本文關鍵詞：c-MET對人食管鱗癌Eca109細胞放射敏感性影響的相關性實驗研究　出處：《山東大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 食管鱗狀細胞癌 c-Met 放射敏感性 分子機制

【摘要】：研究背景與目的:食管癌是最常見的消化道惡性腫瘤之一,全世界每年新發(fā)病例在增加,其中,半數(shù)以上食管癌發(fā)生在中國。據(jù)《2015中國癌癥統(tǒng)計》報道,我國食管癌發(fā)病率已躍居全國惡性腫瘤發(fā)病的第3位,死亡居第4位。手術、放療以及化療等相結(jié)合的多學科綜合治療是食管癌的主要治療模式。雖然近年來各種治療手段都有了很大程度的發(fā)展,但食管癌總體預后仍較差,5年生存率僅15%～25%。另外,患者預后個體差異也較大,這很大程度上取決于食管癌的生物學行為,尤其是不同基因特征對腫瘤增殖、侵襲、轉(zhuǎn)移及治療療效產(chǎn)生至關重要的影響。目前食管癌的特異性分子標志物并不明確。MET(Mesenchymal-epithelial Transition Factor)是肝細胞生長因子(Hepatocyte Growth Factor,HGF)的細胞表面受體,也被叫做c-Met或HGFR(Hepatocyte Growth Factor Receptor),屬于酪氨酸激酶受體(Receptor Tyrosine Kinase,RTK)家族成員,由c-Met基因編碼。異常的RTK信號途徑在腫瘤的發(fā)生、發(fā)展、侵襲、轉(zhuǎn)移以及耐藥的過程中具有重要作用,而c-Met信號通路正屬于RTK信號通路。研究發(fā)現(xiàn)c-Met信號通路的激活與多種腫瘤的發(fā)生、發(fā)展、轉(zhuǎn)移及放化療耐受密切相關,如結(jié)腸癌、胃癌、卵巢癌、頭頸部鱗癌、肺癌、乳頭狀腎癌、三陰性乳腺癌、前列腺癌等。在食管腺癌中,c-Met的表達率為340%～540%,且與預后明顯相關。而在亞裔食管鱗癌中,其同樣存在著較高的表達率,約34%～69%。因此,c-Met可能成為食管鱗癌治療新的靶點。另一方面,放射治療是食管癌的重要治療手段,雖然近年來放療設備和放療技術有了很大的改進,但放療抵抗依然是影響食管鱗癌放療療效的一個重要原因。放療抵抗的產(chǎn)生是一個多基因、多因子、多機制共同作用的復雜過程,與細胞周期阻滯、相關基因改變、腫瘤微環(huán)境變化、自噬性調(diào)節(jié)及腫瘤干細胞的存在等多種因素相關。一些臨床研究顯示c-Met的表達水平與放療療效存在相關性�；A研究表明腫瘤細胞和/或間質(zhì)細胞受到電離輻射后,能夠激活c-Met傳導通路,進而引起腫瘤細胞的放射抵抗。因此,抑制c-Met信號通路有可能減少放療抵抗,從而提高放療療效。綜上所述,研究c-Met與食管鱗癌放射敏感性的相關性具有理論基礎和積極的臨床意義。我們的前期研究已提示c-Met蛋白在食管鱗癌中的表達存在差異,且與患者的預后顯著相關(p=0.001)。亞組分析還提示,在接受術后輔助放療的患者中,c-Met高表達者預后顯著差于低表達者(p=0.002)。在隨后的研究中,我們對其作用機制進行研究,期望為臨床食管鱗癌新的靶向治療藥物的應用及精準的多學科綜合治療的開展提供新思路。材料與方法:在前期研究中,選取在本院行食管癌手術的180例食管鱗癌患者術后腫瘤組織石蠟塊進行免疫組化檢測,分析c-Met蛋白表達與患者腫瘤病理特點及預后的相關性,并分析其對術后輔助放療療效的影響。在隨后的研究中,首先選用c-Met抑制劑BPI-9016M進行干預,探討HGF/c-Met信號通路抑制對人食管鱗癌Eca109細胞株生物學行為及放射敏感性的影響;然后,將Eca109細胞分對照組、BPI-9016M組、輻射組和輻射+BPI-9016M組共4組,采用流式細胞儀技術檢測c-Met抑制劑BPI-9016M對Eca109細胞細胞周期和細胞凋亡的影響,并用蛋白免疫印跡法(Western Blot,WB)檢測細胞凋亡相關蛋白及DNA損傷修復相關蛋白含量的改變,進一步探討其具體的分子作用機制;最后,建立人食管鱗癌裸鼠移植瘤模型,將荷瘤裸鼠分對照組、BPI-9016M組、輻射組和輻射+BPI-9016M組共4組,通過腫瘤體積計算腫瘤生長延緩情況、利用增長系數(shù)(Enhancement Factor,EF)評價c-Met抑制劑BPI-9016M的放射增敏效果。結(jié)果:1.在前期針對臨床病例的免疫組化分析中,我們發(fā)現(xiàn)c-Met蛋白在食管鱗癌中的表達存在差異,且與患者預后顯著相關,高表達者預后差(p=0.001)。亞組分析提示,接受術后輔助放療的患者,c-Met高表達者預后顯著差于低/陰性表達者(p=0.002)。2.體外細胞學實驗顯示,c-Met抑制劑BPI-9016M明顯抑制Eca109人食管鱗癌細胞株的體外增殖生長,存在量-效關系;Eca109細胞的生長受放射線的影響,放射劑量越大,Eca109細胞生長越受限制,并且c-Met抑制劑BPI-9016M對Eca109細胞有放射增敏作用,用多靶單擊模型計算的放射增敏比為1.40。流式細胞儀檢測結(jié)果顯示,c-Met抑制劑BPI-9016M聯(lián)合輻射對Eca109細胞的細胞周期影響不大,但顯著增加其細胞凋亡率(p0.05);蛋白免疫印跡法檢測結(jié)果顯示,與單純輻射組相比較,Eca109細胞經(jīng)BPI-9016M處理后再聯(lián)合輻射,使凋亡相關的蛋白P53和Bcl-2下調(diào)、活化的Caspase 3和Caspase 9上調(diào)。進一步的DNA損傷修復相關蛋白檢測結(jié)果還提示:Eca109細胞經(jīng)放射線照射后,引起了 ATM、ATR、Chk1、Chk2和H2AX的顯著磷酸化,然而各蛋白的磷酸化水平在經(jīng)BPI-9016M預處理后均有所下降,以γ H2AX下降得最為明顯。3.體內(nèi)動物模型研究顯示,Eca109細胞皮下接種于裸鼠成瘤后,分別經(jīng)對照組、BPI-9016M組、輻射組以及輻射+BPI-9016M組的不同處理,從腫瘤生長曲線來看,相較于空白對照組,BPI-9016M組、輻射組以及輻射+BPI-9016M組腫瘤的生長均受到不同程度的抑制,其中輻射+BPI-9016M組較其他治療組均更明顯(p0.05),聯(lián)合組c-Met抑制劑BPI-9016M的放射增敏系數(shù)(EF)為1.51。討論:已有文獻報道,食管癌c-Met蛋白的過表達與腫瘤惡性生物學行為相關。在西方國家的研究中發(fā)現(xiàn)食管腺癌c-Met蛋白的過表達與患者的預后生存相關,而在食管鱗癌患者中,其與預后的相關性尚不清楚。