本文選題：EFEMP2　切入點(diǎn)：子宮內(nèi)膜癌　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究依據(jù)子宮內(nèi)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(nèi)膜癌患者的預(yù)后相對(duì)理想,但是超過(guò)30%的患者在發(fā)現(xiàn)時(shí)已處于腫瘤晚期,并且部分患者伴隨局部、盆腔甚至遠(yuǎn)端轉(zhuǎn)移,因此預(yù)后不良。腫瘤轉(zhuǎn)移是造成患者死亡的主要原因,因此探索子宮內(nèi)膜癌的侵襲、轉(zhuǎn)移機(jī)制,進(jìn)一步闡明腫瘤的發(fā)展過(guò)程顯得尤為迫切。近年來(lái),多種生物標(biāo)記物已被證實(shí)與子宮內(nèi)膜癌的發(fā)展有關(guān),但EFEMP2在子宮內(nèi)膜癌細(xì)胞侵襲轉(zhuǎn)移過(guò)程中的作用尚未見報(bào)道。EFEMP2(EGF containing fibulin-like extracellular matrix protein 2)是一種細(xì)胞外基質(zhì)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(dòng)脈夾層、骨關(guān)節(jié)炎、惡性腫瘤等多種疾病有關(guān)。為了探索EFEMP2在子宮內(nèi)膜癌增殖、侵襲、轉(zhuǎn)移中的作用,本研究首次探討EFEMP2在子宮內(nèi)膜癌組織及細(xì)胞系中的表達(dá)情況及相關(guān)機(jī)制。研究目的1.探究EFEMP2在子宮內(nèi)膜癌組織、細(xì)胞中的表達(dá)及其意義。2.探究慢病毒轉(zhuǎn)染后不同侵襲能力子宮內(nèi)膜癌細(xì)胞的功能轉(zhuǎn)變。3.探究EFEMP2對(duì)子宮內(nèi)膜癌細(xì)胞侵襲轉(zhuǎn)移的作用機(jī)制。研究方法1.免疫組化(IHC)探究EFEMP2在正常子宮內(nèi)膜組織及子宮內(nèi)膜癌組織中的表達(dá)情況;2.單細(xì)胞克隆技術(shù),從KLE、ISK中分離出具有高侵襲能力的克隆細(xì)胞亞系KLE-1、ISK-1和低侵襲能力的克隆細(xì)胞亞系KLE-28、ISK-23;3.逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-qPCR)、免疫印跡(Western blot)、免疫細(xì)胞化學(xué)(ICC)檢測(cè)EFEMP2在正常子宮內(nèi)膜、子宮內(nèi)膜癌細(xì)胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆細(xì)胞亞系中的表達(dá)情況;4.生長(zhǎng)曲線、軟瓊脂克隆形成實(shí)驗(yàn)檢測(cè)子宮內(nèi)膜癌細(xì)胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆細(xì)胞亞系的增殖能力;5.Transwell侵襲轉(zhuǎn)移實(shí)驗(yàn)檢測(cè)子宮內(nèi)膜癌細(xì)胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆細(xì)胞亞系的侵襲轉(zhuǎn)移能力;6.慢病毒介導(dǎo)EFEMP2在高侵襲克隆細(xì)胞亞系過(guò)表達(dá)及RNA干擾沉默EFEMP2在低侵襲克隆細(xì)胞亞系表達(dá),并驗(yàn)證轉(zhuǎn)染效率;7.建立裸鼠移植瘤模型,體內(nèi)模擬子宮內(nèi)膜癌發(fā)展過(guò)程,進(jìn)一步探究EFEMP2在子宮內(nèi)膜癌細(xì)胞增殖、侵襲、轉(zhuǎn)移過(guò)程中的作用;8.Western blot、RT-qPCR檢測(cè)EFEMP2與子宮內(nèi)膜癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化(EMT)之間的關(guān)系。研究結(jié)果1.EFEMP2在正常子宮內(nèi)膜組織中陽(yáng)性表達(dá)率高,在子宮內(nèi)膜癌組織中陽(yáng)性表達(dá)率低(P0.05);EFEMP2的表達(dá)情況與子宮內(nèi)膜癌組織分化、淋巴結(jié)轉(zhuǎn)移、以及不良預(yù)后密切相關(guān)(P0.05);2.EFEMP2在正常子宮內(nèi)膜細(xì)胞中高表達(dá),在子宮內(nèi)膜癌細(xì)胞中低表達(dá);在高侵襲克隆細(xì)胞亞系中低表達(dá),在低侵襲克隆細(xì)胞亞系高表達(dá)(P0.05);3.慢病毒介導(dǎo)EFEMP2過(guò)表達(dá)后高侵襲克隆細(xì)胞增殖能力、侵襲和轉(zhuǎn)移能力減弱;RNA干擾沉默EFEMP2表達(dá)后低侵襲克隆細(xì)胞增殖能力、侵襲和轉(zhuǎn)移能力增強(qiáng)(P0.05);4.裸鼠移植瘤模型:高侵襲克隆細(xì)胞亞系組腫瘤生長(zhǎng)速度快,體積大,低侵襲克隆細(xì)胞亞系組腫瘤生長(zhǎng)速度慢,體積小;EFEMP2過(guò)表達(dá)組成瘤率較低,瘤體體積小,肺轉(zhuǎn)移率低,EFEMP2干擾組成瘤率高,瘤體體積大,肺轉(zhuǎn)移率高(P0.05);5.EFEMP2 過(guò)表達(dá)組 E-cadherin 表達(dá)升高,N-cadherin、Vimentin、Snail、Slug、Twist的表達(dá)降低,抑制EMT;EFEMP2干擾組,E-cadherin表達(dá)降低,Vimentin、Snail、Slug、Twist 的表達(dá)升高,促進(jìn) EMT(P0.05)。研究結(jié)論1.EFEMP2是子宮內(nèi)膜癌的一個(gè)抑癌因子,能夠抑制子宮內(nèi)膜癌細(xì)胞侵襲、轉(zhuǎn)移;2.EFEMP2抑制子宮內(nèi)膜癌細(xì)胞EMT,進(jìn)一步抑制子宮內(nèi)膜癌細(xì)胞侵襲、轉(zhuǎn)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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