發(fā)布時(shí)間：2018-01-13 23:18

  本文關(guān)鍵詞：雙微體的分子細(xì)胞遺傳學(xué)研究　出處：《吉林大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 微陣列比較基因組雜交熒光原位雜交 均質(zhì)染色區(qū) 雙微體

【摘要】：雙微體是游離于染色體外的小片段球狀染色小體，具有獨(dú)特的自我復(fù)制功能，見(jiàn)于乳腺癌、肺癌、卵巢癌、結(jié)腸癌，尤其是神經(jīng)母細(xì)胞瘤等多種人類腫瘤細(xì)胞，是基因擴(kuò)增的一種表現(xiàn)形式，常攜帶耐藥基因或原癌基因，導(dǎo)致腫瘤相關(guān)蛋白的過(guò)表達(dá)，針對(duì)攜帶基因的靶向治療對(duì)腫瘤的預(yù)后和治療具有重要的意義。盡管有關(guān)雙微體的研究很多，但專門針對(duì)各種腫瘤的雙微體染色體來(lái)源的研究很少，我們從ATCC公司篩選所有攜帶雙微體的腫瘤細(xì)胞系，進(jìn)行細(xì)胞培養(yǎng)，并在第3代、8代、13代時(shí)獲取細(xì)胞。利用染色體核型分析技術(shù)檢測(cè)雙微體的數(shù)目及分布情況及粗略的細(xì)胞遺傳學(xué)特征，發(fā)現(xiàn)不同腫瘤細(xì)胞系所攜帶雙微體的數(shù)目及分布相差極大。繼而通過(guò)arrayCGH獲得擴(kuò)增的基因，并明確其斷裂點(diǎn)，采用相應(yīng)的DNA探針，利用熒光原位雜交確認(rèn)基因擴(kuò)增并進(jìn)一步確認(rèn)染色體的來(lái)源及明確擴(kuò)增的形式。我們發(fā)現(xiàn)在10個(gè)細(xì)胞系中，血液腫瘤占10%（HL-60細(xì)胞系），MC-IXC細(xì)胞系，COLO-320DM細(xì)胞系，SW480細(xì)胞系及HL-60細(xì)胞系中由于MYC擴(kuò)增基因所致，雙微體廣泛存在，但其擴(kuò)增區(qū)域的斷裂點(diǎn)不同，其中前2個(gè)細(xì)胞系MYC雙微體與MYC均質(zhì)染色區(qū)共存，后2個(gè)細(xì)胞系僅存在MYC雙微體。SW1783細(xì)胞系、5637細(xì)胞系、CAMA-1細(xì)胞系的擴(kuò)增區(qū)域分別包含PDGFRA基因、RAF1基因及SOX4基因、MYEOV基因及CCND1基因，均以均質(zhì)染色區(qū)的形式存在。T84細(xì)胞系、SNU-1細(xì)胞系及NCI-N87細(xì)胞系僅在部分細(xì)胞中見(jiàn)少量雙微體，arrayCGH未見(jiàn)明顯擴(kuò)增的區(qū)域，未行擴(kuò)增基因FISH檢測(cè)。 得出主要以下結(jié)論： 1.同一細(xì)胞內(nèi)的雙微體及均質(zhì)染色區(qū)可攜帶不同來(lái)源的基因，病理類型相同的腫瘤細(xì)胞可攜帶不同來(lái)源的雙微體。 2.攜帶相同原癌基因的不同腫瘤，雙微體的斷裂點(diǎn)不同； 3.雙微體擁有除均質(zhì)染色區(qū)以外的形成機(jī)制； 4.不同代細(xì)胞間的基因擴(kuò)增未見(jiàn)明顯改變； 5.來(lái)源于不同染色體的基因可以共同交替擴(kuò)增，形成一個(gè)新的混合的擴(kuò)增區(qū)域。 并驗(yàn)證了以下結(jié)論 1.雙微體多見(jiàn)于實(shí)體腫瘤細(xì)胞系中，血液腫瘤中罕見(jiàn)； 2.攜帶雙微體的細(xì)胞多伴有復(fù)雜的染色體核型改變； 3.不同的腫瘤細(xì)胞系攜帶來(lái)源不同的雙微體，數(shù)目相差巨大； 4.在某些腫瘤細(xì)胞系中，，雙微體與均質(zhì)染色區(qū)可以相互轉(zhuǎn)化。 主要?jiǎng)?chuàng)新點(diǎn)： （1）首次篩選不同的包含雙微體的實(shí)體腫瘤細(xì)胞系及血液腫瘤細(xì)胞系共同比較研究。 （2）聯(lián)合應(yīng)用染色體核型分析、arrayCGH及FISH技術(shù)，準(zhǔn)確有效的找出擴(kuò)增基因及擴(kuò)增形式。 （3）利用arrayCGH技術(shù)明確擴(kuò)增區(qū)域的斷裂點(diǎn)。
[Abstract]:Double microbodies are small globular chromatid bodies which are free from chromosomes. They have unique self-replicating function and can be seen in many kinds of human tumor cells such as breast cancer lung cancer ovarian cancer colon cancer especially neuroblastoma and so on. It is a form of gene amplification, often carrying drug resistance genes or proto-oncogenes, resulting in over-expression of tumor-associated proteins. Targeted therapy for carrying genes is of great significance to the prognosis and treatment of cancer. Although there are many studies on double microsomes, there are few studies on the origin of double microsomes specifically for various kinds of tumors. We screened all tumor cell lines with double microsomes from ATCC and cultured them for 8 passages in the third generation. Chromosome karyotype analysis was used to detect the number and distribution of double microbodies