發(fā)布時(shí)間：2018-03-08 06:30

  本文選題：大腸癌　切入點(diǎn)：線粒體　出處：《第四軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：mtDNA的變異包括三大類,即線粒體拷貝數(shù)的變異、胚系突變的異質(zhì)性轉(zhuǎn)換及線粒體體細(xì)胞突變,mtDNA變異與腫瘤的進(jìn)化演變密切相關(guān)。目前為止幾乎在所有類型的腫瘤中都檢測(cè)到mtDNA變異的存在。這些變異可引起細(xì)胞線粒體氧化脅迫,導(dǎo)致惡性循環(huán)。我參與的研究發(fā)現(xiàn)線粒體拷貝數(shù)升高與大腸癌的發(fā)病風(fēng)險(xiǎn)顯著相關(guān),而且mtDNA拷貝數(shù)與大腸癌分級(jí)分期及惡性進(jìn)展也顯著相關(guān)。在此基礎(chǔ)上,本研究擬系統(tǒng)分析mtDNA變異與大腸癌進(jìn)展及預(yù)后的關(guān)系,并深入探討其作用機(jī)制。本研究分為以下三部分實(shí)驗(yàn):第一部分:我們?cè)?20例大腸癌組織樣本中檢測(cè)mtDNA拷貝數(shù)和TFAM的表達(dá)情況,構(gòu)建無(wú)mtDNA以及mtDNA拷貝數(shù)異常改變的細(xì)胞模型,全面檢測(cè)細(xì)胞周期、細(xì)胞增殖、細(xì)胞凋亡、細(xì)胞侵襲遷移能力的變化,并通過(guò)裸鼠皮下成瘤實(shí)驗(yàn)檢測(cè)mtDNA異常改變后細(xì)胞成瘤能力的改變,在此基礎(chǔ)上我們還檢測(cè)了不同mtDNA拷貝數(shù)對(duì)線粒體呼吸功能的影響。結(jié)果發(fā)現(xiàn):(1)在微衛(wèi)星穩(wěn)定的大腸癌組織中mtDNA拷貝數(shù)異常升高,而在微衛(wèi)星不穩(wěn)定的癌組織中則相反,TFAM表達(dá)水平在兩種微衛(wèi)星狀態(tài)的癌組織中也截然相反。(2)mtDNA拷貝數(shù)與TFAM表達(dá)水平具有顯著相關(guān)性,且與大腸癌患者預(yù)后具有一致的趨勢(shì)。(3)異常升高的mtDNA拷貝數(shù)可顯著促進(jìn)大腸癌細(xì)胞增殖、侵襲遷移,同時(shí)抑制細(xì)胞凋亡。(4)過(guò)表達(dá)TFAM使mtDNA拷貝數(shù)升高后,細(xì)胞成瘤能力增強(qiáng)。(5)增加mtDNA拷貝數(shù)可使細(xì)胞轉(zhuǎn)移能力和增殖能力增強(qiáng)。(6)mtDNA拷貝數(shù)異常升高可顯著促進(jìn)細(xì)胞氧耗速率,促進(jìn)細(xì)胞ROS和ATP產(chǎn)生,細(xì)胞膜電位降低。第二部分:我們通過(guò)對(duì)116例大腸癌組織mtDNA深度測(cè)序結(jié)果,結(jié)合公共數(shù)據(jù)庫(kù)數(shù)據(jù),系統(tǒng)分析比較了大腸癌組織中SNP shift數(shù)量、分布、功能及預(yù)后等。結(jié)果發(fā)現(xiàn):(1)大腸癌/癌旁組織中共發(fā)現(xiàn)172個(gè)SNP shift突變位點(diǎn),正常組織/血液中共發(fā)現(xiàn)75個(gè)SNP shift突變位點(diǎn)。(2)癌組織中以正向SNP shift為主。(3)癌組織中SNP shift突變以GA,TC轉(zhuǎn)換形式為主。第三部分:我們通過(guò)對(duì)116例大腸癌組織mtDNA深度測(cè)序結(jié)果,結(jié)合公共數(shù)據(jù)庫(kù)數(shù)據(jù),系統(tǒng)分析比較了大腸癌組織中mtDNA突變數(shù)量、分布、功能及預(yù)后等。結(jié)果發(fā)現(xiàn):(1)大腸癌組織中共發(fā)現(xiàn)110個(gè)mtDNA突變位點(diǎn),平均每個(gè)腸癌患者包含0.95個(gè)突變。(2)按照大腸癌組織mtDNA突變位點(diǎn)位置,繪制腸癌mtDNA突變圖譜。(3)癌組織中complex 1區(qū)域發(fā)生的突變占總體突變的百分比明顯高于正常組織,而正常組織中D-loop區(qū)發(fā)生的突變占總體突變的百分比明顯高于癌組織。(4)癌組織中以GA,TC的轉(zhuǎn)換為主。本研究系統(tǒng)分析了線粒體基因組變異對(duì)大腸癌進(jìn)展及預(yù)后的影響,并初步闡明了mtDNA拷貝數(shù)異常影響大腸癌進(jìn)展及預(yù)后的作用機(jī)制,為系統(tǒng)研究線粒體基因組變異提供了實(shí)驗(yàn)基礎(chǔ),為推進(jìn)臨床大腸癌精準(zhǔn)分子分型體系的構(gòu)建提供了有益的思考。
[Abstract]:The variation of mtDNA consists of three categories, that is, mitochondrial copy number variation. The heterogeneity of blastocyte mutation and mitochondrial somatic mutation / mtDNA mutation are closely related to the evolution of tumor. Up to now, the existence of mtDNA mutation has been detected in almost all types of tumors. Mitochondrial oxidative stress, I participated in a study that found that increased mitochondrial copy numbers were significantly associated with the risk of colorectal cancer, and that mtDNA copy numbers were significantly associated with colorectal cancer grade, stage and progression. This study was intended to systematically analyze the relationship between mtDNA variation and the progression and prognosis of colorectal cancer. This study was divided into the following three parts: the first part: we detected the expression of mtDNA copy number and TFAM in 120 samples of colorectal cancer, and constructed a cell model without abnormal changes of mtDNA and mtDNA copy number. The changes of cell cycle, cell proliferation, cell apoptosis, cell invasion and migration were detected. The change of cell tumorigenesis ability after abnormal mtDNA changes was detected by