【摘要】：目的：(1)初步探討小鼠口腔癌模型骨髓單個(gè)播散細(xì)胞基因組的檢測(cè)方法。(2)初步探討小鼠口腔癌模型舌重度異常增生時(shí)期骨髓播散細(xì)胞RB1CC1基因純合性缺失及其意義。方法：(1)4NQ0飲水法建立小鼠口腔癌淋巴道轉(zhuǎn)移模型。定期處死小鼠,收集雙側(cè)股骨,Ficoll密度梯度離心法提取骨髓單個(gè)核細(xì)胞層的細(xì)胞,并制作細(xì)胞涂片。以細(xì)胞角蛋白(CK)多克隆抗體為一抗免疫組化染色細(xì)胞涂片。小鼠的舌部進(jìn)行常規(guī)HE染色檢測(cè)。(2)收集35只小鼠口腔癌模型舌重度異常增生時(shí)期骨髓單個(gè)核細(xì)胞涂片,激光捕獲顯微切割技術(shù)(LCM)捕獲細(xì)胞涂片上的單個(gè)CK陽(yáng)性(CK+)細(xì)胞。(3)單細(xì)胞全基因組擴(kuò)增技術(shù)(WGA)擴(kuò)增單個(gè)細(xì)胞的DNA。(4)聚合酶鏈反應(yīng)(PCR)檢測(cè)其視網(wǎng)膜母細(xì)胞瘤誘導(dǎo)卷曲蛋白(RB1CC1)基因的第二、第六和第七外顯子純合性缺失情況,并與舌部正常,重度異常增生以及鱗癌組織各4例對(duì)照。結(jié)果：(1)LCM切割46個(gè)CK+細(xì)胞,成功從35只小鼠骨髓涂片各捕獲1個(gè)CK+細(xì)胞,總共35個(gè)細(xì)胞,成功率76%。(2)擴(kuò)增CK+細(xì)胞35個(gè),單細(xì)胞WGA擴(kuò)增成功且PCR成功擴(kuò)增出內(nèi)參GAPDH的單細(xì)胞為3個(gè),成功率8.6%。(3)RB1CC1基因第二外顯子純合性缺失情況分別為單個(gè)CK+細(xì)胞(2/3),舌癌變組織(2/4),舌部正常組織(0/4),重度異常增生組織(0/4)；RB1CC1基因第六外顯子純合性缺失情況分別為單個(gè)CK+細(xì)胞(0/3),舌癌變組織(0/4),舌部正常組織(0/4),重度異常增生組織(0/4)；RB1CC1基因第七外顯子純合性缺失情況分別為單個(gè)CK+細(xì)胞(0/3),舌癌變組織(0/4),舌部正常組織(0/4),重度異常增生組織(0/4)。結(jié)論：(1)LCM技術(shù)結(jié)合單細(xì)胞WGA技術(shù)以及PCR技術(shù)可應(yīng)用于小鼠口腔癌模型骨髓播散細(xì)胞基因突變的分析。(2)小鼠口腔癌模型重度異常增生時(shí)期骨髓CK+細(xì)胞有RB1CC1基因第二外顯子的純合性缺失突變,該時(shí)期骨髓CK+細(xì)胞可能是播散腫瘤細(xì)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.8

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