津白2小鼠自发性乳腺癌的克隆演化研究
发布时间：2018-08-25 19:19
【摘要】：目的:津白2小鼠(Tientsin Albino II,TA2)是天津医科大学培育成功的高发乳腺癌纯系小鼠,是被国际小鼠遗传标准命名委员会(International Committee on Standardized Genetic Nomenclature for Mice,ICLAS)和我国卫生部承认的近交系小鼠。自发性乳腺癌是TA2小鼠的主要特征,不经过任何人工处理,经产TA2母鼠的乳腺癌发生率为81%。与其他类型的小鼠模型相比,TA2自发性乳腺癌最突出的优点是能够反映乳腺癌的自然进程。肿瘤的克隆演化(clonal evolution)是指在自然选择作用下,具有不同基因突变的肿瘤细胞相互竞争,肿瘤由起始的单一克隆,演变为多个亚克隆,这类似于物种的达尔文进化过程。既往研究显示不同类型乳腺癌的基因组的克隆演化具有各自的特征,TA2小鼠的遗传背景相同,其自发性乳腺癌的克隆演化具有什么特点尚不清楚。为了揭示TA2自发性乳腺癌的克隆演化特征,本研究首先明确了TA2自发性乳腺癌的病理学特征和分子分型;对4个TA2自发性乳腺癌标本进行了全基因组重测序,分析比较样本之间的克隆结构和克隆演化特点;另外,在本研究中初步验证了TA2小鼠DNA双链断裂(DNA double-strand breaks,DSBs)修复机制的缺陷。方法:1.收集17例TA2自发性乳腺癌样本,观察小鼠的见瘤时间、产次和肿瘤发生部位;HE染色观察TA2自发性乳腺癌的病理学特点;应用免疫组织化学染色检测ER、PR、HER2、EGFR,确定TA2自发性乳腺癌的分子分型;应用免疫组织化学染色检测Ki67的表达情况,评估TA2自发性乳腺癌的增殖活性。2.选取4例TA2自发性乳腺癌(R01、R02、R03、R04)样本,其中R01和R02来源于同一只TA2小鼠,R01是原发性乳腺癌,R02是颈背部转移性乳腺癌,R03和R04分别来自另外2只TA2,均为原发性乳腺癌;4个样本的病理学类型均为浸润性导管癌,其中R03的组织学分级为3级,其余3个样本均为2级;4个样本的分子分型均为基底细胞样乳腺癌。对TA2自发性乳腺癌和鼠尾DNA进行全基因组重测序,参考基因组为Mouse.C57BL/6J,进行以下生物信息学分析:(1)检测和注释单核苷酸变异(Single Nucleotide Variation,SNV)、小片段插入/缺失(small In Del)、结构变异(Structure Variation,SV)和拷贝数变异(copy number variation,CNV)等遗传学变异;并对变异的基因进行功能注释,筛选出TA2自发性乳腺癌相关的遗传学变化。(2)根据SNV、CNV和LOH等遗传变异,应用Sci Clone方法和EXPANDS方法进行TA2自发性乳腺癌的克隆演化研究。3.原代培养TA2小鼠、TA1低瘤鼠和C57BL/6小鼠胚胎成纤维细胞(Mouse embryonic fibroblasts,MEFs),应用博来霉素处理各MEFs,造成人为的DSBs,应用免疫荧光检测γ-H2AX,比较TA2与其他种系小鼠的DSB修复能力的不同;应用双抗体夹心ELISA法检测TA2、TA1和C57BL/6小鼠的血清Ig A水平,比较TA2与其他种系小鼠的非同源末端连接(non-homologous end joining,NHEJ)能力的不同。结果:1.17例TA2自发性乳腺癌小鼠的平均见瘤鼠龄为315±71天;荷瘤鼠均为经产母鼠,平均分娩次数为3.06±1.12次;肿瘤好发部位依次为右前胸(8例)、右下腹(4例)、左下腹(4例)和左前胸(1例),另有2例合并颈背部肿瘤,其他部位未见肿瘤。17例TA2自发性乳腺癌在组织病理学上均与人乳腺浸润性导管癌类似;大多数样本的组织学分级为2级,除了1例的组织学分级为3级;分子分型均类似于人基底细胞样乳腺癌的表型:ER、PR和HER2均为阴性,EGFR阳性表达;TA2自发性乳腺癌Ki67高表达,细胞增殖活性高。2.对全基因组测序数据进行分析,包括遗传变异分析和克隆演化分析两部分。(1)遗传变异分析:与参考基因组相比,TA2自发性乳腺癌存在一系列的SNVs、In Dels、CNVs和SVs等遗传学变异。全基因组范围内,4个样本的SNVs均以CT或TC为主;韦恩(Venn)分析显示,全基因组范围内,4个样本之间99.02%SNVs是相同的,95.45%In Dels是相同的。对蛋白编码区(coding sequence,CDS)区域发生变异的基因进行功能注释,共分为25类不同功能的基因,4个样本之间每类基因的变异基因数目基本一致。结合基因功能注释,我们对参与DSBs修复的两个经典通路——同源重组(homologous recombination,HR)和NHEJ的基因进行分析,发现4个样本中均发生相同的DSB修复相关基因Brca2、Rad52、Rif1和Trp53bp1的基因突变;结合文献,对16个乳腺癌相关的DNA修复相关基因进行分析,发现4个样本中均发生相同的Trp53、Kmt2c基因突变。(2)克隆演化分析:应用Sci Clone方法分析发现,4个样本均含有6个亚克隆,对4个样本两两比较发现,各个亚克隆的等位基因频率(variant allele frequency,VAF)差别不大,提示4个样本的亚克隆结构类似;应用EXPANDS方法分析发现,4个样本均有11个亚克隆,4个样本各亚克隆的系统发生树均呈“树状”结构,提示4个样本的克隆演化模式相类似。3.(1)博来霉素处理MEFs后,γ-H2AX在细胞核内呈点状聚集(foci)。去除博来霉素4h后,TA2、TA1和C57BL/6 MEFs的γ-H2AX聚集点的平均数目分别为23.96±7.19、21.52±6.80和21.30±6.47,两两比较3种MEFs的γ-H2AX聚集点数目,组间差异没有统计学意义(P0.05)。去除博来霉素24h后,TA2、TA1和C57BL/6 MEFs的γ-H2AX聚集点的平均数目分别为5.98±2.27、1.22±0.98和1.32±0.79,TA1和C57BL/6 MEFs的γ-H2AX聚集点数目的差异没有统计学意义(P0.05);分别与TA1和C57BL/6 MEFs相比,TA2 MEFs的γ-H2AX聚集点数目更多(P0.05),显示TA2 MEFs产生的γ-H2AX聚集点消失的更慢,提示TA2小鼠存在DSB修复缺陷。(2)TA2、TA1和C57BL/6小鼠血清的Ig A浓度分别为418.60±47.18μg/m L、472.69±36.46μg/m L、470.34±37.49μg/m L。TA1和C57BL/6小鼠血清中Ig A水平的差异没有统计学意义(P0.05);分别与TA1和C57BL/6相比,TA2小鼠血清中Ig A水平下降(P0.05),提示TA2小鼠可能存在NHEJ缺陷。结论:本研究初步证实TA2自发性乳腺癌不同个体之间在组织病理学类型、分子分型、遗传变异和克隆演化等方面类似,具体表现在:(1)病理学类型类似于人乳腺浸润性导管癌,大部分的组织学分级为2级;(2)分子分型均为基底细胞样乳腺癌;(3)全基因组的遗传变异类似,具有相同的DSB修复基因突变。(4)克隆演化分析显示TA2自发性乳腺癌的克隆结构和克隆演化类似。基于以上证据,我们推测TA2自发性乳腺癌基因组的克隆演化存在某种相对固定的模式。本研究还初步证实了TA2可能存在DSB修复缺陷,这可能是TA2自发性乳腺癌形成的一个重要原因。
