本文選題：卵巢癌 + E-cadherin��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：研究背景： 卵巢癌是婦科常見腫瘤,也是女性惡性腫瘤主要的死亡原因。由于早期卵巢癌無明顯臨床表現(xiàn),大多數(shù)患者在診斷時已經(jīng)處于晚期腹腔轉(zhuǎn)移階段。晚期卵巢癌患者腹腔中大量難治性腹水的出現(xiàn),不僅加劇了患者的痛苦,也為卵巢癌細(xì)胞產(chǎn)生耐藥和發(fā)生轉(zhuǎn)移提供了必備的存活環(huán)境,由此決定了卵巢癌的不良預(yù)后。多細(xì)胞球體(Multicellular Spheroids, MCS)是卵巢癌細(xì)胞在腹水中的一種主要存在方式,越來越多的研究認(rèn)為MCS的形成是卵巢癌細(xì)胞脫落后在腹水中存活的必要形式,是卵巢癌細(xì)胞成功發(fā)生轉(zhuǎn)移的必要條件。所以,構(gòu)建卵巢癌細(xì)胞的懸浮MCS模型對于研究晚期卵巢癌腹水中的腫瘤細(xì)胞特性,尤其是其對化療藥物的耐受性,具有重要的臨床意義。 作為腫瘤細(xì)胞非錨定依賴性生長的一種重要方式,懸浮MCS的生存和功能很大程度上依賴于細(xì)胞間粘附分子的作用。E-cadherin作為一種細(xì)胞間粘附分子,一度被認(rèn)為是腫瘤抑制蛋白。近年研究發(fā)現(xiàn),E-cadherin的作用并非如此簡單。在乳腺癌中,E-cadherin表達(dá)下降或缺失預(yù)示著腫瘤細(xì)胞的惡性程度較高、預(yù)后較差,但E-cadherin表達(dá)也是乳腺癌細(xì)胞懸浮MCS中細(xì)胞粘附和聚集的必要條件。但需要注意的是,E-cadherin在卵巢癌中的作用與其他腫瘤存在很大差別。E-cadherin在卵巢癌細(xì)胞中的表達(dá)顯著高于正常卵巢上皮細(xì)胞,并能通過介導(dǎo)細(xì)胞粘附激活卵巢癌細(xì)胞內(nèi)的PI3K/AKT及MEK/ERK等信號通路促進卵巢癌細(xì)胞的生長和增殖,這高度提示E-cadherin在卵巢癌細(xì)胞懸浮MCS中可能發(fā)揮獨特而重要的作用。但是,E-cadherin在卵巢癌細(xì)胞中的作用及機制仍存在很大爭議。 目的： 卵巢癌是婦科惡性腫瘤的主要死因,而晚期卵巢癌患者腹水中存在的多細(xì)胞球體(Multicellular Spheroids, MCS)是轉(zhuǎn)移卵巢癌細(xì)胞的主要存在方式。E-cadherin作為一種重要的細(xì)胞粘附分子,我們推測在MCS的形成和存活中發(fā)揮重要作用。因此我們建立高表達(dá)E-cadherin卵巢癌細(xì)胞的立體懸浮培養(yǎng)模型,并對其生長增殖行為和對化療藥物的耐藥性進行初步研究。 方法： 1.通過多聚2-羥乙基甲基丙烯酸脂(2-hydroxylethyl methacrylate, Poly-HEMA)凝膠阻止細(xì)胞貼壁,對E-cadherin表達(dá)水平各異的三株卵巢癌細(xì)胞進行立體懸浮培養(yǎng),觀察、比較其懸浮生長的過程、特點及差別； 2.通過鈣離子剝奪實驗證明E-cadherin在卵巢癌懸浮MCS中的作用； 3.通過CCK8細(xì)胞增殖實驗檢測MCS生長增殖特點； 4.通過CCK8細(xì)胞增殖實驗檢測MCS對化療藥物順鉑的敏感性。 結(jié)果： 1.成功構(gòu)建了高表達(dá)E-cadherin的SKOV-3卵巢癌細(xì)胞立體懸浮生長MCS模型。與低表達(dá)E-cadherin的OVCAR-3和不表達(dá)E-cadherin的SKOV-3形成的懸浮細(xì)胞團相比,高表達(dá)E-cadherin的卵巢癌細(xì)胞形成的MCS體積大、細(xì)胞連接緊密度高、維持時間長。 2.MCS在鈣離子螯合劑EDTA的作用下逐漸分解,最后變?yōu)閱蝹�細(xì)胞或僅含幾個細(xì)胞的微小細(xì)胞團塊；對照組的懸浮MCS用胰酶消化,MCS裂解程度遠(yuǎn)差于EDTA作用的效果,仍呈大細(xì)胞團狀。 3.