基因重组工具在肿瘤学研究中的应用

发布时间：2018-06-23 12:27

  本文选题：转座子 + 体细胞突变　； 参考：《北京协和医学院》2016年博士论文

【摘要】：肿瘤已成为全球首要致死原因,给人类社会带来了沉重的疾病负担。深入具体地认识肿瘤发生的分子机制,将极大地协助大家精准地治疗肿瘤,提高患者生活质量和生存时间。作为一种基因组不稳定的体细胞遗传病,肿瘤之间存在很大异质性,并且本身处于不断“进化”的状态,这些特性使得鉴定、验证更多肿瘤相关基因,并实现靶向治疗,依然是一件充满挑战的研究工作。本博士学位论文包括以下三项研究：1、为发现更多肿瘤抑制基因,我们设计并开展了利用转座子在小鼠体内筛选肿瘤抑制基因的工作。在B1m缺失背景的小鼠体内,我们利用piggyBac转座子介导体细胞发生插入突变,干扰基因正常功能,从而诱发肿瘤生长。接着,我们收集了肿瘤标本,提取肿瘤基因组DNA,利用piggyBac序列标签克隆突变位点,并分析筛选的候选肿瘤基因。我们发现,小鼠体内高频次的转座可以导致小鼠胚胎致死；中等频率的转座会引起小鼠出生后生长发育异常；即使低频次的转座也会使小鼠整体生存周期缩短,并诱发肿瘤。在克隆piggyBac插入位点之后,我们可以看到,相比小鼠尾巴中转座子的整合,肿瘤标本中piggyBac插入位点存在明显的富集现象,提示肿瘤克隆扩增特性；piggyBac插入位点数量及其分布与已有研究比较一致；然而,在目前有限的肿瘤标本及测序分析中,我们尚未发现明确的肿瘤抑制基因。本课题的开展,提示我们利用piggyBac在体筛选肿瘤相关基因是可行；当然,为使研究更深入,限定筛选肿瘤的类型并完善相关专业条件也需要考虑。2、为研究Cdk5rap3在小鼠肝细胞癌发生中的作用,我们在肝细胞特异的Cdk5rap3条件性敲除小鼠中,利用DEN诱发肝细胞癌的生长,通过与野生型小鼠观察比较肝癌发生率及肿瘤生长状态,明确该基因缺失对肝癌发生的影响。观察发现,肝细胞特异地敲除Cdk5rap3显著提高了小鼠肝癌发生率,其肿瘤生长数量和质量均远远高于野生型小鼠；让人意外的是,病理分析发现肿瘤细胞中Cdk5rap3表达明显升高,这些细胞并非来自基因敲除的肝细胞。这些结果提示我们,条件性敲除部分肝细胞中Cdk5rap3可以显著提高小鼠肝细胞癌易感性；这一表型的改变更多地来自肝脏内细胞与细胞或细胞与微环境之间的相互作用。3、我们利用全外显子组测序发现在Flt3-ITD敲入小鼠中存在一个耐药变异(Flt3-ITD c.2076TA)。为验证该变异会引起肿瘤细胞耐药,我们克隆了cDNA,并将其回复为野生型碱基(Flt3-ITD c.2076T),在分别转化了Ba/F3细胞系之后检测了细胞对Quazartinib (AC220)、Sorafenib和Ponatinib的敏感性。与预期一致,我们发现,两个编码序列均可顺利转化Ba/F3细胞,均显示了持续活化的激酶活性；与Flt3-ITD c.2076T相比,Flt3-ITD c.2076TA使得肿瘤细胞对Quazartinib和Sorafenib表现出耐药,而两株细胞对Ponatinib均敏感,与已有报道结果一致。该实验证实了,Flt3-ITD敲入小鼠中发现的变异与人类白血病细胞 FLT3中出现的Gate-keeper耐药突变一样,均会导致肿瘤细胞对Quazartinib和Sorafenib产生耐药性,而Ponatinib可以克服这种耐药。小鼠模型体内存在的耐药突变,提醒我们在使用小鼠模型开展实验研究时需要谨慎设计对照实验,以排除潜在的影响因素。总之,三方面的课题,分别从正向遗传学和反向遗传学的角度,使得自己可以利用多种遗传手段进行肿瘤学相关研究,并对它们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 .
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2
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本文编号：2057123