[Abstract]:OBJECTIVE: Tientsin Albino II (TA2) is a high-risk pure-line breast cancer mouse bred by Tianjin Medical University. It is an inbred mouse recognized by the International Committee on Standardized Genetic Nomenclature for Mice (ICLAS) and the Ministry of Health of China. The main feature of the mice, without any artificial treatment, was that the incidence of breast cancer in TA2-bearing mothers was 81%. Mutant tumor cells compete with each other, and the tumor evolves from a single clone to multiple subclones, which is similar to the Darwinian evolution of species. In order to reveal the clonal evolution of TA2 spontaneous breast cancer, we first clarified the pathological features and molecular typing of TA2 spontaneous breast cancer, sequenced the whole genome of four TA2 spontaneous breast cancer specimens, analyzed and compared the clonal structure and clonal evolution characteristics between the samples; and, in addition, in this study, we studied the clonal evolution of TA2 spontaneous breast cancer. Methods: 1. Seventeen cases of TA2 spontaneous breast cancer were collected to observe the time of tumor occurrence, parity and location of tumor occurrence; HE staining was used to observe the pathological characteristics of TA2 spontaneous breast cancer; immunohistochemical staining was used to detect ER, PR, H. ER2 and EGFR were used to determine the molecular typing of TA2 spontaneous breast cancer; immunohistochemical staining was used to detect the expression of Ki67 and evaluate the proliferative activity of TA2 spontaneous breast cancer. Sexual breast cancer, R03 and R04 were derived from two other TA2 breast cancers, all of them were primary breast cancer; the pathological types of the four samples were invasive ductal carcinoma, of which the histological grade of R03 was grade 3 and the other three samples were grade 2; the molecular typing of the four samples were basal cell-like breast cancer. The following bioinformatics analyses were performed: (1) Detection and annotation of single nucleotide variations (SNV), small in Del, structural variations (SV) and copy number variations (CNV) and other genetic variations. (2) According to the genetic variation of SNV, CNV and LOH, the clonal evolution of TA2 spontaneous breast cancer was studied by Sci Clone method and EXPANDS method. 3. Primary culture of TA2 mice, TA1 hypotumor mice and C57BL/6 mouse embryonic fibroblasts (Mouse embryonic fibroblasts) Blasts, MEFs) were treated with bleomycin to induce artificial DSBs. Immunofluorescence was used to detect gamma-H2AX and compare the repair ability of DSB between TA2 and other strains of mice. The serum Ig A levels of TA2, TA1 and C57BL/6 mice were detected by sandwich ELISA, and the non-homologous End-Joints (non-homolog) between TA2 and other strains of mice were compared. Results: The average age of tumor mice in 17 TA2 spontaneous breast cancer mice was 315 65 17 cases of TA2 spontaneous breast cancer were similar to invasive ductal carcinoma of human breast in histopathology; most of the samples were histologically graded 2, except 1 case was histologically graded 3; the molecular typing was similar to the phenotype of human basal cell-like breast cancer: ER, PR and HER2 were negative, and EGFR was positive; Genetic variation analysis: Compared with reference genome, TA2 spontaneous breast cancer has a series of genetic variations such as SNVs, In Dels, CNVs and SVs. All the SNVs