【摘要】：研究目的宮頸癌是婦科三大惡性腫瘤之一,最新統(tǒng)計(jì)預(yù)測(cè):在美國(guó)一年新發(fā)的宮頸癌約有12360例。宮頸癌的治療主要包括手術(shù)、放療、化療以及綜合治療等。放療是宮頸癌治療的重要手段之一,但放療抵抗成為限制療效的主要障礙。放療抵抗的因素很多:凋亡、環(huán)氧合酶(cyclooxygenases,COXs)、血管新生、低氧以及溫度作用等均可影響放療效果。Kruppel樣因子4(Krüppel-like factor 4,KLF4)是一轉(zhuǎn)錄因子,在人類組織中廣泛表達(dá),在生長(zhǎng)發(fā)育、突變及維持正常組織穩(wěn)態(tài)等生理活動(dòng)中發(fā)揮著重要作用。研究發(fā)現(xiàn)KLF4在放療損傷中表現(xiàn)出抗凋亡作用,通過(guò)協(xié)助DNA修復(fù)、降低細(xì)胞死亡、增加更新能力或三者共同作用等輔助細(xì)胞生存。體內(nèi)研究發(fā)現(xiàn)KLF4在調(diào)節(jié)BMI1+小腸干細(xì)胞內(nèi)穩(wěn)態(tài)和放療損傷后再生能力方面起著重要作用。目前有研究表明,放療后KLF4可導(dǎo)致細(xì)胞周期捕獲、抑制BAX表達(dá),最終降低細(xì)胞凋亡能力;對(duì)小鼠放療后KLF4也表現(xiàn)出抗凋亡作用。再者,我們課題申請(qǐng)前期預(yù)實(shí)驗(yàn)發(fā)現(xiàn)在HeLa細(xì)胞中KLF4表達(dá)與放療敏感性相關(guān)。于是我們推測(cè)KLF4與宮頸癌的放療敏感性可能相關(guān),如果對(duì)調(diào)節(jié)KLF4表達(dá)水平,是否能對(duì)宮頸癌細(xì)胞的放療敏感性產(chǎn)生影響,其分子機(jī)制如何?為了驗(yàn)證這一假設(shè),我們將從臨床資料回顧性研究、分子、細(xì)胞、以及動(dòng)物幾個(gè)層面進(jìn)行研究討論,這將為KLF4參與放療敏感性的機(jī)制研究奠定基礎(chǔ),為提高臨床放療效果提供潛在的藥物靶點(diǎn)。研究方法1.在組織層面,采用免疫組織化學(xué)技術(shù)檢測(cè)KLF4表達(dá),結(jié)合病例資料進(jìn)行相關(guān)性統(tǒng)計(jì)分析。2.在分子層面,使用免疫熒光、qPCR和western-blot檢測(cè)相關(guān)分子表達(dá),應(yīng)用RNAi技術(shù)和過(guò)表達(dá)技術(shù)構(gòu)建慢病毒載體,人源表達(dá)譜芯片檢測(cè)過(guò)表達(dá)KLF4后調(diào)節(jié)的下游基因,雙熒光素酶報(bào)告基因檢測(cè)啟動(dòng)子轉(zhuǎn)錄活性。3.在細(xì)胞層面,細(xì)胞流式術(shù)測(cè)細(xì)胞周期和凋亡,平板克隆測(cè)放療敏感性。4.在動(dòng)物層面,建立宮頸癌裸鼠皮下荷瘤模型,根據(jù)成瘤模型生長(zhǎng)進(jìn)一步觀察KLF4在宮頸癌放療中的作用,HE染色觀察組織形態(tài),免疫組織化學(xué)技術(shù)檢測(cè)增殖、凋亡相關(guān)蛋白表達(dá)水平。研究結(jié)果1.放療敏感性與FIGO分期、腫瘤直徑大小、以及腫瘤分化程度相關(guān);KLF4表達(dá)水平與FIGO分期以及腫瘤分化程度相關(guān);KLF4表達(dá)水平與放療敏感性相關(guān),在放療耐受組中表達(dá)高于敏感組。耐受組中KLF4的高表達(dá)與放療無(wú)反應(yīng)、放療后原位復(fù)發(fā)、遠(yuǎn)處轉(zhuǎn)移相關(guān)。KLF4高表達(dá)組總生存期和腫瘤無(wú)進(jìn)展生存期顯著低于低表達(dá)組。單/多因素回歸分析提示:KLF4表達(dá)水平、FIGO分期、腫瘤直徑大小、以及腫瘤分化程度是影響無(wú)瘤生存期和總生存時(shí)間的高風(fēng)險(xiǎn)因素。2.4株宮頸癌細(xì)胞中KLF4表達(dá)水平,其中以HeLa表達(dá)水平最高,C33A表達(dá)水平最低;比較HeLa和C33A的放療敏感性,C33A放療敏感性高于HeLa;成功構(gòu)建KLF4 RNAi和過(guò)表達(dá)慢病毒;過(guò)表達(dá)KLF4降低宮頸癌細(xì)胞放療敏感性,下調(diào)KLF4增強(qiáng)宮頸癌放療敏感性。3.成功建立宮頸癌裸鼠皮下荷瘤模型,KLF4過(guò)表達(dá)增加宮頸癌瘤體組織的放療抵抗性。下調(diào)KLF4增強(qiáng)宮頸癌瘤體組織的放療敏感性。4.過(guò)表達(dá)KLF4上調(diào)IGF2基因;過(guò)表達(dá)KLF4增強(qiáng)IGF2啟動(dòng)子轉(zhuǎn)錄活性;過(guò)表達(dá)KLF4可激活I(lǐng)GF2作用于PI3K-AKT通路從而參與放療抵抗作用。結(jié)論綜上所述,高表達(dá)KLF4抑制宮頸癌放療敏感性,干預(yù)KLF4表達(dá)水平可影響宮頸癌放療敏感性,KLF4可通過(guò)提高IGF2水平,激活PI3K-AKT通路,從而參與宮頸癌放療抵抗作用。實(shí)驗(yàn)為KLF4參與放療敏感性的機(jī)制研究奠定基礎(chǔ),為提高臨床放療效果提供潛在的藥物靶點(diǎn)。
[Abstract]:Research objective cervical cancer is one of the three major gynecologic malignancies. The latest statistical prediction is that there are about 12360 new cervical cancers in the United States. The treatment of cervical cancer mainly includes surgery, radiotherapy, chemotherapy and comprehensive treatment. Radiotherapy is one of the important methods for the treatment of cervical cancer, but radiation resistance is the main obstacle to the effect. There are many factors of resistance: apoptosis, cyclooxygenases (COXs), angiogenesis, hypoxia, and temperature action, which can affect the effect of radiation therapy,.Kruppel like factor 4 (Kr u ppel-like factor 4, KLF4), a transcription factor, which is widely expressed in human tissues, and plays a role in growth, mutation and maintenance of normal tissue homeostasis. Important role. The study found that KLF4 showed anti apoptotic effect in radiation injury and assisted cell survival by assisting DNA repair, reducing cell death, increasing the ability to renew or three common effects. In vivo studies have found that KLF4 plays an important role in regulating the homeostasis of BMI1+ small intestinal stem cells and regenerative ability after radiation injury. The study showed that KLF4 could lead to cell cycle capture, inhibit BAX expression, and eventually reduce cell apoptosis, and KLF4 also showed anti apoptosis effect on mice after radiotherapy. Furthermore, our preliminary preliminary preliminary experiment found that the expression of KLF4 in HeLa cells was related to the sensitivity of radiotherapy to radiotherapy, so we speculated that KLF4 was sensitive to radiotherapy of cervical cancer. What