本文選題：Cathepsin + L��； 參考：《蘇州大學》2016年博士論文

【摘要】：第一部分突變p53與Cathepsin L表達特征的關系以及在肺癌患者上皮間質轉化中的作用目的:探究78例非小細胞肺癌患者腫瘤組織中p53基因的突變情況與Cathepsin L表達的特征以及EMT相關蛋白表達的相關性,并分析它們的表達變化與臨床病理的關系。方法:收集78例人肺癌及其癌旁組織標本,對腫瘤組織進行p53基因突變檢測,分析其突變規(guī)律與肺癌病理特征的關系;同時采用Western blot、ELISA及免疫組織化學檢測Cathepsin L蛋白的表達情況,分析其與肺癌病理特征的關系;采用Western blot和免疫組織化學檢測肺癌組織中上皮間質轉化標志物E-cadherin和N-cadherin的表達,分析p53基因突變、Cathepsin L與上皮間質轉化的相關性。結果:肺癌組織中p53基因突變率為56%,大多為GC→AT和GC→TA的堿基顛換;p53基因突變與患者的分化程度密切相關。與正常肺組織相比,活性Cathepsin L在腫瘤組織中表達明顯上調;無論在腫瘤細胞中還是病人的血清樣本里,Cathepsin L的表達與肺癌組織的級別密切相關。p53基因突變的肺癌組織中Egr-1激活,Cathepsin L入核現象明顯,而且EMT相關蛋白表達發(fā)生相應的變化。結論:突變p53肺癌組織中EMT通路被激活;Cathepsin L的過度表達參與肺癌的發(fā)生發(fā)展,可以作為肺癌患者預后的指標。第二部分突變p53誘導Cathepsin L在肺癌細胞上皮間質轉化中的調控機制目的:研究野生型p53基因和突變型p53基因誘導Cathepsin L影響上皮間質轉化的調控機制,以此探討突變p53基因獲得性功能在人肺癌侵襲遷移中的作用。方法:體外構建穩(wěn)定感染不同p53基因狀態(tài)慢病毒的細胞株,用電離輻射處理細胞,采用劃痕法、Transwell小室法分別檢測腫瘤細胞的遷移、侵襲能力;鬼筆環(huán)肽染色的方法檢測腫瘤細胞骨架變化;Western blot及免疫熒光檢測上皮間質轉化相關標志蛋白的變化;染色質免疫共沉淀法檢測p53和Egr-1參與調節(jié)下游靶基因Cathepsin L的轉錄激活水平;建立裸小鼠皮下移植瘤模型,觀察不同p53基因狀態(tài)的細胞株在電離輻射治療后的成瘤能力,運用Western blot、免疫組化分析法檢測各組移植瘤Cathepsin L與上皮間質轉化相關標志蛋白的表達情況。結果:在輻照誘導后,穩(wěn)定感染p53基因突變慢病毒的細胞株Cathepsin L和Egr-1表達上調,并且侵襲和遷移能力得到了提高;ChIP結果顯示突變型p53對Cathepsin L的轉錄過程是依賴Egr-1的。在裸小鼠皮下移植瘤模型中,突變組的放療效果卻沒有野生組理想;免疫組化分析顯示,相較于野生組細胞株,突變組細胞株的移植瘤中Cathepsin L定位在細胞核,E-cadherin的表達減少,而N-cadherin的表達則增加,與細胞實驗結果一致。結論:突變p53能通過誘導轉錄因子Egr-1調控Cathepsin L的表達,繼而促進腫瘤細胞的上皮間質轉化,影響腫瘤細胞的侵襲遷移能力。突變型p53和Egr-1協同參與對Cathepsin L的轉錄過程,提示p53基因狀態(tài)和Cathepsin L蛋白在調控腫瘤細胞侵襲遷移方面起著重要的作用。本研究結果將為Cathepsin L成為腫瘤治療的有效靶點提供實驗依據。
[Abstract]:The relationship between the first part of the mutation p53 and the expression of Cathepsin L and the role in the transformation of epithelial mesenchymal transition in lung cancer patients: To explore the mutation of the p53 gene in the tumor tissues of 78 patients with non-small cell lung cancer and the characteristics of the expression of Cathepsin L and the correlation of the expression of EMT related proteins, and to analyze their expression changes and clinical diseases. Methods: 78 cases of human lung cancer and its adjacent tissue specimens were collected, and the p53 gene mutation was detected in the tumor tissue. The relationship between the mutation law and the pathological features of lung cancer was analyzed. The expression of Cathepsin L protein was detected by Western blot, ELISA and immunohistochemistry, and the relationship between the pathological features of the lung cancer and the pathological features of lung cancer was analyzed, and Wes was used in Wes. The expression of epithelial mesenchymal transition markers E-cadherin and N-cadherin in lung cancer tissues was detected by tern blot and immunohistochemistry. The correlation between p53 gene mutation, Cathepsin L and epithelial mesenchymal transition was analyzed. Results: the mutation rate of p53 gene in lung cancer tissues was 56%, most of which were GC, AT and GC to TA. The expression of the active Cathepsin L in the tumor tissue was obviously up-regulated compared with the normal lung tissue. In the tumor cells or in the patient's serum samples, the expression