in the four samples were mainly CT or TC. Venn analysis showed that 99.02% of the SNVs were the same in the whole genome and 95.45% of the in Dels were the same in the four samples. According to the functional annotation, we analyzed the two classical pathways involved in DSBs repair, homologous recombination (HR) and NHEJ, and found that the same DSB repair related genes Brca2, Rad52, Rif1 and Trp53bp1 were mutated in all four samples. Sixteen breast cancer-related DNA repair-related genes were analyzed, and the same Trp53 and Kmt2c gene mutations were found in all four samples. (2) Clonal evolution analysis: Sci Clone analysis showed that all the four samples contained six subclones, and the allele frequ frequencies of each subclone were found by comparing two of the four samples. The results of EXPANDS analysis showed that there were 11 subclones in all four samples, and the phylogenetic trees of each subclone in all four samples showed a "tree" structure, suggesting that the clonal evolution patterns of the four samples were similar. After removing bleomycin for 4 hours, the average number of gamma-H2AX aggregation points of TA2, TA 1 and C57BL/6 MEFs were 23.96 (+ 7.19), 21.52 (+ 6.80) and 21.30 (+ 6.47), respectively. There was no significant difference in the number of gamma-H2AX aggregation points of three MEFs between groups (P 0.05). After removing bleomycin for 24 hours, the aggregation points of TA2, TA 1 and C57BL/6 MEFs were 23.96 (+ 7.19), 21.52 (+ 6.80) and 21.30 (+ 6.47). There was no significant difference in the number of gamma-H2AX aggregation points between TA 1 and C57BL/6 MEFs (P 0.05). Compared with TA 1 and C57BL/6 MEFs, the number of gamma-H2AX aggregation points of TA2 MEFs was more (P 0.05), indicating that the disappearance of gamma-H2AX aggregation points produced by TA2 MEFs was slower, suggesting the presence of DSB in TA2 mice. (2) The serum Ig A levels of TA2, TA1 and C57BL/6 mice were 418.60 (+47.18) ug/m L, 472.69 (+36.46) ug/m L, 470.34 (+37.49) ug/m L.TA1 and C57BL/6 mice, respectively. There was no significant difference in serum Ig A levels between TA2, TA1 and C57BL/6 mice (P 0.05), suggesting that TA2 mice might have lower serum Ig A levels than TA1 and C57BL/6 mice (P 0.05). Conclusion: This study preliminarily confirmed that the histopathological types, molecular typing, genetic variation and clonal evolution of TA2 spontaneous breast cancer were similar among different individuals. The specific manifestations were as follows: (1) Pathological types were similar to human breast invasive ductal carcinoma, most of which were histologically graded to grade 2; (2) Molecular typing was basal. (4) Clonal evolution analysis showed that the clonal structure and clonal evolution of TA2 spontaneous breast cancer were similar. Based on the above evidence, we speculated that there was a relatively fixed pattern in the clonal evolution of TA2 spontaneous breast cancer genome. It is preliminarily confirmed that TA2 may have defective DSB repair, which may be an important cause of spontaneous breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9