我們的前期研究顯示,食管鱗癌患者c-Met蛋白的高表達與其預后差顯著相關,與國內(nèi)一些針對食管鱗癌的免疫組化研究結(jié)果報道相一致,提示c-Met基因有可能是食管鱗癌藥物治療的潛在靶點。我們前期的免疫組化亞組分析還提示,接受術后輔助放療的患者,c-Met高表達者預后顯著差于低表達者,分析原因,與c-Met可能參與多種放療抵抗的機制相關。我們的體外細胞學實驗證實,放射線抑制人食管鱗癌Eca109細胞的生長和增殖,而c-Met抑制劑BPI-9016M增加了 Eca109細胞對放射線的敏感性。流式細胞儀檢測顯示,c-Met抑制劑BPI-9016M聯(lián)合輻射對Eca109細胞的細胞周期影響不大,但顯著增加其細胞凋亡率。蛋白免疫印跡法實驗提示c-Met抑制劑BPI-9016M預處理Eca109細胞后可增加放射后促細胞凋亡相關蛋白的含量。在我們的研究中,BPI-9016M聯(lián)合輻射組活化的Caspase 3和Caspase 9蛋白較其他組別顯著增加,促進了細胞凋亡的發(fā)生。Caspase家族成員大多數(shù)是細胞凋亡的啟動子或效應子,Caspase 9為凋亡啟動子,Caspase 3為凋亡效應子,在細胞凋亡過程中均發(fā)揮重要作用,尤其是Caspase 3在細胞凋亡過程中起關鍵作用,一旦被激活,即發(fā)生下游的級聯(lián)反應,使凋亡不可避免。在我們的研究中,BPI-9016M組Eca109細胞中的P53蛋白稍有減少,輻射組其表達量與對照組接近,而在輻射+BPI-9016M組P53顯著減少,認為BPI-9016M聯(lián)合放射線可通過減少P53的表達來調(diào)控細胞凋亡;在BPI-9016M聯(lián)合輻射組,功能與P53相似的抑制細胞凋亡的Bcl-2也明顯減少。進一步的研究顯示,Eca109細胞經(jīng)輻射處理后,與DNA損傷修復相關的蛋白ATM、ATR、Chk1、Chk2和H2AX的磷酸化水平顯著增加,而細胞預先經(jīng)c-Met抑制劑BPI-9016M處理,再予以輻射,這些DNA損傷相關的關鍵蛋白的磷酸化被不同程度抑制。從機制上解讀,輻射可通過誘導DNA雙鏈斷裂從而導致細胞死亡,放射抵抗的主要原因是由于腫瘤細胞內(nèi)源性或者獲得性的輻射抵抗,主要表現(xiàn)在DNA損傷修復能力的增強。DNA損傷發(fā)生后,細胞可激活相應的修復通路對其進行修復,其反應通路主要由感受因子、傳導通路及效應因子組成。DNA損傷發(fā)生后,感受因子RAD9-HUS1-RAD1復合體可通過一個包含有RAD17的蛋白復合體募集至DNA損傷位點,以促進ATR-介導的效應蛋白激酶Chk1的磷酸化及激活,從而調(diào)節(jié)細胞周期S期的進展及G2/M期的阻滯。另一個感受因子MRE11-RAD50-NBS1(MRN)復合體,可通過檢測雙鏈斷裂位點,在DNA雙鏈斷裂區(qū)域周圍募集ATM并促進ATM介導的組蛋白H2AX磷酸化,隨后一系列的修復相關因子及蛋白招募至DNA損傷區(qū)域,形成損傷點,參與DNA的損傷修復進程。從我們的研究可以推測c-Met抑制劑增加食管鱗癌放射敏感性的機制可能部分是通過抑制ATR-Chk1、ATM-Chk2傳導通路的活化從而抑制細胞DNA損傷修復促進細胞凋亡實現(xiàn)的。我們的研究表明c-Met可能是食管鱗癌放療增敏的重要靶點之一,靶向抑制c-Met聯(lián)合放療在提高臨床食管鱗癌放療療效中具有重要的研究意義。結(jié)論:我們的研究探討了 c-Met抑制劑BPI-9016M聯(lián)合放射線對食管鱗狀細胞癌細胞系Eca109細胞生物學特性的影響,可能能部分解釋c-Met抑制劑對放療的增敏作用機制,對臨床治療方案的選擇亦能提供一定的理論基礎,至于其詳細機制,還有待于進一步研究。
[Abstract]:Background and purpose: esophageal cancer is one of the most common malignant tumors of digestive tract. The number of new cases is increasing every year, and more than half of esophageal cancers occur in China. According to the 2015 China cancer statistics, the incidence of esophageal cancer in China has leaped into the third place of the country's malignant tumor, and the death ranks fourth. Multidisciplinary integrated treatment, combined with surgery, radiotherapy and chemotherapy, is the main mode of treatment for esophageal cancer. Although a variety of treatments have been developed in recent years, the overall prognosis of esophageal cancer is still poor, and the survival rate of 5 years is only 15% to 25%. In addition, the individual differences in prognosis of patients are also large, which largely depends on the biological behavior of esophageal cancer, especially the different genetic characteristics, which have a crucial impact on tumor proliferation, invasion, metastasis and therapeutic effect. The specific molecular markers of esophageal cancer are not clear at present. MET (Mesenchymal-epithelial Transition Factor) is the hepatocyte growth factor (Hepatocyte Growth, Factor, HGF) of the cell surface receptor, also known as c-Met or HGFR (Hepatocyte Growth Factor Receptor), which belongs to