of Cathepsin L was closely related to the activation of Egr-1 in the lung cancer tissues of the.P53 gene mutation, and the Cathepsin L had a obvious nucleation, and EMT. Corresponding changes in the expression of related proteins. Conclusion: the EMT pathway in the mutant p53 lung cancer is activated; the overexpression of Cathepsin L is involved in the development of lung cancer, and it can be used as an indicator of prognosis of lung cancer patients. Second partial mutation p53 induces the mechanism of Cathepsin L in the transformation of epithelial mesenchymal transition of lung cancer cells: the study of wild type p53 The gene and mutant p53 genes induce Cathepsin L to influence the regulatory mechanism of epithelial mesenchymal transition, in order to explore the role of the mutant p53 gene in the invasion and migration of human lung cancer. Methods: to construct a cell line with lentivirus infection of different p53 genes in vitro, use ionizing radiation to treat cells, use scratch method, Transwell chamber method. Detection of tumor cell migration, invasiveness, cytoskeleton changes of tumor cells, Western blot and immunofluorescence detection of epithelial mesenchymal transition related markers; chromatin immunoprecipitation assay p53 and Egr-1 to regulate the transcriptional activation level of Cathepsin L of the downstream target gene; establish nude The mouse subcutaneous transplantation tumor model was used to observe the tumorigenicity of the cell lines with different p53 gene States after ionizing radiation. The expression of Cathepsin L and epithelial mesenchymal transition related markers was detected by Western blot and immunohistochemistry. Results: after irradiated, the p53 gene mutated to the lentivirus. The expression of Cathepsin L and Egr-1 was up-regulated, and the ability to invasion and migration was improved; ChIP results showed that the transcriptional process of mutant p53 to Cathepsin L was Egr-1. In the nude mice model, the radiotherapy effect of the mutant group was not ideal for the wild group; immunohistochemical analysis showed that it was compared to the wild group cell line. In the transplanted tumor of the mutant group, Cathepsin L was located in the nucleus, the expression of E-cadherin decreased, while the expression of N-cadherin increased, which was in agreement with the results of cell experiment. Conclusion: mutation p53 can regulate the expression of Cathepsin L by inducing the transcription factor Egr-1, and then promote the epithelial mesenchymal transformation of the tumor cells and affect the invasion of the tumor cells. Migration ability. Mutational p53 and Egr-1 participate in the transcriptional process of Cathepsin L, suggesting that p53 gene state and Cathepsin L protein play an important role in regulating tumor cell invasion and migration. The results of this study will provide a practical basis for Cathepsin L to be an effective target for cancer treatment.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2

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