高表達(dá)E-cadherin的SKOV-3卵巢癌細(xì)胞懸浮MCS (MCS of SKOV-3with high expression of E-cadherin, SK-H-M)、不表達(dá)E-cadherin的SKOV-3卵巢癌細(xì)胞懸浮MCS (MCS of SKOV-3with no expression of E-cadherin, SK-N-M)和低表達(dá)E-cadherin的OVCAR-3卵巢癌細(xì)胞MCS(MCS of OVCAR-3with low expression of E-cadherin, O V-L-M)在24、48、72h三個時間點的細(xì)胞增殖活性無顯著差異(P0.05)；同一時間點,SK-H-M與SK-N-M和OV-L-M的細(xì)胞增殖活性亦無顯著差異(P0.05)。 4.加入順鉑72h后,三種卵巢癌細(xì)胞懸浮MCS均顯著低于相應(yīng)貼壁細(xì)胞的死亡率(P0.05)；順鉑作用72h后,SK-H-M的死亡率顯著低于SK-N-M和OV-L-M的死亡率(P0.05)；順鉑作用72h后的SK-H-A的死亡率顯著低于SK-N-A和OV-L-A的死亡率(P0.05)。 結(jié)論： 1.成功構(gòu)建了模擬卵巢癌細(xì)胞轉(zhuǎn)移的懸浮MCS模型(SK-H-M)。 2. E-cadherin促進卵巢癌細(xì)胞懸浮MCS的形成和維持。 3.卵巢癌細(xì)胞懸浮MCS處于增殖靜止?fàn)顟B(tài)。 4.卵巢癌細(xì)胞懸浮MCS的耐藥性與E-cadherin表達(dá)水平呈正相關(guān),有可能是晚期卵巢癌治療的重要干預(yù)靶點。
[Abstract]:Research background:
Ovarian cancer is a common gynecologic tumor and also the main cause of death in female malignant tumors. Due to the absence of obvious clinical manifestations of early ovarian cancer, most patients have been in the stage of advanced peritoneal metastasis. The occurrence of a large number of refractory ascites in the abdominal cavity of advanced ovarian cancer patients is not only aggravating the sufferings of the patients, but also for the ovarian cancer cells. Multicellular Spheroids (MCS) is a major mode of existence of ovarian cancer cells in ascites. A growing number of studies suggest that the formation of MCS is the necessary form of ovarian cancer cells to survive in ascites after the ovarian cancer cells fall off. It is a necessary condition for the successful metastasis of ovarian cancer cells. Therefore, the construction of the MCS model of ovarian cancer cells is of great significance in the study of the tumor cell characteristics in the ascites of advanced ovarian cancer, especially the tolerance to chemotherapy drugs.