[Abstract]:OBJECTIVE: Tientsin Albino II (TA2) is a high-risk pure-line breast cancer mouse bred by Tianjin Medical University. It is an inbred mouse recognized by the International Committee on Standardized Genetic Nomenclature for Mice (ICLAS) and the Ministry of Health of China. The main feature of the mice, without any artificial treatment, was that the incidence of breast cancer in TA2-bearing mothers was 81%. Mutant tumor cells compete with each other, and the tumor evolves from a single clone to multiple subclones, which is similar to the Darwinian evolution of species. In order to reveal the clonal evolution of TA2 spontaneous breast cancer, we first clarified the pathological features and molecular typing of TA2 spontaneous breast cancer, sequenced the whole genome of four TA2 spontaneous breast cancer specimens, analyzed and compared the clonal structure and clonal evolution characteristics between the samples; and, in addition, in this study, we studied the clonal evolution of TA2 spontaneous breast cancer. Methods: 1. Seventeen cases of TA2 spontaneous breast cancer were collected to observe the time of tumor occurrence, parity and location of tumor occurrence; HE staining was used to observe the pathological characteristics of TA2 spontaneous breast cancer; immunohistochemical staining was used to detect ER, PR, H. ER2 and EGFR were used to determine the molecular typing of TA2 spontaneous breast cancer; immunohistochemical staining was used to detect the expression of Ki67 and evaluate the proliferative activity of TA2 spontaneous breast cancer. Sexual breast cancer, R03 and R04 were derived from two other TA2 breast cancers, all of them were primary breast cancer; the pathological types of the four samples were invasive ductal carcinoma, of which the histological grade of R03 was grade 3 and the other three samples were grade 2; the molecular typing of the four samples were basal cell-like breast cancer. The following bioinformatics analyses were performed: (1) Detection and annotation of single nucleotide variations (SNV), small in Del, structural variations (SV) and copy number variations (CNV) and other genetic variations. (2) According to the genetic variation of SNV, CNV and LOH, the clonal evolution of TA2 spontaneous breast cancer was studied by Sci Clone method and EXPANDS method. 3. Primary culture of TA2 mice, TA1 hypotumor mice and C57BL/6 mouse embryonic fibroblasts (Mouse embryonic fibroblasts) Blasts, MEFs) were treated with bleomycin to induce artificial DSBs. Immunofluorescence was used to detect gamma-H2AX and compare the repair ability of DSB between TA2 and other strains of mice. The serum Ig A levels of TA2, TA1 and C57BL/6 mice were detected by sandwich ELISA, and the non-homologous End-Joints (non-homolog) between TA2 and other strains of mice were compared. Results: The average age of tumor mice in 17 TA2 spontaneous breast cancer mice was 315 65 17 cases of TA2 spontaneous breast cancer were similar to invasive ductal carcinoma of human breast in histopathology; most of the samples were histologically graded 2, except 1 case was histologically graded 3; the molecular typing was similar to the phenotype of human basal cell-like breast cancer: ER, PR and HER2 were negative, and EGFR was positive; Genetic variation analysis: Compared with reference genome, TA2 spontaneous breast cancer has a series of genetic variations such as SNVs, In Dels, CNVs and SVs. All the SNVs in the four samples were mainly CT or TC. Venn analysis