基因修饰动物微卫星不稳定性发生的生物学效应与机制研究

Genetically modified animals often show phenotypes that are irrelated to the modified genes for unknown reasons. It has been confirmed that there is significant relationship between tumor and microsatellite instability (MSI), of which MSI was closely related to the prognosis of tumor and to the therapy effects of medicines. In our previous study, MSI was found to be existed in genetically modified mice and chemical induced mutant mice. In addition, the changed expression patterns of related genes could be detected when MSI occurred in genetically modified mice. So we hypothesize that: genetic modification or tumor may induce MSI. MSI has additional effects on related genes and induce unexpected phenotypes or tumor features in animals. In present study, in order to investigate the regulatory mechanism of MSI occurrence, we will detect MSI in knockout mice by microsatellite loci, which were prone to be MSI as demonstrated in our previous study. We will detect microsatellite loci that are linked to genes knocked out in those mice as well. Based on this, we will study the influence of MSI on the expression of related genes, and on the methylation and phosphorylation levels of these genes by RT-PCR, Western-blot, point mutation and inducing tumor by chemicals in vivo and in vitro. Furthermore, we will clarify the effect of MSI on the related gene function and characteristics of tumor. The results of the present study will be a theoretical basement of assessing on biological property of genetically modified animals and provide a creative method to treatment and evaluation on prognosis of tumor.

基因修饰动物常出现与修饰基因无关的表型变化，原因不明。大量研究证实肿瘤伴有微卫星不稳定（MSI）发生，且MSI与肿瘤的预后和药物敏感性高度相关。我们前期研究发现在基因修饰动物和化学物致突变动物中均存在MSI，MSI的发生可引起其他相关基因的表达变化。据此提出假说：基因修饰或肿瘤导致MSI发生，MSI作用于其他相关基因，从而引起该动物出现"预料之外"的表型性状或肿瘤特征改变。本研究拟采用前期筛选出的易发MSI的微卫星位点和敲除基因连锁的微卫星位点对基因敲除动物进行MSI检测，探讨MSI发生规律；在此基础上，利用RT-PCR、Western-blot、点突变、诱发肿瘤等技术手段，从体内和细胞水平研究MSI对相关基因表达水平的影响及其甲基化和磷酸化水平的调控，进一步阐明MSI对相关基因功能及肿瘤特征的影响，为基因修饰动物的生物学特性评价提供理论依据，为肿瘤的治疗和预后判断提供新思路。

大量报道已证实微卫星不稳定（microsatellite instability,MSI）和肿瘤高度相关，其发生机制却不明了。我们的前期研究证明在基因修饰动物和化学物致突变动物中均存在MSI，因此我们假设：基因修饰致使基因改变，导致MSI发生，MSI作用于相关基因，从而引起肿瘤发生、基因敲除动物出现敲除基因控制以外的性状。通过实验我们发现18种胚胎干细胞敲除动物中2种基因敲除小鼠有连锁位点的MSI现象（11.1%），MSI发生率为0.9%，且Cckar敲除小鼠MSI位点周围基因蛋白表达水平有变化；17种CRISPR/Cas9敲除动物有8种连锁位点发生了MSI（47.1%），MSI发生率为8.9%；21种CRISPR/Cas9敲除动物19种（89.5%）在42个易突变位点发生MSI，8个（19%）位点表现在MSI阳性，分析发现CRISPR/Cas9技术更易造成随机位点和TG连锁位点MSI的发生。我们利用MNU诱导C57BL/6J小鼠建立了胸腺淋巴瘤模型，成模率为61.3%，通过检测发现MNU诱导可以导致易突变位点和肿瘤相关基因内位点发生MSI，且MSI发生与肿瘤发生及呈负相关（P < 0.0001）。肿瘤相关基因表达水平及其甲基化水平的变化显示，某些CpG位点甲基化状态的改变与肿瘤相关基因的表达相关。我们分别筛选了人结直肠癌及胃癌肿瘤相关基因内部微卫星61个和91个，初步发现胃癌中P53基因内MSI发生于P53基因外显子突变呈负相关（P=0.0416）。本课题首次通过分析连锁位点和随机位点MSI的发生，研究了胚胎干细胞和CRISPR/Cas9两种基因敲除技术对基因组稳定性的影响，为基因修饰动物的生物特性评价提供理论依据。通过研究MNU诱导小鼠胸腺淋巴瘤模型MSI的发生及与肿瘤发生的相关性为接下来从临床上进一步阐明MSI与肿瘤发生发展、肿瘤的治疗和预后判定奠定了基础。