and the rough cytogenetic characteristics. It was found that the number and distribution of double microbodies carried by different tumor cell lines varied greatly. Then the amplified genes were obtained by arrayCGH and the breakpoints were identified and the corresponding DNA probes were used. Fluorescence in situ hybridization (Fish) was used to confirm the origin of chromosome and the form of chromosome amplification. We found that blood tumor accounted for 10 HL-60 cell lines out of 10 cell lines. The MC-IXC cell line, COLO-320DM cell line, SW480 cell line and HL-60 cell line, due to the MYC amplification gene, the presence of double microbodies is widespread. However, the breakpoints of the amplified region were different. The first two cell lines, MYC double microbodies and MYC homogenized staining region, only existed MYC double microbody. SW1783 cell lines. The amplified region of 5637 cell line, CAMA-1, contains PDGFRA gene, RAF1 gene, SOX4 gene, MYEOV gene and CCND1 gene, respectively. There were only a few double microbodies in some cells of SNU-1 cell line and NCI-N87 cell line in the form of homogeneous staining region. There was no obvious amplified region in arrayCGH, and no amplification gene FISH was detected. The main conclusions are as follows: 1. Double microbodies and homogenous staining regions in the same cell can carry genes from different sources, while tumor cells with the same pathological type can carry double microbodies from different sources. 2. Different tumors carrying the same proto-oncogene had different breakpoints. 3. The double microbodies have the formation mechanism except the homogeneous staining area. 4. There was no obvious change in gene amplification between different generations. 5. Genes from different chromosomes can be amplified alternately to form a new mixed region. And verify the following conclusions 1. Double microbodies were found mostly in solid tumor cell lines, but rare in blood tumors. 2. The cells carrying double microbodies were often accompanied by complex chromosome karyotype changes; 3. Different tumor cell lines carry different double microbodies, and the number of tumor cells varies greatly. 4. In some tumor cell lines, the double microsomes and the homogenized staining region can transform each other. Major innovations: For the first time, different solid tumor cell lines containing double microbodies and blood tumor cell lines were screened for the first time. (2) using chromosome karyotype analysis and FISH technique, we can find out the amplified genes and their forms accurately and effectively. ArrayCGH technique was used to identify the breakpoints of the amplified region.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R394

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