the tyrosine kinase receptor (Receptor Tyrosine, Kinase, RTK) encoding by c-Met gene family members. Abnormal RTK signaling pathway plays an important role in tumor occurrence, development, invasion, metastasis and drug resistance. C-Met signaling pathway is a RTK signaling pathway. It is found that activation of c-Met signaling pathway is closely related to the occurrence, development, metastasis and chemoradiation tolerance of multiple tumors, such as colon cancer, gastric cancer, ovarian cancer, head and neck squamous cell carcinoma, lung cancer, papillary renal cell carcinoma, three negative breast cancer, prostate cancer and so on. In the adenocarcinoma of the esophagus, the expression rate of c-Met is 340% ~ 540%, and it is closely related to the prognosis. In Asian esophageal squamous cell carcinoma, it also has a high rate of expression, about 34% to 69%. Therefore, c-Met may be a new target for the treatment of squamous cell carcinoma of the esophagus. On the other hand, radiotherapy is an important treatment for esophageal cancer. Although radiotherapy equipment and radiotherapy technology have been greatly improved in recent years, radiotherapy resistance is still an important reason that affects the radiotherapy efficacy of esophageal squamous cell carcinoma. The emergence of radiation resistance is a complex process of multiple genes, multiple factors and multiple mechanisms, which is related to cell cycle arrest, related gene alterations, tumor microenvironment changes, autophagy regulation and the existence of cancer stem cells. Some clinical studies have shown that there is a correlation between the expression level of c-Met and the therapeutic effect of radiotherapy. Basic studies have shown that tumor cells and / or interstitial cells can activate the c-Met pathway after ionizing radiation and then cause radiation resistance of tumor cells. Therefore, inhibition of c-Met signaling pathway may reduce radiation resistance and improve the therapeutic effect of radiotherapy. To sum up, the study of the correlation between c-Met and radiosensitivity of squamous cell carcinoma of the esophagus has a theoretical basis and positive clinical significance. Our previous studies have suggested that the expression of c-Met protein in esophageal squamous cell carcinoma is different and has a significant correlation with the prognosis of the patients (p=0.001). Subgroup analysis also suggested that in patients with postoperative adjuvant radiotherapy, the prognosis of c-Met high expression was significantly lower than that of low expression (p=0.002). In subsequent studies, we studied the mechanism of action, hoping to provide new ideas for the application of new targeted therapy drugs and precise multidisciplinary comprehensive treatment for esophageal squamous cell carcinoma. Materials and methods: in previous studies, selected in our hospital surgery of esophageal cancer