【摘要】：背景：宮頸癌是最常見的婦科惡性腫瘤之一,居女性癌癥患者死亡原因的第4位。近年來發(fā)病率逐漸增高,尤其是發(fā)展中國家。除HPV感染外,宿主與細胞生長分化相關(guān)的基因表達異常在宮頸癌的發(fā)病機制中同樣具有重要作用。近年來,臨床前基礎(chǔ)研究表明宮頸癌基因治療是很有潛力的治療手段,其中包括在細胞周期控制和凋亡信號中具有重要作用的p53基因治療、宮頸癌基因治療中研究較多的Bax基因治療、HPVE2和p21基因以及針對癌基因的反義RNA技術(shù)治療和宮頸癌治療的理想候選基因FHIT和PTEN基因的研究。我們課題組前期研究利用RNA-Seq技術(shù),發(fā)現(xiàn)細胞周期蛋白家族中的cyclin O在宮頸癌組織中的表達水平明顯高于癌旁組織,提示其編碼基因CCNO可能與宮頸癌的發(fā)生發(fā)展相關(guān)。目的：研究下調(diào)CCNO基因表達對宮頸癌Hela細胞株體外增殖、細胞周期及凋亡的影響,為研究宮頸癌生物治療提供理論依據(jù)。材料：病理組織切片來源于北京協(xié)和醫(yī)學(xué)院婦產(chǎn)科宮頸癌患者,包括癌灶組織及癌旁組織樣本,剔除術(shù)前接受放射治療、化學(xué)治療以及未能獲取滿意存檔石蠟組織標本者；Hela細胞株,為人類宮頸腺癌細胞株,購自上海細胞庫(TCHu187,中國上海細胞典藏委員會)。方法1.應(yīng)用免疫熒光檢測CCNO在宮頸癌組織中的表達情況；2.設(shè)計靶向CCNO小干擾RNA (CCNO-siRNA),將其轉(zhuǎn)染入體外培養(yǎng)的宮頸癌Hela細胞株細胞中,沉默細胞內(nèi)源性CCNO基因表達；3.應(yīng)用Western blotting方法檢測CCNO-siRNA的轉(zhuǎn)染效率,確定CCNO基因被沉默,可用于后續(xù)實驗；4.應(yīng)用CCK-8方法檢測CCNO對宮頸癌細胞增殖的影響；5.應(yīng)用流式細胞分析技術(shù)檢測CCNO對細胞周期轉(zhuǎn)化、細胞凋亡的影響。結(jié)果1.免疫熒光檢測宮頸癌中CCNO基因表達高于癌旁組織；2.轉(zhuǎn)染CCNO-siRNA可特異高效地下調(diào)宮頸癌Hela細胞CCNO蛋白表達水平；3.在轉(zhuǎn)染72 h、96 h后,CCK-8法顯示CCNO-siRNA轉(zhuǎn)染實驗組生長速率明顯低于對照組,差異有統(tǒng)計學(xué)意義(P0.05,P0.01)；4.細胞周期檢測顯示,Hela細胞轉(zhuǎn)染CCNO-siRNA實驗組G1期細胞較對照組增多均7%左右(P0.05),而S期細胞比例則減少12%左右(P0.05)；5.細胞凋亡檢測顯示,Hela細胞轉(zhuǎn)染CCNO-siRNA實驗組較對照組凋亡細胞增加約17%左右(P0.05)。結(jié)論1. CCNO在宮頸癌組織中的表達高于癌旁組織；2.靶向CCNO-siRNA可抑制宮頸腺癌Hela細胞中內(nèi)源性CCNO的表達；3. CCNO對體外培養(yǎng)的宮頸腺癌Hela細胞具有促進細胞增殖、促進細胞周期轉(zhuǎn)化和抑制凋亡作用。
[Abstract]:Background: cervical cancer is one of the most common gynecological malignancies and is the fourth leading cause of death in female cancer patients. In recent years, the incidence of disease has gradually increased, especially in developing countries. In addition to HPV infection, the abnormal expression of host genes associated with cell growth and differentiation also plays an important role in the pathogenesis of cervical cancer. In recent years, preclinical basic studies have shown that gene therapy for cervical cancer is a potential therapy, including p53 gene therapy, which plays an important role in cell cycle control and apoptosis signal. Bax gene therapy, HPVE2 and p21 gene, antisense RNA therapy for oncogene and ideal candidate genes FHIT and PTEN for cervical cancer therapy were studied. Our previous study using RNA-Seq technique showed that the expression of cyclin O in the cyclin family was significantly higher than that in the adjacent tissues, suggesting that the CCNO encoding gene might be related to the occurrence and development of cervical cancer. Aim: to study the effect of down-regulation of CCNO gene expression on proliferation, cell cycle and apoptosis of cervical cancer Hela cell line in vitro. Materials: histopathological sections were obtained from cervical cancer patients in obstetrics and gynecology of Peking Union Medical College, including samples of cancer focus and adjacent tissues. Those receiving preoperative radiotherapy, chemotherapy and failing to obtain satisfactory archived paraffin tissue specimens were excluded. Hela cell line, a human cervical adenocarcinoma cell line, was purchased from Shanghai Cell Bank (TCHu187, Shanghai Cell Collection Committee). Method 1. Immunofluorescence was used to detect the expression of CCNO in cervical carcinoma. 2. Small interfering RNA (CCNO-siRNA) targeting CCNO was designed and transfected into cervical cancer Hela cell line in vitro, and the expression of endogenous CCNO gene was silenced. 3. Western blotting method was used to detect the transfection efficiency of CCNO-siRNA and to confirm that the CCNO gene was silenced and could be used in subsequent experiments. 4. CCK-8 assay was used to detect the effect of CCNO on the proliferation of cervical cancer cells. Flow cytometry was used to detect the effect of CCNO on cell cycle transformation and apoptosis. Result 1. The expression of CCNO gene in cervical carcinoma was higher than that in adjacent tissues by immunofluorescence. Transfection of CCNO-siRNA can specifically and efficiently down-regulate the expression of CCNO protein in cervical cancer Hela cells. CCK-8 assay showed that the growth rate of the experimental group transfected with CCNO-siRNA was significantly lower than that of the control group at 72 h or 96 h after transfection, and the difference was statistically significant (P0.05, P0.01). Cell cycle analysis showed that compared with the control group, the G1 phase of Hela cells transfected with CCNO-siRNA increased by about 7% (P0.05), while the percentage of S phase cells decreased by 12% (P0.05); Apoptosis assay showed that Hela cells transfected with CCNO-siRNA increased by about 17% compared with control group (P0.05). Conclusion 1. The expression of CCNO in cervical cancer tissues was higher than that in paracancerous tissues. Targeted CCNO-siRNA could inhibit the expression of endogenous CCNO in cervical adenocarcinoma Hela cells. CCNO can promote cell proliferation, promote cell cycle transformation and inhibit apoptosis of cervical adenocarcinoma Hela cells cultured in vitro.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.33
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本文編號：2275211