环巴胺抑制子宫内膜癌细胞生长及机制探讨

发布时间：2018-04-20 14:48

  本文选题：子宫内膜癌 + 环巴胺　； 参考：《河北北方学院》2017年硕士论文

【摘要】：子宫内膜癌是常见的妇科恶性肿瘤之一,治疗方法主要包括手术、放疗、化疗、激素等,其中化疗是治疗子宫内膜癌的重要辅助治疗手段。目前常用的治疗方案是以铂类为基础的联合化疗,虽然在一定程度上可延长患者生存期,但肿瘤细胞产生的耐药性及化疗不良反应,是导致子宫内膜癌复发及治疗失败的主要原因,因此,探索治疗子宫内膜癌的靶向药物具有重要意义。环巴胺(cyclopamine,CYP)是Shh(Sonic hedgehog)信号通路的特异性阻断剂,通过改变Shh通路组分Smo(smoothened)的空间结构而抑制Smo的活性,直接阻断Shh信号通路的激活,阻止其下游靶基因表达。目前,已在多种肿瘤中证实CYP可抑制肿瘤细胞增殖。有报道证实CYP可抑制子宫内膜癌细胞系Ishikawa及HHUA细胞的增殖,但CYP对人子宫内膜癌HEC-1A细胞的抑制作用及相应调控机制尚无报道。本实验通过体外培养人子宫内膜癌细胞HEC-1A,采用0、5、10、20、40μmol·L~(-1)不同剂量的CYP处理HEC-1A细胞24 h,48 h或72 h后,倒置显微镜及瑞氏吉姆萨染色观察细胞形态改变,AO/EB双染法观察死亡细胞,CCK-8法检测细胞增殖,流式细胞术AnnexinⅤ-FITC/PI法检测细胞发生的凋亡率,Q-PCR法检测Bax和Bcl-2基因的表达水平,划痕实验检测细胞迁移能力,流式细胞术检测细胞周期及细胞自噬相关蛋白LC3-B的表达,蛋白质印迹法(Western blot)检测LC3-B蛋白表达水平。结果表明:不同剂量的CYP处理HEC-1A细胞可使细胞形态发生明显改变,且具有CYP时间浓度依赖性;AO/EB染色结果表明,CYP可诱发HEC-1A细胞大量死亡;CCK-8结果显示,CYP可显著抑制HEC-1A细胞的增殖(P0.05);流式细胞术检测结果发现CYP可导致HEC-1A细胞凋亡,且随着CYP剂量的升高HEC-1A细胞发生凋亡的比率显著提高(P0.05);Q-PCR检测凋亡相关基因表达,结果显示,CYP处理可诱导HEC-1A细胞上调Bax基因表达,下调Bcl-2基因表达;CYP处理组HEC-1A细胞的迁移能力显著下降(P0.05);CYP可使HEC-1A细胞周期阻滞于G_0/G1期;并可诱导HEC-1A细胞内LC3-B蛋白聚集,且呈时间-剂量依赖性;LC3-B蛋白表达水平随CYP浓度增加而增高。结论提示:CYP可抑制人子宫内膜癌细胞HEC-1A的存活并诱导其发生凋亡,同时可阻滞细胞周期运转,引发细胞自噬并抑制迁移。
[Abstract]:Endometrial carcinoma is one of the most common gynecological malignancies. The main treatment methods include surgery, radiotherapy, chemotherapy, hormones and so on. Among them, chemotherapy is an important adjuvant treatment for endometrial carcinoma. At present, the commonly used treatment is platinum-based combination chemotherapy. Although it can prolong the patient's survival to a certain extent, the drug resistance and adverse reaction of chemotherapy are produced by tumor cells. It is the main cause of recurrence and failure in treatment of endometrial carcinoma. Therefore, it is of great significance to explore targeted drugs for the treatment of endometrial carcinoma. Cyp is a specific inhibitor of Shh(Sonic hedgehog signaling pathway. Cyp inhibits the activity of Smo by changing the spatial structure of Shh pathway component, thus directly blocking the activation of Shh signaling pathway and blocking its downstream target gene expression. At present, CYP has been proved to inhibit the proliferation of tumor cells in a variety of tumors. It has been reported that CYP can inhibit the proliferation of endometrial carcinoma cell lines Ishikawa and HHUA, but the inhibitory effect of CYP on human endometrial carcinoma HEC-1A cells and the corresponding regulatory mechanism have not been reported. In this study, human endometrial cancer cell HEC-1A was cultured in vitro. HEC-1A cells were treated with different doses of CYP for 24 h or 72 h. The morphologic changes of cells were observed by inverted microscope and Giemsa staining. The cell proliferation was detected by CCK-8 method and the expression of Bax and Bcl-2 gene was detected by flow cytometry (Annexin 鈪，

本文编号：1778206