in 180 cases of patients with esophageal squamous cell carcinoma after resection of tumor tissue paraffin block immunohistochemical analysis of c-Met protein expression correlated with the pathological characteristics and prognosis of patients with cancer, and to analyze the effect of postoperative adjuvant radiotherapy curative effect. In a subsequent study, first select c-Met inhibitor BPI-9016M intervention, to investigate the inhibitory effects of HGF/c-Met signaling pathway on the biological behavior of human esophageal squamous cell carcinoma cell line Eca109 and Radiosensitivity; then, Eca109 cells were divided into control group, BPI-9016M group, radiation group and radiation group +BPI-9016M 4 group, the effect of c-Met inhibitor BPI-9016M flow detection cytometry on Eca109 cell cycle and apoptosis, and by Western blotting (Western Blot, WB) detection of apoptosis related protein and DNA repair related protein content changes, further explore its molecular mechanism; finally, to establish the xenograft model of human esophageal squamous cell carcinoma in nude mice bearing tumor the nude mice were divided into control group, BPI-9016M group, radiation group and radiation group +BPI-9016M 4 group, through the calculation of tumor volume tumor growth delay, using growth coefficient (E Nhancement Factor, EF) was used to evaluate the radiosensitization effect of c-Met inhibitor BPI-9016M. Results: 1.. In the early stage of immunohistochemical analysis of clinical cases, we found that the expression of c-Met protein in esophageal squamous cell carcinoma was different, and was significantly correlated with the prognosis of patients. The prognosis of patients with high expression was poor (p=0.001). Subgroup analysis showed that the prognosis of c-Met high expression patients was significantly lower than that of low / negative expression (p=0.002) patients who received postoperative adjuvant radiotherapy. 2. in vitro experiments, c-Met inhibitor BPI-9016M significantly inhibited the proliferation of Eca109 human esophageal squamous carcinoma cell line in vitro, the dose effect relationship; influence the growth of Eca109 cells by radiation, the radiation dose, Eca109 cell growth is restricted, and the c-Met inhibitor BPI-9016M has radiosensitizing effect on Eca109 cells, calculation multi target single hit model radiosensitization ratio is 1.40. Flow cytometry showed that c-Met inhibitor BPI-9016M combined with radiation on the cell cycle of Eca109 cells is not affected, but significantly increased the apoptosis rate (P0.05); the detection results of Western blotting showed that compared with radiation group, Eca109 cells treated by BPI-9016M combined with radiation, the apoptosis related protein P53 and the down-regulation of Bcl-2 and activation of Caspase 3 and C
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.1

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