本文選題：轉(zhuǎn)座子 + 體細(xì)胞突變��； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：腫瘤已成為全球首要致死原因,給人類(lèi)社會(huì)帶來(lái)了沉重的疾病負(fù)擔(dān)。深入具體地認(rèn)識(shí)腫瘤發(fā)生的分子機(jī)制,將極大地協(xié)助大家精準(zhǔn)地治療腫瘤,提高患者生活質(zhì)量和生存時(shí)間。作為一種基因組不穩(wěn)定的體細(xì)胞遺傳病,腫瘤之間存在很大異質(zhì)性,并且本身處于不斷“進(jìn)化”的狀態(tài),這些特性使得鑒定、驗(yàn)證更多腫瘤相關(guān)基因,并實(shí)現(xiàn)靶向治療,依然是一件充滿(mǎn)挑戰(zhàn)的研究工作。本博士學(xué)位論文包括以下三項(xiàng)研究：1、為發(fā)現(xiàn)更多腫瘤抑制基因,我們?cè)O(shè)計(jì)并開(kāi)展了利用轉(zhuǎn)座子在小鼠體內(nèi)篩選腫瘤抑制基因的工作。在B1m缺失背景的小鼠體內(nèi),我們利用piggyBac轉(zhuǎn)座子介導(dǎo)體細(xì)胞發(fā)生插入突變,干擾基因正常功能,從而誘發(fā)腫瘤生長(zhǎng)。接著,我們收集了腫瘤標(biāo)本,提取腫瘤基因組DNA,利用piggyBac序列標(biāo)簽克隆突變位點(diǎn),并分析篩選的候選腫瘤基因。我們發(fā)現(xiàn),小鼠體內(nèi)高頻次的轉(zhuǎn)座可以導(dǎo)致小鼠胚胎致死；中等頻率的轉(zhuǎn)座會(huì)引起小鼠出生后生長(zhǎng)發(fā)育異常；即使低頻次的轉(zhuǎn)座也會(huì)使小鼠整體生存周期縮短,并誘發(fā)腫瘤。在克隆piggyBac插入位點(diǎn)之后,我們可以看到,相比小鼠尾巴中轉(zhuǎn)座子的整合,腫瘤標(biāo)本中piggyBac插入位點(diǎn)存在明顯的富集現(xiàn)象,提示腫瘤克隆擴(kuò)增特性；piggyBac插入位點(diǎn)數(shù)量及其分布與已有研究比較一致；然而,在目前有限的腫瘤標(biāo)本及測(cè)序分析中,我們尚未發(fā)現(xiàn)明確的腫瘤抑制基因。本課題的開(kāi)展,提示我們利用piggyBac在體篩選腫瘤相關(guān)基因是可行；當(dāng)然,為使研究更深入,限定篩選腫瘤的類(lèi)型并完善相關(guān)專(zhuān)業(yè)條件也需要考慮。2、為研究Cdk5rap3在小鼠肝細(xì)胞癌發(fā)生中的作用,我們?cè)诟渭?xì)胞特異的Cdk5rap3條件性敲除小鼠中,利用DEN誘發(fā)肝細(xì)胞癌的生長(zhǎng),通過(guò)與野生型小鼠觀察比較肝癌發(fā)生率及腫瘤生長(zhǎng)狀態(tài),明確該基因缺失對(duì)肝癌發(fā)生的影響。觀察發(fā)現(xiàn),肝細(xì)胞特異地敲除Cdk5rap3顯著提高了小鼠肝癌發(fā)生率,其腫瘤生長(zhǎng)數(shù)量和質(zhì)量均遠(yuǎn)遠(yuǎn)高于野生型小鼠；讓人意外的是,病理分析發(fā)現(xiàn)腫瘤細(xì)胞中Cdk5rap3表達(dá)明顯升高,這些細(xì)胞并非來(lái)自基因敲除的肝細(xì)胞。這些結(jié)果提示我們,條件性敲除部分肝細(xì)胞中Cdk5rap3可以顯著提高小鼠肝細(xì)胞癌易感性；這一表型的改變更多地來(lái)自肝臟內(nèi)細(xì)胞與細(xì)胞或細(xì)胞與微環(huán)境之間的相互作用。3、我們利用全外顯子組測(cè)序發(fā)現(xiàn)在Flt3-ITD敲入小鼠中存在一個(gè)耐藥變異(Flt3-ITD c.2076TA)。為驗(yàn)證該變異會(huì)引起腫瘤細(xì)胞耐藥,我們克隆了cDNA,并將其回復(fù)為野生型堿基(Flt3-ITD c.2076T),在分別轉(zhuǎn)化了Ba/F3細(xì)胞系之后檢測(cè)了細(xì)胞對(duì)Quazartinib (AC220)、Sorafenib和Ponatinib的敏感性。與預(yù)期一致,我們發(fā)現(xiàn),兩個(gè)編碼序列均可順利轉(zhuǎn)化Ba/F3細(xì)胞,均顯示了持續(xù)活化的激酶活性；與Flt3-ITD c.2076T相比,Flt3-ITD c.2076TA使得腫瘤細(xì)胞對(duì)Quazartinib和Sorafenib表現(xiàn)出耐藥,而兩株細(xì)胞對(duì)Ponatinib均敏感,與已有報(bào)道結(jié)果一致。該實(shí)驗(yàn)證實(shí)了,Flt3-ITD敲入小鼠中發(fā)現(xiàn)的變異與人類(lèi)白血病細(xì)胞 FLT3中出現(xiàn)的Gate-keeper耐藥突變一樣,均會(huì)導(dǎo)致腫瘤細(xì)胞對(duì)Quazartinib和Sorafenib產(chǎn)生耐藥性,而Ponatinib可以克服這種耐藥。小鼠模型體內(nèi)存在的耐藥突變,提醒我們?cè)谑褂眯∈竽Ｐ烷_(kāi)展實(shí)驗(yàn)研究時(shí)需要謹(jǐn)慎設(shè)計(jì)對(duì)照實(shí)驗(yàn),以排除潛在的影響因素。總之,三方面的課題,分別從正向遺傳學(xué)和反向遺傳學(xué)的角度,使得自己可以利用多種遺傳手段進(jìn)行腫瘤學(xué)相關(guān)研究,并對(duì)它們有了一定的理解和掌握。
[Abstract]:In order to find out more tumor suppressor genes , we designed and carried out the work of using transposon to screen tumor suppressor genes in mice . These characteristics make it possible to identify and validate more tumor - related genes and to achieve targeted therapy .