showed that 99.02% of the SNVs were the same in the whole genome and 95.45% of the in Dels were the same in the four samples. According to the functional annotation, we analyzed the two classical pathways involved in DSBs repair, homologous recombination (HR) and NHEJ, and found that the same DSB repair related genes Brca2, Rad52, Rif1 and Trp53bp1 were mutated in all four samples. Sixteen breast cancer-related DNA repair-related genes were analyzed, and the same Trp53 and Kmt2c gene mutations were found in all four samples. (2) Clonal evolution analysis: Sci Clone analysis showed that all the four samples contained six subclones, and the allele frequ frequencies of each subclone were found by comparing two of the four samples. The results of EXPANDS analysis showed that there were 11 subclones in all four samples, and the phylogenetic trees of each subclone in all four samples showed a "tree" structure, suggesting that the clonal evolution patterns of the four samples were similar. After removing bleomycin for 4 hours, the average number of gamma-H2AX aggregation points of TA2, TA 1 and C57BL/6 MEFs were 23.96 (+ 7.19), 21.52 (+ 6.80) and 21.30 (+ 6.47), respectively. There was no significant difference in the number of gamma-H2AX aggregation points of three MEFs between groups (P 0.05). After removing bleomycin for 24 hours, the aggregation points of TA2, TA 1 and C57BL/6 MEFs were 23.96 (+ 7.19), 21.52 (+ 6.80) and 21.30 (+ 6.47). There was no significant difference in the number of gamma-H2AX aggregation points between TA 1 and C57BL/6 MEFs (P 0.05). Compared with TA 1 and C57BL/6 MEFs, the number of gamma-H2AX aggregation points of TA2 MEFs was more (P 0.05), indicating that the disappearance of gamma-H2AX aggregation points produced by TA2 MEFs was slower, suggesting the presence of DSB in TA2 mice. (2) The serum Ig A levels of TA2, TA1 and C57BL/6 mice were 418.60 (+47.18) ug/m L, 472.69 (+36.46) ug/m L, 470.34 (+37.49) ug/m L.TA1 and C57BL/6 mice, respectively. There was no significant difference in serum Ig A levels between TA2, TA1 and C57BL/6 mice (P 0.05), suggesting that TA2 mice might have lower serum Ig A levels than TA1 and C57BL/6 mice (P 0.05). Conclusion: This study preliminarily confirmed that the histopathological types, molecular typing, genetic variation and clonal evolution of TA2 spontaneous breast cancer were similar among different individuals. The specific manifestations were as follows: (1) Pathological types were similar to human breast invasive ductal carcinoma, most of which were histologically graded to grade 2; (2) Molecular typing was basal. (4) Clonal evolution analysis showed that the clonal structure and clonal evolution of TA2 spontaneous breast cancer were similar. Based on the above evidence, we speculated that there was a relatively fixed pattern in the clonal evolution of TA2 spontaneous breast cancer genome. It is preliminarily confirmed that TA2 may have defective DSB repair, which may be an important cause of spontaneous breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9
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7 马骥;赵庆利;刘文超;;RhoA在缺氧诱导的乳腺癌新生血管形成中的功能及其机制研究[A];中华医学会肿瘤学分会第七届全国中青年肿瘤学术会议——中华医学会肿瘤学分会“中华肿瘤 明日之星”大型评选活动暨中青年委员全国遴选论文汇编[C];2011年
8 严枫;谭旭艳;耿尧;鞠q，
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