is the molecular mechanism of the effect of KLF4 expression level on radiation sensitivity of cervical cancer cells? In order to verify this hypothesis, we will discuss the clinical data in a retrospective study, molecular, cell, and animal studies, which will be a mechanism for the involvement of KLF4 in radiation sensitivity. Lay the foundation and provide potential drug targets for improving the effect of clinical radiotherapy. Method 1. at the tissue level, the expression of KLF4 was detected by immunohistochemical technique, and the correlation statistical analysis of the case data was carried out at the molecular level of.2.. Immunofluorescence, qPCR and Western-blot were used to detect the expression of related molecules, and RNAi technology and overwatch were used. The lentivirus vector was constructed by technology. The downstream gene was detected by human source expression chip, and the double luciferase reporter gene was used to detect the promoter transcriptional activity.3. at the cell level. Cell flow cytometry was used to detect cell cycle and apoptosis. The sensitivity of.4. at the animal level was measured by flat clones, and the subcutaneous tumor bearing model of cervical cancer in nude mice was established. The role of KLF4 in the radiotherapy of cervical cancer was further observed on the basis of the tumor growth model. HE staining was used to observe tissue morphology, immunohistochemical technique to detect proliferation and expression of apoptosis related proteins. Results 1. the results of radiotherapy sensitivity and FIGO staging, tumor diameter, and the degree of tumor differentiation were correlated with the level of KLF4 expression and FIGO staging. The level of tumor differentiation was related, the expression of KLF4 was associated with the sensitivity of radiotherapy. The expression of KLF4 was higher in the radiotherapy tolerance group than in the sensitive group. The high expression of the tumor in the tolerance group was not reacted with the radiotherapy, the recurrence in situ after radiotherapy, the total survival time and the progression free survival period of the distant metastasis related.KLF4 high expression group were significantly lower than that of the low expression group. The expression level of KLF4, FIGO stage, tumor diameter, and the degree of tumor differentiation were the high risk factors of KLF4 expression in.2.4 strain of.2.4 strain, which had the highest level of HeLa expression and the lowest level of C33A expression, compared with the radiotherapy sensitivity of HeLa and C33A, and the sensitivity of C33A radiotherapy was higher than that of HeLa and C33A. HeLa; successful construction of KLF4 RNAi and overexpression of lentivirus; overexpression of KLF4 to reduce the sensitivity of cervical cancer cell radiation, down regulation of KLF4 to enhance the sensitivity of cervical cancer radiation sensitivity.3. to establish a subcutaneous tumor bearing model of cervical cancer in nude mice, KLF4 overexpression to increase the radiation resistance of cervical cancer tissue. Down regulation of KLF4 to enhance the sensitivity of cervical cancer tissue to radiotherapy .4. overexpression of KLF4 up-regulated IGF2 gene, overexpressed KLF4 to enhance IGF2 promoter activity, overexpression of KLF4 can activate IGF2 to act on PI3K-AKT pathway and thus participate in radiation resistance. Conclusion high expression of KLF4 inhibits radiotherapy sensitivity of cervical cancer, and intervention of KLF4 expression can affect radiotherapy sensitivity of cervical cancer, KLF4 can be improved by improving The level of IGF2 activates the PI3K-AKT pathway and participates in the radiation resistance of cervical cancer. The experiment lays the foundation for the study of the mechanism of KLF4's involvement in radiation sensitivity, and provides potential drug targets for improving the effect of clinical radiotherapy.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33
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本文編號(hào)：2169816