本文選題：EFEMP2　切入點：子宮內膜癌　出處：《山東大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究依據(jù)子宮內膜癌(endometrial carcinoma,EC)是在女性生殖系統(tǒng)發(fā)生的惡性腫瘤,盡管早期子宮內膜癌患者的預后相對理想,但是超過30%的患者在發(fā)現(xiàn)時已處于腫瘤晚期,并且部分患者伴隨局部、盆腔甚至遠端轉移,因此預后不良。腫瘤轉移是造成患者死亡的主要原因,因此探索子宮內膜癌的侵襲、轉移機制,進一步闡明腫瘤的發(fā)展過程顯得尤為迫切。近年來,多種生物標記物已被證實與子宮內膜癌的發(fā)展有關,但EFEMP2在子宮內膜癌細胞侵襲轉移過程中的作用尚未見報道。EFEMP2(EGF containing fibulin-like extracellular matrix protein 2)是一種細胞外基質蛋白,主要參與彈性纖維的合成與排列。EFEMP2與皮膚松弛癥、主動脈夾層、骨關節(jié)炎、惡性腫瘤等多種疾病有關。為了探索EFEMP2在子宮內膜癌增殖、侵襲、轉移中的作用,本研究首次探討EFEMP2在子宮內膜癌組織及細胞系中的表達情況及相關機制。研究目的1.探究EFEMP2在子宮內膜癌組織、細胞中的表達及其意義。2.探究慢病毒轉染后不同侵襲能力子宮內膜癌細胞的功能轉變。3.探究EFEMP2對子宮內膜癌細胞侵襲轉移的作用機制。研究方法1.免疫組化(IHC)探究EFEMP2在正常子宮內膜組織及子宮內膜癌組織中的表達情況;2.單細胞克隆技術,從KLE、ISK中分離出具有高侵襲能力的克隆細胞亞系KLE-1、ISK-1和低侵襲能力的克隆細胞亞系KLE-28、ISK-23;3.逆轉錄-聚合酶鏈反應(RT-qPCR)、免疫印跡(Western blot)、免疫細胞化學(ICC)檢測EFEMP2在正常子宮內膜、子宮內膜癌細胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆細胞亞系中的表達情況;4.生長曲線、軟瓊脂克隆形成實驗檢測子宮內膜癌細胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆細胞亞系的增殖能力;5.Transwell侵襲轉移實驗檢測子宮內膜癌細胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆細胞亞系的侵襲轉移能力;6.慢病毒介導EFEMP2在高侵襲克隆細胞亞系過表達及RNA干擾沉默EFEMP2在低侵襲克隆細胞亞系表達,并驗證轉染效率;7.建立裸鼠移植瘤模型,體內模擬子宮內膜癌發(fā)展過程,進一步探究EFEMP2在子宮內膜癌細胞增殖、侵襲、轉移過程中的作用;8.Western blot、RT-qPCR檢測EFEMP2與子宮內膜癌細胞上皮間質轉化(EMT)之間的關系。研究結果1.EFEMP2在正常子宮內膜組織中陽性表達率高,在子宮內膜癌組織中陽性表達率低(P0.05);EFEMP2的表達情況與子宮內膜癌組織分化、淋巴結轉移、以及不良預后密切相關(P0.05);2.EFEMP2在正常子宮內膜細胞中高表達,在子宮內膜癌細胞中低表達;在高侵襲克隆細胞亞系中低表達,在低侵襲克隆細胞亞系高表達(P0.05);3.慢病毒介導EFEMP2過表達后高侵襲克隆細胞增殖能力、侵襲和轉移能力減弱;RNA干擾沉默EFEMP2表達后低侵襲克隆細胞增殖能力、侵襲和轉移能力增強(P0.05);4.裸鼠移植瘤模型:高侵襲克隆細胞亞系組腫瘤生長速度快,體積大,低侵襲克隆細胞亞系組腫瘤生長速度慢,體積小;EFEMP2過表達組成瘤率較低,瘤體體積小,肺轉移率低,EFEMP2干擾組成瘤率高,瘤體體積大,肺轉移率高(P0.05);5.EFEMP2 過表達組 E-cadherin 表達升高,N-cadherin、Vimentin、Snail、Slug、Twist的表達降低,抑制EMT;EFEMP2干擾組,E-cadherin表達降低,Vimentin、Snail、Slug、Twist 的表達升高,促進 EMT(P0.05)。研究結論1.EFEMP2是子宮內膜癌的一個抑癌因子,能夠抑制子宮內膜癌細胞侵襲、轉移;2.EFEMP2抑制子宮內膜癌細胞EMT,進一步抑制子宮內膜癌細胞侵襲、轉移。
[Abstract]:On the basis of endometrial carcinoma (endometrial, carcinoma, EC) in the occurrence of the female reproductive system malignant tumors, although the early prognosis of patients with endometrial cancer is relatively ideal, but more than 30% of patients in the discovery has been in advanced cancer, and some patients with local, pelvic even distant metastasis, poor prognosis. Therefore tumor metastasis is the result of the main cause of death in patients, so the exploration of endometrial cancer invasion, metastasis, and further clarify the process of tumor development is particularly urgent. In recent years, a variety of biological markers have been linked to the development of endometrial cancer, but EFEMP2 in endometrial cancer cell invasion and metastasis process has not been reported in.EFEMP2 (EGF containing fibulin-like extracellular matrix protein 2) is an extracellular matrix protein, mainly involved in the synthesis and arrangement of elastic fibers and skin.EFEMP2 Skin relaxation, aortic dissection, osteoarthritis, malignant tumors and other diseases. In order to explore EFEMP2 invasion in endometrial carcinoma, proliferation, metastasis, this is the first study to investigate the expression of EFEMP2 in endometrial cancer tissues and cells and the related mechanism. The purpose of the study is to explore 1. EFEMP2 in endometrial carcinoma the organization, function change of.3..2. expression and significance of the research of lentiviral transfection after invasion of endometrial