小鼠口腔癌模型骨髓播散细胞RB1CC1基因纯合性缺失的初步研究
发布时间：2018-10-08 16:33
【摘要】：目的：(1)初步探讨小鼠口腔癌模型骨髓单个播散细胞基因组的检测方法。(2)初步探讨小鼠口腔癌模型舌重度异常增生时期骨髓播散细胞RB1CC1基因纯合性缺失及其意义。方法：(1)4NQ0饮水法建立小鼠口腔癌淋巴道转移模型。定期处死小鼠,收集双侧股骨,Ficoll密度梯度离心法提取骨髓单个核细胞层的细胞,并制作细胞涂片。以细胞角蛋白(CK)多克隆抗体为一抗免疫组化染色细胞涂片。小鼠的舌部进行常规HE染色检测。(2)收集35只小鼠口腔癌模型舌重度异常增生时期骨髓单个核细胞涂片,激光捕获显微切割技术(LCM)捕获细胞涂片上的单个CK阳性(CK+)细胞。(3)单细胞全基因组扩增技术(WGA)扩增单个细胞的DNA。(4)聚合酶链反应(PCR)检测其视网膜母细胞瘤诱导卷曲蛋白(RB1CC1)基因的第二、第六和第七外显子纯合性缺失情况,并与舌部正常,重度异常增生以及鳞癌组织各4例对照。结果：(1)LCM切割46个CK+细胞,成功从35只小鼠骨髓涂片各捕获1个CK+细胞,总共35个细胞,成功率76%。(2)扩增CK+细胞35个,单细胞WGA扩增成功且PCR成功扩增出内参GAPDH的单细胞为3个,成功率8.6%。(3)RB1CC1基因第二外显子纯合性缺失情况分别为单个CK+细胞(2/3),舌癌变组织(2/4),舌部正常组织(0/4),重度异常增生组织(0/4)；RB1CC1基因第六外显子纯合性缺失情况分别为单个CK+细胞(0/3),舌癌变组织(0/4),舌部正常组织(0/4),重度异常增生组织(0/4)；RB1CC1基因第七外显子纯合性缺失情况分别为单个CK+细胞(0/3),舌癌变组织(0/4),舌部正常组织(0/4),重度异常增生组织(0/4)。结论：(1)LCM技术结合单细胞WGA技术以及PCR技术可应用于小鼠口腔癌模型骨髓播散细胞基因突变的分析。(2)小鼠口腔癌模型重度异常增生时期骨髓CK+细胞有RB1CC1基因第二外显子的纯合性缺失突变,该时期骨髓CK+细胞可能是播散肿瘤细胞。
[Abstract]:Objective: (1) to explore the method of detecting the genome of bone marrow single disseminated cell in mouse oral carcinoma model. (2) to explore the homozygous deletion of RB1CC1 gene in bone marrow diffusing cells during severe dysplasia of tongue in mouse oral carcinoma model. Methods: (1) 4NQ0 drinking water method was used to establish lymphatic metastasis model of oral cancer in mice. The cells of bone marrow mononuclear cell layer were collected by Ficoll density gradient centrifugation, and cell smears were prepared. Cytokeratin (CK) polyclonal antibody was used as an anti-immunohistochemical staining cell smear. The tongue of mice was detected by routine HE staining. (2) Bone marrow mononuclear cell smears were collected from 35 mice with oral cancer during severe dysplasia of tongue. Laser capture microdissection technique (LCM) capture cell smear single CK positive (CK) cell. (3) single cell whole genome amplification technique (WGA) amplification of single cell DNA. (4) polymerase chain reaction (PCR) detection of its retinoblastoma induced crimp The second protein (RB1CC1) gene, The homozygous deletions of exon 6 and exon 7 were compared with those of normal tongue, severe dysplasia and squamous cell carcinoma. Results: (1) 46 CK cells were incised by LCM, one CK cell was successfully captured from 35 mouse bone marrow smears, 35 cells were successfully obtained, and the success rate was 76.5%. (2) 35 CK cells were successfully amplified, and 3 of them were successfully amplified by single cell WGA and successfully amplified by PCR. (3) homozygous deletion of exon 2 of RB1CC1 gene was found in single CK cell (2 / 3), tongue carcinoma (2 / 4), normal tongue (0 / 4) and severe dysplasia (0 / 4). Homozygous deletion of exon 6 of RB1CC1 gene was found in single CK cell (0 / 3), tongue carcinoma (0 / 4), normal tongue (0 / 4) and severe dysplasia (0 / 4). Homozygous deletions of exon 7 of RB1CC1 gene were found in single CK cell (0 / 3), tongue carcinoma (0 / 4), normal tongue (0 / 4) and severe dysplasia (0 / 4). Conclusion: (1) LCM technique combined with single cell WGA technique and PCR technique can be used to analyze gene mutation of bone marrow diffuser cells in mouse oral carcinoma model. (2) there is the second RB1CC1 gene in bone marrow CK cells during severe dysplasia of mouse oral carcinoma model. Homozygous deletion mutations in exons, Bone marrow CK cells may be disseminated tumor cells during this period.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.8
本文编号：2257474
[Abstract]:Objective: (1) to explore the method of detecting the genome of bone marrow single disseminated cell in mouse oral carcinoma model. (2) to explore the homozygous deletion of RB1CC1 gene in bone marrow diffusing cells during severe dysplasia of tongue in mouse oral carcinoma model. Methods: (1) 4NQ0 drinking water method was used to establish lymphatic metastasis model of oral cancer in mice. The cells of bone marrow mononuclear cell layer were collected by Ficoll density gradient centrifugation, and cell smears were prepared. Cytokeratin (CK) polyclonal antibody was used as an anti-immunohistochemical staining cell smear. The tongue of mice was detected by routine HE staining. (2) Bone marrow mononuclear cell smears were collected from 35 mice with oral cancer during severe dysplasia of tongue. Laser capture microdissection technique (LCM) capture cell smear single CK positive (CK) cell. (3) single cell whole genome amplification technique (WGA) amplification of single cell DNA. (4) polymerase chain reaction (PCR) detection of its retinoblastoma induced crimp The second protein (RB1CC1) gene, The homozygous deletions of exon 6 and exon 7 were compared with those of normal tongue, severe dysplasia and squamous cell carcinoma. Results: (1) 46 CK cells were incised by LCM, one CK cell was successfully captured from 35 mouse bone marrow smears, 35 cells were successfully obtained, and the success rate was 76.5%. (2) 35 CK cells were successfully amplified, and 3 of them were successfully amplified by single cell WGA and successfully amplified by PCR. (3) homozygous deletion of exon 2 of RB1CC1 gene was found in single CK cell (2 / 3), tongue carcinoma (2 / 4), normal tongue (0 / 4) and severe dysplasia (0 / 4). Homozygous deletion of exon 6 of RB1CC1 gene was found in single CK cell (0 / 3), tongue carcinoma (0 / 4), normal tongue (0 / 4) and severe dysplasia (0 / 4). Homozygous deletions of exon 7 of RB1CC1 gene were found in single CK cell (0 / 3), tongue carcinoma (0 / 4), normal tongue (0 / 4) and severe dysplasia (0 / 4). Conclusion: (1) LCM technique combined with single cell WGA technique and PCR technique can be used to analyze gene mutation of bone marrow diffuser cells in mouse oral carcinoma model. (2) there is the second RB1CC1 gene in bone marrow CK cells during severe dysplasia of mouse oral carcinoma model. Homozygous deletion mutations in exons, Bone marrow CK cells may be disseminated tumor cells during this period.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.8
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