本文選題：子宮內(nèi)膜癌 + 環(huán)巴胺��； 參考：《河北北方學(xué)院》2017年碩士論文

【摘要】：子宮內(nèi)膜癌是常見的婦科惡性腫瘤之一,治療方法主要包括手術(shù)、放療、化療、激素等,其中化療是治療子宮內(nèi)膜癌的重要輔助治療手段。目前常用的治療方案是以鉑類為基礎(chǔ)的聯(lián)合化療,雖然在一定程度上可延長患者生存期,但腫瘤細(xì)胞產(chǎn)生的耐藥性及化療不良反應(yīng),是導(dǎo)致子宮內(nèi)膜癌復(fù)發(fā)及治療失敗的主要原因,因此,探索治療子宮內(nèi)膜癌的靶向藥物具有重要意義。環(huán)巴胺(cyclopamine,CYP)是Shh(Sonic hedgehog)信號通路的特異性阻斷劑,通過改變Shh通路組分Smo(smoothened)的空間結(jié)構(gòu)而抑制Smo的活性,直接阻斷Shh信號通路的激活,阻止其下游靶基因表達(dá)。目前,已在多種腫瘤中證實CYP可抑制腫瘤細(xì)胞增殖。有報道證實CYP可抑制子宮內(nèi)膜癌細(xì)胞系Ishikawa及HHUA細(xì)胞的增殖,但CYP對人子宮內(nèi)膜癌HEC-1A細(xì)胞的抑制作用及相應(yīng)調(diào)控機制尚無報道。本實驗通過體外培養(yǎng)人子宮內(nèi)膜癌細(xì)胞HEC-1A,采用0、5、10、20、40μmol·L~(-1)不同劑量的CYP處理HEC-1A細(xì)胞24 h,48 h或72 h后,倒置顯微鏡及瑞氏吉姆薩染色觀察細(xì)胞形態(tài)改變,AO/EB雙染法觀察死亡細(xì)胞,CCK-8法檢測細(xì)胞增殖,流式細(xì)胞術(shù)AnnexinⅤ-FITC/PI法檢測細(xì)胞發(fā)生的凋亡率,Q-PCR法檢測Bax和Bcl-2基因的表達(dá)水平,劃痕實驗檢測細(xì)胞遷移能力,流式細(xì)胞術(shù)檢測細(xì)胞周期及細(xì)胞自噬相關(guān)蛋白LC3-B的表達(dá),蛋白質(zhì)印跡法(Western blot)檢測LC3-B蛋白表達(dá)水平。結(jié)果表明:不同劑量的CYP處理HEC-1A細(xì)胞可使細(xì)胞形態(tài)發(fā)生明顯改變,且具有CYP時間濃度依賴性;AO/EB染色結(jié)果表明,CYP可誘發(fā)HEC-1A細(xì)胞大量死亡;CCK-8結(jié)果顯示,CYP可顯著抑制HEC-1A細(xì)胞的增殖(P0.05);流式細(xì)胞術(shù)檢測結(jié)果發(fā)現(xiàn)CYP可導(dǎo)致HEC-1A細(xì)胞凋亡,且隨著CYP劑量的升高HEC-1A細(xì)胞發(fā)生凋亡的比率顯著提高(P0.05);Q-PCR檢測凋亡相關(guān)基因表達(dá),結(jié)果顯示,CYP處理可誘導(dǎo)HEC-1A細(xì)胞上調(diào)Bax基因表達(dá),下調(diào)Bcl-2基因表達(dá);CYP處理組HEC-1A細(xì)胞的遷移能力顯著下降(P0.05);CYP可使HEC-1A細(xì)胞周期阻滯于G_0/G1期;并可誘導(dǎo)HEC-1A細(xì)胞內(nèi)LC3-B蛋白聚集,且呈時間-劑量依賴性;LC3-B蛋白表達(dá)水平隨CYP濃度增加而增高。結(jié)論提示:CYP可抑制人子宮內(nèi)膜癌細(xì)胞HEC-1A的存活并誘導(dǎo)其發(fā)生凋亡,同時可阻滯細(xì)胞周期運轉(zhuǎn),引發(fā)細(xì)胞自噬并抑制遷移。
[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，

本文編號：1778206