subcutaneous tumorigenesis assay in nude mice. On this basis, we also examined the effects of different mtDNA copy numbers on mitochondrial respiratory function. The results showed that the mtDNA copy number increased abnormally in microsatellite stable colorectal cancer tissues. On the contrary, the expression of TFAM in microsatellite unstable cancer tissues was significantly correlated with the expression of TFAM in two kinds of microsatellite cancer tissues, and the number of copies of mtDNA was significantly correlated with the expression of TFAM in two kinds of microsatellite carcinomas, and the expression level of TFAM was significantly correlated with the expression of TFAM. The abnormal increase of mtDNA copy number could significantly promote the proliferation, invasion and migration of colorectal cancer cells, and inhibit the overexpression of TFAM, which resulted in the increase of mtDNA copy number. Increasing the copy number of mtDNA can enhance the ability of cell metastasis and proliferation. The abnormal increase of copy number of mtDNA can significantly promote the rate of cell oxygen consumption and the production of ROS and ATP. The second part: we analyzed and compared the number and distribution of SNP shift in colorectal cancer tissue by deep sequencing of mtDNA in 116 cases of colorectal cancer, combined with the data of common database, and compared the number and distribution of SNP shift in colorectal cancer tissue. Function and prognosis. The results showed that 172 SNP shift mutation sites were found in colorectal carcinoma / paracancerous tissues. A total of 75 SNP shift mutation sites were found in normal tissue / blood.) positive SNP shift was the main mutation of SNP shift in cancer tissues. In the third part, we analyzed the results of mtDNA deep sequencing in 116 colorectal cancer tissues. The number, distribution, function and prognosis of mtDNA mutations in colorectal cancer tissues were systematically analyzed and compared with common database data. The results showed that 110 mtDNA mutation sites were found in colorectal cancer tissues. An average of 0.95 mutations per patient with colorectal cancer.) according to the location of mtDNA mutation sites in colorectal cancer tissues, the percentage of mutations occurring in complex 1 region in colorectal cancer tissues was significantly higher than that in normal tissues, according to the location of mtDNA mutation loci in colorectal cancer tissues, and the percentage of mutations in complex 1 region in cancer tissues was significantly higher than that in normal tissues. However, the percentage of mutations in D-loop region in normal tissues was significantly higher than that in cancer tissues. In this study, the effect of mitochondrial genome mutation on the progression and prognosis of colorectal cancer was systematically analyzed. The mechanism of the abnormal copy number of mtDNA affecting the progression and prognosis of colorectal cancer was elucidated, which provided the experimental basis for systematic study of mitochondrial genome variation, and provided useful thoughts for the construction of accurate molecular typing system for clinical colorectal cancer.
【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)】：R735.34
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本文編號(hào)：1582795