As an important way of non anchorage dependent growth of tumor cells, the survival and function of suspended MCS depend largely on the role of intercellular adhesion molecules as an intercellular adhesion molecule, which was once considered as a tumor suppressor. In recent years, the study found that the role of E-cadherin is not so simple. In breast cancer, it is found that the role of.E-cadherin is not so simple. The decrease or deletion of E-cadherin indicates that the malignant degree of tumor cells is higher and the prognosis is poor, but E-cadherin expression is also a necessary condition for cell adhesion and aggregation in MCS of breast cancer cells. However, it should be noted that the role of E-cadherin in ovarian cancer is very different from that of other swollen tumors,.E-cadherin in ovarian cancer cells. The expression is significantly higher than normal ovarian epithelial cells, and can promote the growth and proliferation of ovarian cancer cells by mediating cell adhesion and activating the PI3K/AKT and MEK/ERK signaling pathways in ovarian cancer cells. This suggests that E-cadherin may play a unique and important role in ovarian cancer cell suspension of MCS. However, E-cadherin is in ovarian cancer. There is still a lot of controversy in the role and mechanism of cells.
Objective:
Ovarian cancer is the main cause of death in gynecologic malignancies, and the Multicellular Spheroids (MCS) in the ascites of patients with advanced ovarian cancer is the main way to transfer ovarian cancer cells.E-cadherin as an important cell adhesion molecule. We speculate that it plays an important role in the formation and survival of MCS. Therefore, we should play an important role in the formation and survival of MCS. A three-dimensional suspension culture model of ovarian cancer cells with high expression of E-cadherin was established, and the growth and proliferation behavior and drug resistance of chemotherapeutic drugs were preliminarily studied.
Method:
1. 2- hydroxyethyl methacrylate (Poly-HEMA) gel (2-hydroxylethyl methacrylate, Poly-HEMA) gel was used to prevent cell adhesion, and the three-dimensional suspension culture of ovarian cancer cells with different E-cadherin expression levels was carried out, and the process, characteristics and difference of the suspension growth were compared.
2. the role of E-cadherin in ovarian cancer suspension MCS was demonstrated by calcium deprivation test.
3. the growth and proliferation characteristics of MCS were detected by CCK8 cell proliferation assay.
4. the sensitivity of MCS to cisplatin was detected by CCK8 cell proliferation assay.
Result:
1. the solid suspension growth model of SKOV-3 ovarian cancer cells with high expression of E-cadherin was successfully constructed. Compared with the OVCAR-3 of low expression E-cadherin and the suspended cell group that did not express the SKOV-3 of E-cadherin, the ovarian cancer cells with high expression of E-cadherin have a large MCS volume, high cell connection density and long maintenance time.
2.MCS was gradually decomposed under the action of calcium ion chelating agent EDTA, and finally became a single cell or small cell mass with only a few cells. The control group's levitation MCS was digested with trypsin, and the degree of MCS fragmentation was far worse than the effect of EDTA, but it still showed large cell mass.
3. SKOV-3 ovarian cancer cells with high expression of E-cadherin (MCS of SKOV-3with high expression of E-cadherin, SK-H-M). There was no significant difference in cell proliferation activity between ession of E-cadherin and O V-L-M at three time points of 24,48,72h (P0.05), and there was no significant difference in cell proliferation activity between SK-H-M and SK-N-M and OV-L-M at the same time point (P0.05).
After 4. cisplatin 72h, the suspension of MCS in ovarian cancer cells was significantly lower than that of the corresponding adherent cells (P0.05). After cisplatin action 72h, the mortality of SK-H-M was significantly lower than that of SK-N-M and OV-L-M (P0.05), and the mortality of SK-H-A after cisplatin action 72h was significantly lower than that of SK-N-A and OV-L-A.
Conclusion:
1. a suspension MCS model (SK-H-M) for ovarian cancer cell migration was successfully constructed.
2. E-cadherin promotes the formation and maintenance of MCS in ovarian cancer cells.
3. ovarian cancer cell suspension MCS is in a static state of proliferation.
4. the drug resistance of ovarian cancer cell suspension MCS is positively correlated with the expression level of E-cadherin. It may be an important intervention target for the treatment of advanced ovarian cancer.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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