cancer cells to explore the mechanism of action of EFEMP2 on invasion and metastasis of endometrial carcinoma cells. Methods: 1. immunohistochemical (IHC) expression of EFEMP2 in normal endometrium and endometrial carcinoma; 2. single cell cloning technique from KLE, isolation and cloning of KLE-1 cells with high invasive ability of ISK, cloning cell lines KLE-28, ISK-1 and ISK-23 3. low invasion; reverse transcriptase Polymerase chain reaction (RT-qPCR) and immunoblotting (Western blot), immunocytochemistry (ICC) detection of EFEMP2 in normal endometrium, endometrial carcinoma cell lines (KLE, ISK, HEC-1A, HEC-1B) and cloned cell lines in expression; 4. growth curve, soft agar colony formation assay of cancer cells endometrial lines (KLE, ISK, HEC-1A, HEC-1B) and clonal cell sublines proliferation of cancer cells; experimental detection of endometrial 5.Transwell invasion and metastasis (KLE, ISK, HEC-1A, HEC-1B) and cloning cell line invasion and metastasis ability; 6. lentivirus mediated EFEMP2 overexpression and RNA interference to silence EFEMP2 in highly invasive cell cloning expression in low invasive clone cell line, and to verify the transfection efficiency; 7. nude mice in vivo, simulation and development of endometrial cancer, further research on the EFEMP2 in the invasion and proliferation of endometrial cancer cells, metastasis The role of 8.Western blot, RT-qPCR EFEMP2; and the detection of endometrial cancer cell epithelial mesenchymal transition (EMT). The relationship between the results of 1.EFEMP2 in normal endometrial tissues. The positive expression rate is high, the positive expression in endometrial carcinoma was low (P0.05); the expression and differentiation of EFEMP2 in endometrial carcinoma the lymph node metastasis, and the prognosis is closely related (P0.05); the expression of 2.EFEMP2 in normal endometrial cells, low expression in endometrial cancer cells; low expression in highly invasive clonal cell sublines, high expression in low invasive clone cell line (P0.05); 3. lentivirus mediated overexpression of EFEMP2 after the high invasive cell proliferation clone, weaken the ability of invasion and metastasis; RNA interference silencing EFEMP2 expression after low invasive clonal cell proliferation ability, enhance the ability of invasion and metastasis (P0.05); xenograft model in nude mice: 4. grams of high invasion Lung cell line tumor growth speed, large volume, low invasive clonal cell line tumor growth is slow, the volume is small; the over expression of EFEMP2 of tumor formation rate is low, the tumor volume is small, the low rate of lung metastasis, EFEMP2 interference of tumor formation rate, tumor volume and lung metastasis rate (P0.05); 5.EFEMP2 overexpression increased the expression of E-cadherin group N-cadherin, Vimentin, Snail, Slug, Twist expression decreased, the inhibition of EMT; EFEMP2 interference group, decreased expression of E-cadherin, Vimentin, Snail, Slug, increased the expression of Twist, promote EMT (P0.05). Conclusions 1.EFEMP2 is a tumor suppressor gene in endometrial cancer, can inhibit the invasion of endometrial cancer cell metastasis; 2.EFEMP2 inhibits endometrial cancer cell EMT and further inhibit the endometrial cancer cell invasion and metastasis.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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