Moderate frequency transposition can cause abnormal growth and development of mice after birth ;
Even though low - frequency transposition can shorten the whole life cycle of mice and induce the tumor , we can see that , compared with the integration of the transposon in the tail of the mouse , there is a significant enrichment phenomenon in the insertion site in the tumor specimen , which suggests that the amplification characteristic of the tumor is cloned ;
The number of insertion sites and their distribution were consistent with those of existing studies .
However , in the present limited tumor specimen and sequencing analysis , we have not found a clear tumor suppressor gene .
To study the role of Cdk5rap3 in the carcinogenesis of hepatocellular carcinoma in mice , the effects of the deletion of Cdk5rap3 on hepatocellular carcinoma were studied .
It was surprising that the expression of Cdk5rap3 in tumor cells was significantly increased by pathological analysis , and these cells were not from knock - out hepatocytes . These results suggested that Cdk5rap3 could significantly improve the susceptibility to hepatocellular carcinoma in mice .
The alteration of this phenotype is more derived from the interaction between cells and cells or cells and microenvironment in the liver . 3 . We have found that there is a resistance variation ( Flt3 - itd c.2076TA ) in the Flt3 - Ds knock - in mice using full exon sequencing . In order to verify that the mutation causes resistance to tumor cells , we cloned cDNA and responded to wild - type bases ( Flt3 - itd c.2076T ) . After transformation of the Ba / F3 cell line , the sensitivity of the cells to Quazartinib ( AC220 ) , Sorbate and Ponds was detected . As expected , we found that both coding sequences were successfully transformed into Ba / F3 cells , showing a continuously activated kinase activity ;
Compared with Flt3 - itd c . 2076T , Flt3 - itd c . 2076TA results in the resistance of tumor cells to Quazartinib and Sorrow , which is consistent with the reported results . The experiments confirm that the mutations found in the mouse model are the same as those in human leukemia cells FLT3 , which can overcome the potential influence factors . In conclusion , the three aspects are from the perspective of forward genetics and reverse genetics , so that they can use a variety of genetic means to carry out oncology related research , and have some understanding and control .
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R730.2
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本文編號(hào)：2057123