E-cadherin相关卵巢癌细胞立体悬浮生长模型的构建及其特征

发布时间：2018-07-05 13:16

本文选题：卵巢癌 + E-cadherin　；参考：《山东大学》2014年硕士论文

【摘要】：研究背景：卵巢癌是妇科常见肿瘤,也是女性恶性肿瘤主要的死亡原因。由于早期卵巢癌无明显临床表现,大多数患者在诊断时已经处于晚期腹腔转移阶段。晚期卵巢癌患者腹腔中大量难治性腹水的出现,不仅加剧了患者的痛苦,也为卵巢癌细胞产生耐药和发生转移提供了必备的存活环境,由此决定了卵巢癌的不良预后。多细胞球体(Multicellular Spheroids, MCS)是卵巢癌细胞在腹水中的一种主要存在方式,越来越多的研究认为MCS的形成是卵巢癌细胞脱落后在腹水中存活的必要形式,是卵巢癌细胞成功发生转移的必要条件。所以,构建卵巢癌细胞的悬浮MCS模型对于研究晚期卵巢癌腹水中的肿瘤细胞特性,尤其是其对化疗药物的耐受性,具有重要的临床意义。作为肿瘤细胞非锚定依赖性生长的一种重要方式,悬浮MCS的生存和功能很大程度上依赖于细胞间粘附分子的作用。E-cadherin作为一种细胞间粘附分子,一度被认为是肿瘤抑制蛋白。近年研究发现,E-cadherin的作用并非如此简单。在乳腺癌中,E-cadherin表达下降或缺失预示着肿瘤细胞的恶性程度较高、预后较差,但E-cadherin表达也是乳腺癌细胞悬浮MCS中细胞粘附和聚集的必要条件。但需要注意的是,E-cadherin在卵巢癌中的作用与其他肿瘤存在很大差别。E-cadherin在卵巢癌细胞中的表达显著高于正常卵巢上皮细胞,并能通过介导细胞粘附激活卵巢癌细胞内的PI3K/AKT及MEK/ERK等信号通路促进卵巢癌细胞的生长和增殖,这高度提示E-cadherin在卵巢癌细胞悬浮MCS中可能发挥独特而重要的作用。但是,E-cadherin在卵巢癌细胞中的作用及机制仍存在很大争议。目的：卵巢癌是妇科恶性肿瘤的主要死因,而晚期卵巢癌患者腹水中存在的多细胞球体(Multicellular Spheroids, MCS)是转移卵巢癌细胞的主要存在方式。E-cadherin作为一种重要的细胞粘附分子,我们推测在MCS的形成和存活中发挥重要作用。因此我们建立高表达E-cadherin卵巢癌细胞的立体悬浮培养模型,并对其生长增殖行为和对化疗药物的耐药性进行初步研究。方法： 1.通过多聚2-羟乙基甲基丙烯酸脂(2-hydroxylethyl methacrylate, Poly-HEMA)凝胶阻止细胞贴壁,对E-cadherin表达水平各异的三株卵巢癌细胞进行立体悬浮培养,观察、比较其悬浮生长的过程、特点及差别； 2.通过钙离子剥夺实验证明E-cadherin在卵巢癌悬浮MCS中的作用； 3.通过CCK8细胞增殖实验检测MCS生长增殖特点； 4.通过CCK8细胞增殖实验检测MCS对化疗药物顺铂的敏感性。结果： 1.成功构建了高表达E-cadherin的SKOV-3卵巢癌细胞立体悬浮生长MCS模型。与低表达E-cadherin的OVCAR-3和不表达E-cadherin的SKOV-3形成的悬浮细胞团相比,高表达E-cadherin的卵巢癌细胞形成的MCS体积大、细胞连接紧密度高、维持时间长。 2.MCS在钙离子螯合剂EDTA的作用下逐渐分解,最后变为单个细胞或仅含几个细胞的微小细胞团块；对照组的悬浮MCS用胰酶消化,MCS裂解程度远差于EDTA作用的效果,仍呈大细胞团状。 3.高表达E-cadherin的SKOV-3卵巢癌细胞悬浮MCS (MCS of SKOV-3with high expression of E-cadherin, SK-H-M)、不表达E-cadherin的SKOV-3卵巢癌细胞悬浮MCS (MCS of SKOV-3with no expression of E-cadherin, SK-N-M)和低表达E-cadherin的OVCAR-3卵巢癌细胞MCS(MCS of OVCAR-3with low expression of E-cadherin, O V-L-M)在24、48、72h三个时间点的细胞增殖活性无显著差异(P0.05)；同一时间点,SK-H-M与SK-N-M和OV-L-M的细胞增殖活性亦无显著差异(P0.05)。 4.加入顺铂72h后,三种卵巢癌细胞悬浮MCS均显著低于相应贴壁细胞的死亡率(P0.05)；顺铂作用72h后,SK-H-M的死亡率显著低于SK-N-M和OV-L-M的死亡率(P0.05)；顺铂作用72h后的SK-H-A的死亡率显著低于SK-N-A和OV-L-A的死亡率(P0.05)。结论： 1.成功构建了模拟卵巢癌细胞转移的悬浮MCS模型(SK-H-M)。 2. E-cadherin促进卵巢癌细胞悬浮MCS的形成和维持。 3.卵巢癌细胞悬浮MCS处于增殖静止状态。 4.卵巢癌细胞悬浮MCS的耐药性与E-cadherin表达水平呈正相关,有可能是晚期卵巢癌治疗的重要干预靶点。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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