發(fā)布時間：2018-05-03 16:54

  本文選題：腎細胞癌 + 原肌球蛋白　； 參考：《吉林大學》2015年博士論文

【摘要】：原肌球蛋白（tropomyosin, TPM）是組成細胞骨架結構微絲的重要成分，主要調(diào)節(jié)肌細胞的收縮和非肌細胞的細胞骨架穩(wěn)定性。以往的觀點都認為這些TPM蛋白與多種良性肌肉病變的發(fā)生密切相關，比如重癥肌無力和家族性肥厚性心肌病等。但近年，越來越多的研究開始關注TPM1在腫瘤的發(fā)生發(fā)展過程中的作用，進而發(fā)現(xiàn)TPM1在乳腺癌等腫瘤中扮演了腫瘤抑制基因（tumorsuppressor gene, TSG）的角色。這種抑癌作用具體表現(xiàn)在，TPM1參與了細胞變形、侵襲、遷移、抗凋亡等一系列惡性腫瘤的生物學行為。對其作用和調(diào)控機理的深入認識會使TPM1運用到腫瘤的診斷和治療，并將使之發(fā)展為新的腫瘤標志物或藥物治療靶點成為可能。 最近研究發(fā)現(xiàn)TPM1可能是miRNA-21的靶基因之一，而miRNA-21是在各種實體腫瘤中研究得最多的miRNA，它在各種實體腫瘤包括腎癌中都是高表達的。然而，對于TPM1在腎細胞癌中的表達水平如何以及TPM1在腎癌中是否也發(fā)揮抑癌基因的作用等問題，由于以前沒有進行過系統(tǒng)的研究，目前這些情況仍然是不清楚的。但根據(jù)現(xiàn)有的事實與理論推測TPM1在腎細胞癌中應該是被下調(diào)的，并且在腎細胞癌中發(fā)揮著抑癌基因的作用，可能參與了腫瘤細胞的轉移和侵襲等惡性生物學行為。 為了證實以上推測，本實驗運用實時定量PCR、免疫蛋白印記（Westernblotting）和免疫組織化學染色的方法檢測了TPM1在腎癌組織和腎癌細胞系OSRC-2及786-O中的表達水平。結合腎細胞癌患者的臨床資料，分析了TPM1的表達水平與病理類型、腫瘤分期、腫瘤核分級和術后生存率等臨床特征的相關性。構建pcDNA3.1-TPM1重組真核表達質(zhì)粒，運用lipofectamine2000做媒介瞬時轉染OSRC-2和786-O腎癌細胞，研究TPM1在腎癌中的腫瘤生物學功能。使用CCK-8試劑盒檢測TPM1轉染后的細胞增殖狀態(tài)；利用劃痕實驗以及Transwell遷移實驗檢測TPM1對腎癌細胞遷移能力的影響；使用Transwell基質(zhì)膠侵襲實驗檢測TPM1轉染前后腎癌細胞侵襲能力的變化；使用流式細胞術PI/AnnexinV雙染色和PI單染色法分別檢測了TPM1對腎癌細胞凋亡和細胞周期的調(diào)控作用。 實驗結果顯示：與正常腎組織相比，，TPM1在腎細胞癌中的mRNA和蛋白質(zhì)表達水平顯著且特異的下調(diào)。TPM1的表達水平與腎細胞癌患者的腫瘤大小、Fuhrman核分級和疾病特異性生存率相關。在腎癌細胞系中增加TPM1的表達會顯著地抑制腫瘤細胞的遷移和侵襲，TPM1還可以促進腎細胞癌細胞的凋亡和非特異性的細胞周期阻滯，這些都提示TPM1是腎細胞癌中潛在的腫瘤抑制基因。
[Abstract]:Tropomyosin (TPM) is an important component of cytoskeletal microfilaments, which mainly regulates the contraction of myocytes and the cytoskeleton stability of non-myocytes. It has been suggested that these TPM proteins are closely related to the occurrence of various benign muscle diseases, such as myasthenia gravis and familial hypertrophic cardiomyopathy. However, in recent years, more and more studies have begun to focus on the role of TPM1 in tumorigenesis and development, and it has been found that TPM1 plays the role of tumor suppressor gene in breast cancer and other tumors. TPM1 is involved in the biological behavior of a series of malignant tumors, such as cell deformation, invasion, migration, anti-apoptosis and so on. Further understanding of its role and regulation mechanism will make it possible for TPM1 to be used in the diagnosis and treatment of tumors and to develop into new tumor markers or drug therapy targets. Recent studies have found that TPM1 may be one of the target genes of miRNA-21, while miRNA-21 is the most widely studied miRNAs in various solid tumors, including renal cell carcinoma. However, the expression level of TPM1 in renal cell carcinoma and whether TPM1 also plays the role of tumor suppressor gene in renal cell carcinoma have not been systematically studied before, so these conditions are still unclear. However, according to the existing facts and theories, we speculate that TPM1 may be down-regulated in renal cell carcinoma and play an important role in tumor suppressor gene, which may be involved in malignant biological behavior such as metastasis and invasion of tumor cells. In order to confirm the above conjecture, the expression of TPM1 in renal cell carcinoma (RCC) and renal cell lines (OSRC-2 and 786-O) was detected by real-time quantitative PCR, Western blotting and immunohistochemical staining. Based on the clinical data of renal cell carcinoma (RCC), the correlation between the expression of TPM1 and pathological type, tumor stage, tumor nuclear grade and postoperative survival rate was analyzed. PcDNA3.1-TPM1 recombinant eukaryotic expression plasmid was constructed and transient transfection of OSRC-2 and 786-O RCC cells was carried out using lipofectamine2000 as a medium to study the tumor biological function of TPM1 in RCC. CCK-8 kit was used to detect the cell proliferation after TPM1 transfection, and the effect of TPM1 on the migration ability of renal cancer cells was detected by scratch test and Transwell migration test. Transwell matrix gel invasion assay was used to detect the invasion ability of renal cancer cells before and after TPM1 transfection, and the effects of TPM1 on apoptosis and cell cycle of renal cell carcinoma cells were detected by flow cytometry PI/AnnexinV double staining and Pi single staining, respectively. The results showed that the expression level of mRNA and protein in renal cell carcinoma was significantly and specifically down-regulated compared with normal renal tissue. The expression level of TPM1 was correlated with the tumor size and the disease specific survival rate in renal cell carcinoma patients. Increasing the expression of TPM1 in renal cell carcinoma cell lines significantly inhibited the migration and invasion of tumor cells and promoted the apoptosis and non-specific cell cycle arrest of renal cell carcinoma cells. These results suggest that TPM1 is a potential tumor suppressor gene in renal cell carcinoma.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.11

【參考文獻】

相關博士學位論文 前1條

1 王勇;組蛋白乙�；D移酶MYST1與腎細胞癌關系的研究[D];吉林大學;2013年

本文編號：1839306