并挖掘CaSR在其中扮演的角色,闡明子宮內膜癌EMT的發(fā)生及其有關的調節(jié)因子,為后續(xù)如何阻斷這一機制的發(fā)生和尋找抑制子宮內膜癌復發(fā)轉移的方法提供新思路。[方法]1.IHC法檢測正常和子宮內膜癌組織中HIF-1α和EMT標志分子E-cadherin、 vimentin的表達水平；2.IF法觀察缺氧前后子宮內膜癌細胞系Ishikawa、Kle的細胞骨架及E-cadherin、vimentin的定位和表達水平：3.瞬時轉染干擾HIF-1 α表達;4. Transwell模型觀察缺氧或質粒轉染細胞后其遷移和侵襲能力;5. RT-PCR和Western blotting檢測缺氧或轉染后細胞HIF-1α、E-cadherin、 vimentin、Snail和CaSR轉錄表達變化；6.構建慢病毒轉染細胞沉默及過表達CaSR,熒光染料Fluo-2/AM示蹤監(jiān)測細胞內鈣離子變化以驗證CaSR表達量改變后的功能變化;RT-PCR、Western blotting和IF法分析轉染前后細胞EMT相關蛋白變化及功能變化;7.CCK-8法檢測細胞凋亡。1.與正常之宮內膜組織相比較,子宮內膜癌中HIF-1α和vimentin表達增高(P0.05),而E-cadherin降低,但無統(tǒng)計學意義(P0.05);E-cadherin表達與HIF-1α、vimentin呈負相關(P0.05),提示缺氧可能與EMT具有相關性。2.缺氧后細胞骨架重建,細胞之間連接變稀疏,間隙增寬,部分細胞伸出偽足,運動能力增強,向梭形樣改變。同時E-cadherin表達降低,vimenti n表達增高,結果顯示缺氧后可以促進子宮內膜癌細胞呈EMT表型變化。3.缺氧后HIF-1α、EMT相關分子vimentin、Snail轉錄表達增高(P0.05),而E-cadherin轉錄表達降低,CaSR的轉錄表達也明顯增高(P0.01),可見缺氧后子宮內膜癌發(fā)生EMT改變,并伴隨著CaSR表達的增加。4.缺氧后HIF-1α表達增加,質粒轉染可以成功干擾HIF-1α表達;HIF-1α的增加可以促進子宮內膜癌EMT改變,但沉默HIF-1α表達后可以抑制缺氧引起的EMT表型產(chǎn)生,同時伴有腫瘤細胞侵襲能力的減弱(P0.05),但受到CaSR的負性調控。5.沉默及過表達子宮內膜癌細胞CaSR后細胞內鈣離子內流分別相應的減少、增加;沉默CaSR表達促進細胞增殖,過表達CaSR抑制細胞的增殖；CaSR可以抑制H IF-1α表達,同時抑制EMT形成(E-cadherin增高及Vimentin降低),其伴隨著β-catenin的表達增加及定位的改變。[結論]缺氧后子宮內膜癌可以發(fā)生EMT,其通路至少部分依賴HIF-1 α的增加,但受CaSR負性調節(jié)。缺氧還可以促進CaSR表達,進而抑制子宮內膜癌細胞的增殖及改變β-catenin胞膜／胞漿定位,參與抑制EMT發(fā)生,但其內在機制還有待進一步深究,以為干擾子宮內膜癌EMT調控提供更可靠的理論基礎。
[Abstract]:[objective] Myometrium infiltration and distant invasion and metastasis are the main factors influencing the prognosis of endometrial carcinoma. In recent years, more and more studies have shown that EMT plays an important role in the in situ invasion and distant metastasis of endometrial carcinoma. The study also found that hypoxia can induce the process of EMT, in which calcium sensitive receptor CaSR may play a key role. Therefore, we explore the relationship between hypoxia and EMT in endometrial carcinoma. And excavate the role of CaSR in it to elucidate the occurrence of EMT in endometrial carcinoma and its related regulatory factors. To provide a new way to block the occurrence of this mechanism and to find a way to inhibit the recurrence and metastasis of endometrial carcinoma. [methods] 1.IHC method was used to detect the expression of E-cadherin and vimentin in normal and endometrial carcinoma tissues. The cytoskeleton of endometrial carcinoma cell line Ishikawaa Kle and the localization and expression level of E-cadherin vimentin were observed before and after hypoxia. Transient transfection interfered with the expression of HIF-1 偽. The Transwell model was used to observe the ability of migration and invasion after hypoxia or plasmid transfection. 5. RT-PCR and Western blotting. To detect the transcriptional changes of HIF-1 偽 偽 -E-cadherin, vimentininSnail and CaSR after hypoxia or transfection. To construct lentivirus-transfected cells for silencing and overexpression of CaSR, fluorescent dye Fluo-2/AM tracer was used to monitor the changes of intracellular calcium in order to verify the functional changes of RT-PCRG after the changes of CaSR expression. Blotting and if methods were used to analyze the changes of EMT related proteins and their functions before and after transfection. The apoptosis of cells was detected by CCK-8 method and compared with normal endometrial tissue. In endometrial carcinoma, the expression of HIF-1 偽 and vimentin increased and E-cadherin decreased, but the expression of E-cadherin was negatively correlated with HIF-1 偽 vimentin, suggesting that hypoxia may have a correlation with EMT. 2. After hypoxia, the cytoskeleton was reconstructed, the connections between cells became sparse, and the gap widened. Some of the cells extended pseudopodia, increased their motor ability and changed to spindle shape. Meanwhile, the expression of E-cadherin decreased, and the expression of vimenti n increased. The results showed that hypoxia could promote the expression of EMT phenotypic changes in endometrial carcinoma cells. After hypoxia, the expression of HIF-1 偽 -EMT-related molecule vimentininSnail was increased (P0.05A), while the expression of E-cadherin decreased significantly (P 0.01). The EMT changes in endometrial carcinoma after hypoxia were observed. With the increase of CaSR expression, the expression of HIF-1 偽 was increased after hypoxia. Plasmid transfection could successfully interfere with the expression of HIF-1 偽 and increase the expression of HIF-1 偽, but silencing the expression of HIF-1 偽 could inhibit the phenotypic production of EMT induced by hypoxia. At the same time, the invasion ability of tumor cells was weakened (P 0.05), but it was regulated negatively by CaSR. 5. The intracellular calcium influx decreased and increased after silencing and overexpression of endometrial cancer cells CaSR, and the silencing of CaSR expression promoted cell proliferation. Overexpression of CaSR inhibits cell proliferation and CaSR can inhibit the expression of H IF-1 偽. At the same time, the increase of E-cadherin and the decrease of Vimentin were inhibited by EMT, which was accompanied by the increase of 尾 -catenin expression and the change of localization. [conclusion] EMTs may occur in endometrial carcinoma after hypoxia, and its pathway depends at least in part on the increase of HIF-1 偽. However, under the negative regulation of CaSR, hypoxia can also promote the expression of CaSR, thus inhibit the proliferation of endometrial cancer cells and change the localization of 尾 -catenin membrane / cytoplasm, and participate in the inhibition of EMT, but the underlying mechanism remains to be further studied. To provide a more reliable theoretical basis for interfering with EMT regulation of endometrial carcinoma.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.33

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